Research

Methodology
in Life Sciences
Volume I

Ranjit Singh
Copyright © 2024 by Ranjit Singh

All rights reserved.

ISBN: 978-81-19538-27-0
 Dedication

My Father Shri Yatindra Nath Singh

 Preface
Embark on a captivating journey into the intricate world of research methodology in life
sciences with 'Research Methodology in Life Sciences, Volume I.' Crafted with the intent
of igniting passion and curiosity, this textbook bridges fundamental principles with
dynamic lab practices, preparing students for research on living organisms. Rather than
a mere collection of facts, it presents research methodology as a narrative, unveiling the
fascinating stories of molecules and their roles in the tapestry of life. Adopting a
storytelling approach, it weaves interconnected pathways of research, engaging students
and making the subject matter relatable. Rich illustrations, diagrams, and molecular
models enhance comprehension and instill an appreciation for the beauty and complexity
of research methodology. As you embark on this adventure, may this volume spark your
curiosity, illuminate the wonders of the research world, and equip you with the knowledge
and skills to appreciate the intricate balance that sustains our curiosity. This book is
equally useful for B.Sc., M.Sc., Ph.D. Course work Students and Research scholars. Stay
tuned for the next volume in this exciting series!
 Acknowledgement
I am highly indebted to my parents, Shri Y. N. Singh and Smt. Basanti Singh, my wife, Dr. Suman Singh, my
daughters, Riddhima and Siddhika Singh, my niece Er. Aditya Singh, sister Rekha Singh and my brother
Advocate, Ajit Singh for their Patience and continuous love, support, encouragement, and of course, persuasion
to continue to study and understood the true value of education and rewards of the perseverance.

I would like to express my deepest gratitude to my colleagues, co-writers Dr. Amit Kumar Singh, Assistant
Professor, Department of Botany, University of Lucknow, Dr. Saumya Mishra, Assistant Professor, Department
of Botany, Kashi Naresh Government P. G. College, Gyanpur, Bhadohi. Dr. Soni Kumari, Assistant Professor,
Department of Botany, P. P. K. College, Ranchi University, Jharkhand, Dr. Maya Yadav, Assistant Professor,
Department of Botany, Dr. Shyama Prasad Mukherjee Government College, Bhadohi for suggesting the book
topic, his valuable writings, guidance and constant encouragement throughout the period of book writing.

I express my indebtedness to Prof. (Dr.) P. N. Dongre, Principal, Vishram Singh P. G. College, Chunar, Mirzapur
for encouragement to do such work for academic profile.

I am highly grateful to my Principal Dr. Shyama Prasad Mukherjee Government College, Bhadohi, Prof. (Dr.)
Shahid Pervez, for their critical evaluation, comments and valuable suggestion to write this book.

I am highly thankful to Dr. Rashmi Singh, Dr. K. A. Siddiqui, Dr. R. K. Pandey, Dr. Shefali Singh, Dr. Ravi Yadav,
Dr. Geeta Yadav and Dr. Suman Gupta, Department of Botany, K. N. Government P. G. College, Gyanpur,
Bhadohi for regular encouragement to write book on this topic.

My heartiest thanks to Dr. Shweta Singh, HOD, Assistant Professor, Department of Chemistry, Dr. Anurag Singh,
HOD, Dept. of Physics, Dr. Ashutosh Kumar Srivastava, HOD, Department of Zoology, Dr. Aneesh K. Mishra,
HOD, Dept. Of Geography, Dr. Rustam Ali, Dept. Of Zoology, Dr. R. K. Yadav, Dept. of Physics, Dr. Sujeet K. Singh,
HOD, Dept. Of Sociology, Dr. Shikha Tiwari, HOD, Dept. of Hindi, Dr. Ritwick Ranjan, Dept. of Sociology, Dr.
Poonam Dwivedi, Dept of Sociology, Dr Ashish Jaysawal, Dept of Chemistry, Dr Amit K. Tiwari, HOD, Dept of
English Dr. B. K. Maurya Dept of Zoology, GDC, Palhipatti, Dr. Sarveshanand dept. of Mathematics, Dr Bhawana
Singh, HOD, dept. of Mathematics and Shashank for their utmost cooperation, suggestion and selfless help.

Thanks to all other who have not been mentioned by name but have given invaluable helps in their own ways.

Ranjit Singh

Assistant Professor

Department of Botany

Dr. Shyama Prasad Mukherjee Government P. G. College, Bhadohi, Uttar Pradesh, India-221401
Table of Content
CHAPTER: 1 ................................................................................................................................................ 1
CHAPTER: 2 .............................................................................................................................................. 11
CHAPTER: 3 .............................................................................................................................................. 22
CHAPTER: 4 .............................................................................................................................................. 30
CHAPTER: 5 .............................................................................................................................................. 53
CHAPTER: 6 .............................................................................................................................................. 57
CHAPTER: 7 .............................................................................................................................................. 62
CHAPTER: 8 .............................................................................................................................................. 73
CHAPTER: 9 .............................................................................................................................................. 87
CHAPTER: 10 .......................................................................................................................................... 114
CHAPTER: 11 .......................................................................................................................................... 121
CHAPTER: 12 .......................................................................................................................................... 133
CHAPTER: 13 .......................................................................................................................................... 174
CHAPTER: 14 .......................................................................................................................................... 183
CHAPTER: 15 .......................................................................................................................................... 202
CHAPTER: 16 .......................................................................................................................................... 207
CHAPTER: 17 .......................................................................................................................................... 216
CHAPTER: 18 .......................................................................................................................................... 228
CHAPTER: 19 .......................................................................................................................................... 248
Chapter 1: Experimental Designs: Meaning, Principles and Factors

Chapter 2: Sterilization Techniques

Chapter 3: Isolation of pure culture

Chapter 4: Measurement and Quantification of microbial growth

Chapter 5: Maintenance and preservation of microbes (pure culture)

Chapter 6: Introduction to cell and tissue culture

Chapter 7: Culture types and Isolation of culture.

Chapter 8: Tissue culture media (composition and Preparation)

Chapter 9: Design and laboratory setup of tissue culture lab

Chapter 10: General Design and setup of Laboratory

Chapter 11: Chromatography techniques and its Applications

Chapter 12: Spectroscopy

Chapter 13: Molecular Techniques Comet assay analysis

Chapter 14: Real Time-Polymerase chain Reaction [RT-PCR]

Chapter 15: Random Amplified Polymorphic DNA (RAPD) Marker

Chapter 16: Restriction Fragment Length Polymorphism (RFLPs)

Chapter 17: ARDRA

Chapter 18: Fluorescence in Situ Hybridization (FISH)

Chapter 19: Principles and Design of experiments

 CHAPTER: 1
Experimental Designs: Meaning, Principles and Factors

Meaning of Experimental Designs:

Experimental designs are various types of plot arrangements which are used to test a set of
treatments to draw valid conclusions about a particular problem. Before dealing with experimental
designs, it is necessary to define experiment, treatment and experimental unit. A scientifically
planned method is called an experiment and various objects of comparison are known as
treatments. The group of material to which a treatment is applied in a single trial of experiment is
known as experimental unit. It may a plot of land, a patient in a hospital, a group of cattle in a
dairy, etc. Experimental designs are useful to a researcher in several ways as given below:

1. In reducing the soil heterogeneity and thereby experimental error to a considerable extent.

2. In testing the significance of difference among various objects of comparison.

3. In screening of various treatments in a scientific manner.

4. In partitioning of variation into different components.

5. In the assessment of variances and co-variances.

6. In proper interpretation of scientific results and drawing valid conclusions.

Principles of Experimental Designs:

There are three basic principles of experimental designs, viz., replication, randomization and local
control or error control. All these principles help in reducing the experimental error and thus make
the experiment more efficient.

These principles are briefly-described below:

1. Replication:

Repetition of treatments under investigation is known as replication. There are two main
advantages of replications:

i. Increase in replication increases the precision by reducing the error to a great extent. The increase
in precision results in two ways. In case of field experiments standard error is calculated as VrVE
/ r where VE = error variance and r = number of replications. Thus, it is clear that the standard
error of mean decreases as the number of replication increases. Increase in replication increases

1
the degree of freedom of error which decreases the value of t due to decrease in confidence interval.
The shortening of confidence interval is a good proof of increased precision.

ii. The error of experiment arises from the difference between the plots of the same treatment.
Thus, without replications estimate of error is not possible and without the estimate of error
comparisons are not possible.

Another way of reducing the experimental error is to increase the plot size. But the plot size cannot
be increased beyond 0.1 acre, because further increase in plot will increase the heterogeneity within
the plot and affect the advantage obtained by increasing the plot size.

As the smaller plots are homogeneous in fertility, it is better to keep the experimental area the
same and increase the number of replications by shortening the plot size.

2. Randomization:

Allocation of treatments of different plots by a random process is known as randomization of

treatments. Randomization gives equal chance to all the treatments for being allotted to a more
fertile plot as well as to a less fertile plot.

3. Local Control or Error Control:

The principle of making use of greater homogeneity in groups of experimental units for reducing
the experimental error is known as local control.

The fertility of the field may be of two types:

i. A major fertility variation which is usually marked by a fertility gradient.

ii. Sporadic or scattered fertility variations which are not systematic but are distributed in patches.

The first type of fertility effects are reduced by dividing the field into homogeneous blocks or
replications and the effects of second type of fertility are minimized by randomization of
treatments within the block. The experimental error should be reduced as far as possible because
lower error helps in determining the small real differences between the treatments.

Factors of Experimental Designs

The choice of experimental designs depends on three main factors, viz:

(1) Number and nature of treatments under study,

(2) Objective of the experiment, and

(3) Available resources.

2
Experimental Error:

The variation due to environmental factors or uncontrolled factors is called experimental error.

Correction Factor:

If refers to the square of grand total divided by the number of observations. Thus, correction factor
= (Grand total) 2/number of observations.

Critical Difference:

The least significant difference, greater than which all the differences are significant is known as
critical difference (CD).

Critical Difference = SE difference x t

S.E. difference = / r where VE = error variance, r = replications, t = table value at error degrees of
freedom.

Critical difference is used to compare the observed differences among different treatments. If the
difference is greater than critical difference, it is considered as significant and vice versa.

F Test:

It is a test of significance which is used for testing the significance of differences among several
treatments. It differs from z-test and t-test which are applied to test the significance of difference
between two treatment means or between sample mean and population mean. For F test first we
have to calculate F value.

F Value:

It is the ratio between the treatment variance and error variance.

It is also known as variance ratio and is estimated as given below:

F value = treatment variance/error variance:

The observed value of F is compared with the table value of F at appropriate degrees of freedom
and at desired level of probability (5% or 1%). If the observed value of F is more than the table
value, the differences among treatments are considered as significant and vice versa.

3
Comparison of Various Experimental Designs

Main 6 Types of Experimental Designs

The following points highlight the top six types of experimental designs. The types are:

1. Completely Randomized Design

2. Randomized Block Design

3. Latin Square Design

4. Split Plot Design

5. Lattice Design

6. Augmented Designs.

Experimental Design:

Type 1: Completely Randomized Design (CRD):

The design which is used when the experimental material is limited and homogeneous is known
as completely randomized design. This design is specially used for pot culture experiments.

The important characteristics of this design are given below:

The whole field is divided into plots of similar shape and size. The number of plots is equal to the
product of treatments and replications. These plots are then serially numbered.

ii. Replications: There is no restriction on the number of replications in this design. The number
of replications can vary from treatment to treatment. Normally, the number of replications for
different treatments should be equal to get the estimates of treatment effects with same precision.
The number of replication depends on the availability of experimental material and level of
precision required.

If the experimental material for some treatments is available in limited quantities, the number of
replications in such case can be reduced. If the estimates of certain treatments effects are required
with more precision, the number of their replications is increased. This type of flexibility in the
choice of replications is present in this design only.

4
iii. Randomization: The randomization is done treatment wise with the help of random table. First
random numbers equal to the number of plots are taken from the random table. From these random
numbers each treatment is assigned numbers as per number of replications. Then random

Numbers of each treatment are allotted to the plots bearing the serial number similar to the random
number.

iv. Local Control: The principle of local control or error control is not adopted in this design. The
error variance can only be reduced by selecting a homogeneous set of experimental units. This is
possible when the experimental area is limited. When the number of treatments is large, it may not
always be possible to get a large homogeneous set of experimental units. Under such situations, it
is hot desirable to adopt CRD.

v. Variation: In this design, the total variation is split into two components, namely variation due
to treatments and variation due to error.

vi. Standard Error:

The standard error is calculated as, where, VE = pooled error variance and r1 and r2 are number
of replications. If r1 = r2.

Merits:

I. Layout: The layout of this design is simple. In this design, any number of treatments
and replications can be used.
II. Analysis: The analysis of this design is simple. The analysis remains simple even when
some of the observations are missing or rejected.
III. Accuracy: This design provides maximum number of degrees of freedom for the
estimation of error as compared with other designs for a given number of treatments
and experimental units.
Demerits:

I. There is a basic assumption about the homogeneity of plots or experimental units which
is rarely met in field experiments. Hence this design is not suitable for field
experiments.
II. This design is not suitable when there is fertility variation in the field because no local
control is provided in this design.
Uses:

I. This design is useful for pot culture experiments where it is possible to obtain
homogeneous material.
II. This design is recommended when some of the experimental units will fail to respond
or likely to be destroyed.

5
Type 2 Randomized Block Design (RBD):

The experimental design which controls the fertility variation in one direction only is known as
randomized block design (RBD). Adoption of this design is useful when the variation between the
blocks is significant.

The main features of this design are briefly presented below :

I. Layout: First the experimental field is divided into homogeneous groups equal to the
number of replications. These homogeneous groups are known as blocks. Then each
block is further divided into plots of similar shape and size equal to the number of
treatments.
II. Replications: There is no restriction on the number of replications. However, all the
treatments should have equal number of replications.
III. Randomization: The treatments are allotted to the plots in each block by a random
process. Separate randomization is used in each block.
IV. Local Control: The principle of local control or error control is adopted in this design
by forming homogeneous blocks.
V. Variation: In this design, the total variation is split into three components, namely
blocks, treatments and error.
VI. Standard Error: The standard error is calculated as clip_image002_thumb1. If some
treatments have extra replications, the SE will be calculated as
clip_image004_thumb1.Where, r1 and r2 are the number of replications of the
treatment to be compared.
Merits:

I. Layout: The layout and analysis of this design are very simple. The data can be analysed
even when observations of some plots are missing on account of damage caused by
animals, insects or flood. In such case one or two damaged replications can be
discarded and analysis can be performed with the data of remaining replications.
II. Efficiency: A large number of treatments can be tested by this design, but the efficiency
of error control decreases beyond 20 due to increase in heterogeneity within the block.
III. Accuracy: This design provides more accurate results than CRD due to formation of
homogeneous blocks. This design controls the fertility variation in one direction.
Demerits :

I. When the missing values are numerous, this design is less convenient than CRD
because the analysis becomes more complicated.
II. The efficiency of error control decreases when the number of treatments becomes more
than 20, because increase in treatment increases the heterogeneity within the block.

6
Uses:

This design can be used when the fertility variation moves in one direction. In this design error is
reduced by forming homogeneous blocks.

Type 3. Latin Square Design (LSD):

The experimental design which simultaneously controls the fertility variation in two directions is
called Latin square design (LSD). In other words, Latin square designs are adopted for eliminating
the variation of two factors which are generally called rows and columns.

The main features of Latin square design are briefly described below:

I. Layout: In this design the field is divided into homogeneous blocks in two ways. The
blocks in one direction are commonly known as rows and the blocks in other direction
as columns. The number of plots in each row is the same as the number of plots in each
column. This number is equal to the number of treatments.
II. Replications: In this design, the number of replications per treatment is always equal
to the number of treatments.
III. Randomization: The treatments are assigned in such a way that every treatment occurs
once and only once in each row and each column. The easiest way of obtaining such
randomization is to start with reduced Latin square, which is the one in which the first
row and first column are arranged in alphabetical order, and then reshuffling the rows,
columns and treatments with the help of random numbers.
IV. Local Control: The principle of local control is adopted in this design. This design
controls the experimental error by forming rows and columns.
V. Variation: In this design, the total variation is separated into four components, namely
rows, columns, treatments and error.
VI. Standard Error: The standard error is calculated as clip_image002_thumb2 , where VE
= error variance and n = number of replications.
Merits:

I. Layout and Analysis: The layout and analysis of this design are simple.
II. Accuracy: This design provides more accurate results than CRD and RBD because the
fertility variation is controlled in two directions.
III. Efficiency: This design is useful for comparing a small group of treatments. The
maximum number of treatments for which we can use Latin square design is 12.
IV. When data from some of the plots are missing, Yates’ missing plot technique can be
used for analysis of data.
Demerits:

I. This design is less flexible and does not permit any number of replications for any
treatment. The number of replications is always equal to the number of treatments.

7
 II. This design can be used only for small number of treatments (maximum 12) because
the number of experimental units required for this design increases rapidly with the
increase in the number of treatments.
III. In the field experiments, it is easier to manage RBD than LSD because RBD can be
used even when the field is in strips or rectangular, while LSD requires square shaped
field.
Uses:

This design is used when fertility moves in two directions.

Type 4 Split Plot Design (SPD):

The experimental design in which experimental plots are split or divided into main plots, sub-plots
and ultimate-plots is called split plot design (SPD). In this design several factors are studied
simultaneously with different levels of precision. The factors are such that some of them require
larger plots like irrigation, depth of ploughing and sowing dates, and others require smaller plots.
The important characteristics of SPD are briefly described below:

I. Layout: The layout of this design consists of four steps as given below:

(a) First the experimental field is divided into homogeneous blocks equal to the number of
replications

(b) Then each block is divided into a number of plots equal to the number of levels of the first
factor. These plots are known as main plots.

(c) Then each main plot is divided into a number of sub-plots equal to the number of levels of
second factor.

(d) Then each sub-plot is divided into a number of ultimate plots equal to the number of levels of
third factor.

II. Replications: There is no restriction on the number of replications unlike LSD. However, the
number of replications should be uniform for all the treatments.

III. Randomization: The levels of the first factor are applied to the main plots of each block by the
random process. The sub-plot treatments are then allotted at random to the sub-plots within each
main plot. A separate randomization is carried out for each main plot. The levels of third factor
are allotted to the ultimate plots in each sub-plot by a separate randomization.

IV. Local Control: The principle of local control is adopted in this design by forming homogeneous
blocks.

V. Variation: In this design, the total variation is divided into several components, i.e., blocks, first
factor, second factor, third factor, their combinations and error a, b and c (Table 36.6).

8
Merits:

I. Accuracy: The effects of main plot, sub-plot and ultimate plots are estimated with
increasing trends of precision, while in RBD main effects and interactions are estimated
with the same precision.
II. Efficiency:
III. Several factors out of which some require larger plots and others smaller plots can be
tested in a single experiment with a very little extra cost.
IV. By inclusion of more factors in a experiment we can get additional information without
spending much for the same.
Demerits:

I. The layout and analysis of this design are more complicated as compared to that of
RBD and LSD.
II. This design provides lesser degrees of freedom for the estimation of error variance than
RBD.
Uses:

I. This design is useful when all the factors are not of equal importance, i.e., some of them
require larger plots and others require smaller plots.
II. When some of the factors have small amount of material, they can be used as sub-plots
or ultimate plots in this design.
Type 5 Lattice Design:

Lattice designs are incomplete block designs in which the number of varieties or treatments forms
a square. The important characteristics of lattice design are given below:

I. Layout: The experimental field is divided into homogeneous parts equal to the number
of replications. Each part is further divided into plots of equal size in such a way that
the number of plots should form a square and each replication has equal plots in each
direction (i.e., equal rows and columns).
II. Replications: There is no restriction on the number of replications. It would be better if
the number replication is 4 because it would make the layout compact.
III. Randomization: The treatments are randomized replication wise. Separate
randomization is done in each replication.
IV. Variation: The total variation is divided into five components, namely replications,
rows, columns, treatments and error (Table 36.7).
Demerits:

This design can only be used when the number of treatments to be used forms a square. Moreover,
the analysis of variance is complicated.

9
Type 6 Augmented Designs:

The concept of augmented design was developed by Federer (1956). This is an experimental design
which is used to test a large number of germplasm lines in a limited area. The important
characteristics of this design are given below:

I. Layout: In this design, standard or check varieties are replicated among the cultures.
Thus, standards are replicated and cultures are non-replicated. The number of check
varieties should be at least 4.
II. Replications: The number of replications depends on the number of check varieties to
be used. For example, if we have 4 check varieties, we should have a minimum of 5
replications to get more than 10 error degrees of freedom.
III. Randomization: The check varieties are first randomly allocated to the plots of each
block, and the germplasm lines are then randomly allocated to the remaining plots of
each block.
Merits:

This is an ideal and efficient design for testing a very large number of germplasm lines at a time
with small quantity of seed, on a limited piece of land.

Demerits:

The analysis of augmented design is very much complicated. In this design, the comparison of
cultures within a replication is easy but to compare culture of one replication with that of another
is very tedious job. In this design, there is no proper check of fertility variation.

10
 CHAPTER: 2
Sterilization Techniques

Sterilization, which is any practice, physical or chemical, that abolishes all forms of life, which is
used especially to destroy microorganisms, spores, propagules, Archaea and viruses. Specifically,
sterilization is the complete destruction of all forms of microorganisms by a suitable chemical
agent or by physical agents such as heat, either wet steam. under pressure at 120 °C (250 °F) or
more for at least 15 minutes, or dry heat at 160 to 180 °C (320 to 360 °F) for three hours.

Sterilization is indispensable for the complete destruction or removal of all microorganisms

(including spore-forming and non-spore forming bacteria, viruses, fungi, and protozoa) that could
contaminate pharmaceuticals or other materials and thereby constitute a health hazard. Since the
achievement of the absolute state of sterility cannot be demonstrated, the sterility of a
pharmaceutical preparation can be defined only in terms of probability. The efficacy of any
sterilization process will depend on the nature of the product, the extent and type of any
contamination, and the conditions under which the final product has been prepared. The
requirements for Good Manufacturing Practice should be observed throughout all stages of
manufacture and sterilization.

Classical sterilization techniques using saturated steam under pressure or hot air are the most
reliable and should be used whenever possible. Other sterilization methods include filtration,
ionizing radiation (gamma and electron-beam radiation), and gas (ethylene oxide, formaldehyde).

For products that cannot be sterilized in the final containers, aseptic processing is necessary.
Materials and products that have been sterilized by one of the above processes are transferred to
pre sterilized containers and sealed, both operations being carried out under controlled aseptic
conditions.

Whatever method of sterilization is chosen, the procedure must be validated for each type of
product or material, both with respect to the assurance of sterility and to ensure that no adverse
change has taken place within the product. Failure to follow precisely a defined, validated process
could result in a non-sterile or deteriorated product. A typical validation programed for steam or
dry-heat sterilization requires the correlation of temperature measurements, made with sensory
devices to demonstrate heat penetration and heat distribution, with the destruction of biological
indicators, i.e. preparations of specific microorganisms known to have high resistance to the
particular sterilization process. Biological indicators are also used to validate other sterilization
methods (see specific methods), and sometimes for routine control of individual cycles. Periodic

11
revalidation is recommended.

Sterilization

Chemical method
Physical method
Heat Radiation Filtration

Heat as physical method of sterilization is done either by dry heat or moist heat. In dry heat method,
Red heat, Flaming, Hot air oven and Infra-red radiation utilized as heating process to destroy
microorganisms. The Second physical method i.e. Radiation method, non-ionizing radiation viz.
ultraviolet, infrared radiation and in ionizing radiation, x-ray, gamma ray are used as sterilizing
agent used frequently. Third method, under the categories of physical method Filtration method
can be done. With Birchfield filter, chamberlain filter, Seitz filter, sintered glass filter, laminar
airflow, cellulose membrane filters are used as sterilization of microorganism

Heating in an autoclave (steam sterilization)

Under steam sterilization exposure of microorganisms to saturated steam under pressure in an

autoclave leads to their destruction by the permanent denaturation of enzymes and structural
proteins. The temperature at which denaturation occurs varies inversely with the volume of water
present. Sterilization in saturated steam thus necessitates precise control of time, temperature, and
pressure. As shift of the air by steam is dubious to be readily achieved, the air should be evacuated
from the autoclave before admittance of steam. This method should be used whenever promising
for aqueous preparations and for surgical dressings and medical equipment.

Autoclave

12
The references for sterilization in an autoclave are 15 minutes at 121-124 °C (200 kPa). The
temperature should be used to regulate and monitor the process; the pressure is mostly used to
obtain the required steam temperature. Other conditions, with different arrangements of time and
temperature, are given below.

1 atm. = 101 325 Pa = 101.325 K Pa

Temperature Approximate corresponding pressure (kPa) Minimum

(°C) sterilization time
(min)
126-129 250 (~2.5 atm.) 10
134-138 300 (~3.0 atm.) 5
Minimum sterilization time should be measured from the moment when all the materials to be
sterilized have reached the required temperature throughout. Monitoring the physical conditions
within the autoclave during sterilization is essential. To provide the required information,
temperature-monitoring probes should be inserted into representative containers, with additional
probes placed in the load at the potentially coolest parts of the loaded chamber (as established in
the course of the validation program). The conditions should be within ±2 °C and ±10 kPa (±0.1
atm.) of the required values. Each cycle should be recorded on a time-temperature chart or by other
suitable means.

Aqueous solutions in glass containers usually reach thermal equilibrium within 10 minutes for
volumes up to 100 mL and 20 minutes for volumes up to 1000 mL.

Porous loads, such as surgical dressings and related products, should be processed in an apparatus
that ensures steam penetration. Most dressings are adequately sterilized by maintaining them at a
temperature of 134 - 138 °C for 5 minutes. In certain cases, glass, porcelain, or metal articles are
sterilized at 121 - 124 °C for 20 minutes. Oils and fats may be sterilized at 121 °C for 2 hours but,
whenever possible, should be sterilized by dry heat.

In some cases (e.g. in thermolabile substances), sterilization may be carried out at temperatures
below 121 °C, provided that the chosen combination of time and temperature has been validated.
Lower temperatures offer a different level of sterilization; if this is evaluated in combination with
the known microbial burden of the material before sterilization, the lower temperatures may be
satisfactory. Specific conditions of temperature and time for certain preparations are stated in
individual monographs.

The bio indicator strain proposed for validation of this sterilization process is: spores of Bacillus
stearothermophilus (e.g. ATCC7953 or CIP 52.81) for which the D-value (i.e. 90% reduction of
the microbial population) is 1.5-2 minutes at 121 °C, using about 106 spores per indicator.

13
Dry-heat sterilization:

In dry-heat processes, the primary lethal process is considered to be oxidation of cell constituents.
Dry-heat sterilization requires a higher temperature than moist heat and a longer exposure time.
The method is, therefore, more convenient for heat-stable, non-aqueous materials that cannot be
sterilized by steam because of its deleterious effects or failure to penetrate. Such materials include
glassware, powders, oils, and some oil-based injectable.

Preparations to be sterilized by dry heat are filled in units that are either sealed or temporarily
closed for sterilization. The entire content of each container is maintained in the oven for the time
and at the temperature given in the table below. Other conditions may be necessary for different
preparations to ensure the effective elimination of all undesirable microorganisms.

Temperature (In degree) Minimum sterilization time (in minutes)

160 180
170 60
180 30

The oven should normally be equipped with a forced air system to ensure even distribution of heat
throughout all the materials processed. This should be controlled by monitoring the temperature.
Containers that have been temporarily closed during the sterilization procedure are sealed after
sterilization using aseptic techniques to prevent microbial recontamination.

The bio indicator strain proposed for validation of the sterilization process is: spores of Bacillus
subtilis (e.g. var. niger ATCC 9372 or CIP 77.18) for which the D-value is 5-10 minutes at 160 °C
using about 106 spores per indicator.

14
Filtration:

Sterilization by filtration is engaged mainly for thermolabile solutions. These may be sterilized by
channel through sterile bacteria-retaining filters, e.g. membrane filters (cellulose derivatives, etc.),
plastic, porous ceramic, or suitable sintered glass filters, or combinations of these. Asbestos-
containing filters should not be used.

Membrane
filter

Suitable measures should be taken to avoid loss of solute by adsorption onto the filter and to
prevent the release of contaminants from the filter. Suitable filters will prevent the passage of
microorganisms, but the filtration must be followed by an aseptic handover of the sterilized
solution to the final containers which are then instantly sealed with excessive care to exclude any
recontamination. Usually, membranes of not greater than 0.22 μm nominal pore size should be
used. The effectiveness of the filtration method must be validated if larger pore sizes are employed.
To confirm the integrity of filters, both before and after filtration, a bubble point or similar test
should be used, in agreement with the filter manufacturer's instructions. This test pays a prescribed
pressure to force air bubbles through the intact membrane previously wetted with the product, with
water, or with a hydrocarbon liquid.

15
All filters, tubes, and equipment used "downstream" must be sterile. Filters capable of
withstanding heat may be sterilized in the assembly before use by autoclaving at 121 °C for 15 -
45 minutes depending on the size of the filter assembly. The effectiveness of this sterilization
should be validated. For filtration of a liquid in which microbial growth is possible, the same filter
should not be used for procedures lasting longer than one working day.

Exposure to ionizing radiation:

Sterilization of certain active ingredients, drug products, and health devices in their finishing
container or package may be accomplished by exposure to ionizing radiation in the form of gamma
radiation from a suitable radio isotopic source such as 60Co (cobalt 60) or of electrons energized
by a suitable electron accelerator. Laws and regulations for protection against radiation must be
respected.

Gamma radiation and electron beams are used to influence ionization of the molecules in
organisms. Mutations are thus formed in the DNA and these reactions alter replication. These
processes are very hazardous and only well-trained and skilled staff should decide upon the
desirability of their use and should confirm monitoring of the processes. Specifically designed and
purpose-built installations and equipment requisite to be used. It is usual to select an absorbed
radiation level of 25 kGy1 (2.5 M rad) 2, although other levels may be engaged provided that they
have been authenticated.

1 kilo gray

2 mega rad

Radiation doses should be monitored with specific dosimeters during the entire process.
Dosimeters should be calibrated against a standard source on receipt from the supplier and at
appropriate intervals thereafter. The radiation system should be reviewed and validated whenever
the source material is changed and, in any case, at least once a year. The bio indicator strains
proposed for validation of this sterilization process are: spores of Bacillus pumilus (e.g. ATCC
27142 or CIP 77.25) with 25 kGy (2.5 Mrad) for which the D-value is about 3 kGy (0.3 Mrad)
using 107-108 spores per indicator; for higher doses, spores of Bacillus cereus (e.g. SSI C 1/1) or
Bacillus sphaericus (e.g. SSl C1A) are used.

Gas sterilization:

The active agent of the gas sterilization process can be ethylene oxide or another highly volatile
substance. The highly flammable and potentially explosive nature of such agents is a disadvantage
unless they are mixed with suitable inert gases to reduce their highly toxic properties and the
possibility of toxic residues remaining in treated materials. The whole process is difficult to control
and should only be considered if no other sterilization procedure can be used. It must only be
carried out under the supervision of highly skilled staff.

16
The sterilizing efficiency of ethylene oxide depends on the concentration of the gas, the humidity,
the time of exposure, the temperature, and the nature of the load. In particular, it is necessary to
ensure that the nature of the packaging is such that the gas exchange can take place. It is also
important to maintain sufficient humidity during sterilization. Records of gas concentration and of
temperature and humidity should be made for each cycle. Appropriate sterilization conditions must
be determined experimentally for each type of load. After sterilization, time should be allowed for
the elimination of residual sterilizing agents and other volatile residues, which should be confirmed
by specific tests. Because of the difficulty of controlling the process, efficiency must be monitored
each time using the proposed bio indicator strains: spores of Bacillus subtilis (e.g. var. niger ATCC
9372 or CIP 77.18) or of Bacillus stearothermophilus, (e.g. ATCC 7953 or CIP 52.81). The same
quantity of spores should be used as for "Heating in autoclave “and” Dry-heat sterilization".

Process Agent Technique

Physical process (A) Heat- 1. Hot air oven
- Dry heat 2. flaming
3. incineration
- Moist heat 1.steam under pressure
(autoclaving)
2.boiling
3. pasteurization

(B) Radiation
Non-ionizing 1.UV rays
2. Infrared

Ionizing 1. high energy electrons

2. gamma rays
(C) Filtration 1. Membrane filter
2.candle filter
3.asbestos pads
Chemical agents (A) Liquid 1. spraying
1. phenols 2.dusting
2. surface active agents 3, aqueous treatments
3. alcohols
4. halogens
5. metallic salts
6. dyes
7. aldehydes
(B) gaseous agents Fumigation
1. ethylene oxide
2. formaldehyde
3. beta-propinolactone

17
Sterilization of the Culture Vessels and Instruments:

For sterilization of culture vessels, glassware, cotton plugs, gauze, plastic caps, lab wares, filters
and pipettes it is better to use steam sterilization techniques i.e., by autoclaving at high pressure
(15 psi) and high temperature (121°C) for 15-20 mins. All the items should be properly covered
with aluminum foil before sterilization. Glassware metal instruments can also be sterilized by
exposure to dry hot air oven (160°-180°C) for 2-4 hrs. The metal instruments like forceps, scalpels,
needles, and spatula are further sterilized by flame sterilization technique before use by dipping in
95% alcohol, and followed by flaming and cooling. Metal equipment’s are generally not sterilized
by using autoclave to avoid rusting. Now-a-days flame sterilization is replaced by glass bead
sterilizer’s for safety purposes.

Sterilization of Nutrient Media:

The nutrient media used in tissue culture are commonly sterilized by autoclaving and filter
sterilization. Macro-, micronutrients, double distilled water and other stable compound mixtures
are autoclaved, whereas the thermolabile compounds are filter-sterilized separately and mixed with
the media whenever necessary. Some vitamins, amino acids, plant extracts and hormones are
thermolabile, they require filter sterilization. The solutions are passed through a bacterial
membrane filter under positive pressure. A Millipore or Seitz filter (pore size 0.2 µm) is used for
filter sterilization. The sterilized compound is then mixed with the autoclaved media.

Sterilization of Culture Rooms and Transfer-Area:

The floor and walls of culture rooms are cleaned by gently washing with detergent then by wiping
them with 2% sodium hypochlorite solution and then using 95% ethanol. The process of surface
sterilization of culture rooms should be done at regular intervals.

The transfer-area is also sterilized once or twice a month by washing with commercial brand of
antifungal liquid/powder. Transfer rooms which are large are further sterilised by UV light. UV
radiation is harmful for eyes, so UV radiation should be done before performing any laboratory
work. Time of sterilization varies according to the size of the room. Where laminar air flow cabinet
is used for transfer, the surface is cleaned by wiping with 95% ethanol 20 min before initiating the
operation and chamber is sterilised by UV light before work in progress. Then the sterile air is
flowed through HEPA filter during works.

Sterilization of Explant:

All different kinds of plant materials or explants should be surface sterilised by a variety of
chemicals before using in tissue culture. It is the eradication of surface microorganisms with the
help of different chemicals. The plants and organ tissues are surface sterilized to eliminate bacteria
and fungi only and they should not lose their biological activity. The type and concentration of
different chemical sterilant to be used for sterilization of different types of explants and exposure

18
time must be decided experimentally. Some common disinfectants used for sterilizing plant
materials are listed below Table Sometimes the sterilization procedure may lead to lethality to
plant tissue, so the use of disinfectants should be tested. Here are few disinfectants which are used
commonly for surface sterilization of different explants.

Table: Disinfectants, concentration and duration of treatment for surface sterilization of explants

Disinfectant Concentration Duration of treatment(min)

Sodium hypochlorite 0.5-5% 5-30
Calcium hypochlorite 9-10% 5-30
Ethyl alcohol 75-95% 2
Hydrogen peroxide 3-12% 5-15
Mercuric chloride 0.1-1.0% 2-10

Some common disinfectant used are given below-

➢ 1% solution of Sodium hypochlorite (NaClO), commercial bleach having 5% active

chlorine can be used.
➢ 4%-l0% solution of Calcium hypochlorite [Ca (ClO)2] can be used, it enters within the
plant tissue slower than sodium hypochlorite.
➢ 1% solution of Bromine-water.
➢ 0.01-0.1% solution of (HgCl2) which is an extremely toxic substance for plant tissue,
repeated rinsing with water is very much essential.
➢ 70% Ethyl alcohol is used for sterilization of plant material dipping them for 30 sec-2 mins
➢ 10% Hydrogen peroxide (H2O2) solution is effective for end surface sterilization
All these sterilizing agents should be washed out properly before using the explant as the retention
of these chemical substances may affect the establishment of successful tissue culture. But in most
of the cases it becomes difficult to determine the optimal conditions for each kind of tissue. So to
avoid this problem the explants can be taken from aseptically grown plants developed from the
surface sterilised seeds as these seeds are more resistant to chemicals due to presence of seed coat.
For this purpose, the seeds are surface sterilised and then cultured aseptically in basal nutrient
media. These give rise to aseptic seedlings from which the different explants can be used. Explants
from such seedlings need no further sterilization. But for anther culture and shoot tip culture the
explants are collected from outside grown plant. For these kinds of explants addition of few drops
of surfactant (Trito X or Tween20) to the solution or treating the plant material in a solution of
Catalon for 2 min. before exposing to sterilant may enhance sterilization efficiency.

Aseptic Transfer of Explant/Sub-Culturing:

Control of contamination largely depends upon the operator’s technique while transferring the
sterilized explant/sub-culturing into the sterile culture vial containing nutrient media under aseptic
condition. Dust from the surface, hair, hands and clothes are the potential sources of

19
contamination. Before starting the transfer procedure, the surface of transfer area and hands should
be wiped with 95% ethanol; sterile clothes (aprons) should be used. All the metallic equipment’s
used for transfer (inoculating needle, forceps, and scalpel) should be dipped into 95% ethanol and
then flamed and cooled. The tissue material should not touch the edge of culture vessel during
transfer.

Precautions taken during aseptic manipulation in tissue culture laboratory:

1. Laboratory should be entered wearing lab shoes and lab coats and keeping long hair tied
back.

2. Avoid handling alcohols around open flames.

3. on no occasion pipette by mouth.

4. Mask should be worn during inoculation.

5. Examine all equipment and media for visible contamination before use.

6. Stay planned as possible—label everything and set up all of your ingredients before getting
started.

7. Do not pass your hands/arms over open bottles, plate or tube.

8. Wipe done working surface with ethanol and turn on UV lamp for 10 min after finishing the
experiment.

Media Sterilization – Plant Tissue Culture Protocol

Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05 kg/cm 2 (15-
20 psi). The time required for sterilization depends upon the volume of medium in the vessel. The
minimum times required for sterilization of different volumes of medium are listed below. It is
advisable to dispense medium in small aliquots whenever possible as many media components are
broken down on prolonged exposure to heat. There is evidence that medium exposed to
temperatures in excess of 121 °C may not properly gel or may result in poor cell growth.
Table for minimum autoclaving time for plant tissue culture medium:

Medium volume per vessel (in Minimum autoclaving time (in

ml.) min.)
25 20
50 25
100 28
250 31

20
 500 35
1000 40
2000 48

Minimum autoclaving time is the time required for the volume of liquid to reach the sterilization
temperature 1210c and 15 min. at 121degree Celsius (Burger, 1988). Time may vary with make of
Autoclave.
Several medium constituents are considered thermolabile and should not be autoclaved. Stock
solutions of the heat labile components are prepared and filter sterilized through a 0.22 µm filter
into a sterile container. The filtered solution is aseptically added to the culture medium, which has
been autoclaved and allowed to cool to nearly 35-45 °C. The medium is then distributed under
sterile conditions. Investigation in accordance with your system is recommended.

21
 CHAPTER: 3
ISOLATION OF PURE CULTURE

Pure culture is a laboratory culture, having a particular species of organism. A pure

culture is usually derived from a mixed culture (one having many species) by
transferring a lesser sample into new, sterile growth medium in such a way as to
distribute the individual cells across the medium surface or by dilution of the
sample manifold before inoculating the new medium. Both methods separate the
individual cells so that, when they multiply, each will form a discrete colony,
which may then be used to inoculate more medium, with the guarantee that only
one type of organism will be present. Isolation of a pure culture may be enriched
by providing a mixed inoculum with a medium favoring the growth of one
organism to the exclusion of others.

A pure culture ideally contains a single bacterial species. There are a number of
ways available for the isolation of pure cultures from mixed populations. A pure
culture may be isolated by the use of distinctive media with specific chemical or
physical agents that permit the enrichment or selection of one organism over
another. The differential and selective procedures will be utilized. Modest methods
for isolation of a pure culture include:

➢ Spread plating on moderate solid agar medium with a glass spreader and
➢ Streak plating with a loop. The resolution of spread plating and streak
plating is to isolate individual bacterial cells (colony-forming components)
on a nutrient medium.
Both processes (spread plating and streak plating) require the aseptic culture
technique. Asepsis can be defined as the lack of infectious microorganisms.
However, the word is usually applied to any technique planned to keep unwanted
microorganisms from contaminating sterile materials.

ISOLATION OF PURE CULTURES

Material required-

1. Seven 9ml dilution tubes of sterile saline

2. Seven nutrient agar plates

22
3. 1.0 ml and 0.1 ml pipets

4. Glass spreader aka “hockey stick”

5. 95% ethyl alcohol in glass beaker (Keep alcohol away from flame)

6. Mixed overnight broth culture of Staphylococcus aureus and Serratia

marcescens

Procedure: (work in pairs)

A. Spread Plate Technique

In this technique, the number of bacteria per unit volume of sample is reduced by
serial dilution before the sample is spread on the surface of an agar plate.

1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix
the nutrient broth tubes before each serial transfer. Transfer 0.1 ml of the final
three dilutions (10-5, 10-6, 10-7) to each of three nutrient agar plates, and label the
plates.

2. Position the beaker of alcohol containing the glass spreader away from the
flame. Remove the spreader and very carefully pass it over the flame just once.
This will ignite the excess alcohol on the spreader and effectively sterilize it.

3. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient
agar plates until the medium no longer appears moist. Return the spreader to the
alcohol.

23
4. Repeat the flaming and spreading for each of the remaining two plates.

5. Invert the three plates and incubate at room temperature until the next lab
period.

B. Streak Plate Technique

The streak plating technique isolates individual bacterial cells (colony-forming

units) on the surface of an agar plate using a wire loop. The streaking patterns
shown in the figure below result in continuous dilution of the inoculum to give
well separated surface colonies. Once again, the idea is to obtain isolated colonies
after incubation of the plate.

1. Label two nutrient agar plates’ No. 1 and No. 2.

2. Prepare two streak plates by following two of the 3 streaking patterns shown in
the figure below. Use the 10-1 dilution as inoculum.

3. Invert the plates and incubate at room temperature until the next lab period.

C. Exposure Plates

Exposure of sterile media to the environment will demonstrate the importance of

aseptic technique.

1. Label two nutrient agar plates as "Exposure I" and "Exposure II."

2. Uncover the plate marked "Exposure I" and allow it to remain exposed in the lab
for about 5 minutes.

24
3. Expose the plate marked "Exposure II" to a source of possible contaminants.
Use your imagination: cough or sneeze, place your fingers on the surface of the
agar, etc.

4. Invert the plates and incubate at room temperature until the next lab period.

Estimation of microorganisms:
1. Colony counter

Procedure:

A. Spread Plate Technique

1. Count the number of colonies on each plate and record.

DILUTION Red Colonies White Colonies Total Number

10-6

10-7

10-8

B. Streak Plate Technique

1. Observe plates. Did you obtain isolated colonies on the agar plates which were
streaked with Serratia marcescens? Which streaking technique do you prefer? If
you did not obtain isolated colonies, what changes should you make in your
technique to ensure isolated colonies?

Procedure:

A. Spread Plate Technique One very important method in microbiology is to

isolate a single type of bacteria from a source that contains many. The most
effective way to do this is the streak plate method, which dilutes the individual
cells by spreading them over the surface of an agar plate (see Figure). Single cells
reproduce and create millions of clones, which all pile up on top of the original
cell. The piles of bacterial cells observed after an incubation period are called

25
colonies. Each colony represents the descendants of a single bacterial cell, and
therefore, all of the cells in the colonies are clones. Therefore, when you transfer a
single colony from the streak plate to new media, you have achieved a pure culture
with only one type of bacteria.

In this technique, the number of bacteria per unit volume of sample is reduced

by serial dilution before the sample is spread on the surface of an agar plate.

1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix the

nutrient broth tubes before each serial transfer. Transfer 0.1 ml of the final

three dilutions (10-5, 10-6, 10-7) to each of three nutrient agar plates, and label

the plates.

2. Position the beaker of alcohol containing the glass spreader away from the

26
flame. Remove the spreader and very carefully pass it over the flame just once

(lab instructor will demonstrate). This will ignite the excess alcohol on the

spreader and effectively sterilize it.

3. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrients

agar plates until the medium no longer appears moist. Return the spreader to

the alcohol.

4. Repeat the flaming and spreading for each of the remaining two plates.

5. Invert the three plates and incubate at room temperature until the next lab

period.

B. Streak Plate Technique Separation of a mixed culture into individual colonies

that can be subcultured to make pure cultures depends on how well the streak plate
is prepared. The goal of streak plate method is to dilute the cells by spreading them
out over the surface of the agar. This is accomplished in stages, as will be
demonstrated in lab before you try it yourself. Use the simulated agar surface
below to practice the streak pattern using a pen or pencil.

Obtain two TSA plates, and write your name on the bottom half (the half
containing the media) around the edge and following the curve (so the writing
won’t hide your view of the bacterial colonies once they grow). Also write M.
luteus on one plate (the name of the bacteria you will subculture to this plate). On
the other, write “mixed” to indicate that you’re subculturing from the mixed
culture broth to this plate.

As demonstrated, use a sterilized inoculating loop to pick up one M. luteus colony

(or a piece of a colony) and transfer it to the surface of the agar plate. Spread the
bacteria over approximately a quarter of the plate, edge to edge. Consider this step

Flame the loop and cool it in the agar. Overlap the step 1 streak 3-4 times to pull
out a reduced number of bacteria, and spread them out down the side of the plate.
Consider this step 2. Flame the loop and cool it in the agar. Overlap the step 2

27
streak 3-4 times and spread over the surface. Continue this process, flaming the
loop in between each step, until the entire surface of the agar plate is covered.

After performing this with the M. luteus culture for practice, repeat the process
with a drop of the mixed culture broth that you transfer to the plate with a sterile
inoculating loop. Place the streak plate subcultures in an incubator at the
temperature and time specified by your instructor.

The streak plating technique isolates individual bacterial cells (colony-forming

units) on the surface of an agar plate using a wire loop. The streaking patterns
shown in the figure below result in continuous dilution of the inoculum to give
well separated surface colonies. Once again, the idea is to obtain isolated colonies
after incubation of the plate.

1. Label two nutrient agar plates No. 1 and No. 2.

2. Prepare two streak plates by following two of the 3 streaking patterns shown in
the figure below. Use the 10-1 dilution as inoculum.

3. Invert the plates and incubate at room temperature until the next lab period.

C. Exposure Plates

Exposure of sterile media to the environment will demonstrate the importance

of aseptic technique.

1. Label two nutrient agar plates as "Exposure I" and "Exposure II."

2. Uncover the plate marked "Exposure I" and allow it to remain exposed in the

lab for about 5 minutes.

3. Expose the plate marked "Exposure II" to a source of possible contaminants.

Use your imagination: cough or sneeze, place your fingers on the surface of

the agar, etc.

4. Invert the plates and incubate at room temperature until the next lab period.

28
Robert Koch, in full Robert Heinrich Hermann Koch, (born Dec. 11, 1843,
Clausthal, Hannover [now Clausthal-Zellerfeld, Ger.]—died May 27, 1910, Baden-
Baden, Ger.), German physician and one of the founders of bacteriology. He
discovered the anthrax disease cycle (1876) and the bacteria responsible for
tuberculosis (1882) and cholera (1883). For his discoveries in regard to
tuberculosis, he received the Nobel Prize for Physiology or Medicine in 1905.

References
[1]. Tischerr. (1965). Pure culture of Anubuenaflos-aquae.Nature, Lond. 205, 419

[2]. Gradmann, Christoph. (2001). Isolation, Contamination, and Pure Culture: Monomorphism and Polymorphism
of Pathogenic Micro-Organisms as Research Problem 1860–1880. Perspectives on science : historical,
philosophical, social. 9. 147-72. 10.1162/106361401317447264.

[3].https://www.britannica.com/science/pure-culture

[4].https://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol346_W06/labman_week4.pdf

29
 CHAPTER: 4
MEASUREMENT AND QUANTIFICATION OF
MICROBIAL GROWTH

Microorganisms specifically anaerobes are extremely diverse with respect to their natural
distribution on Globe. Due to their anoxic habit, they occupied specific terrestrial areas on Earth
that provide restricted substrates diversity. Probably, this niche adaptation leads to the great
metabolic versatility that anaerobes possess. Their metabolic versatility makes anaerobes
interesting nominees for the claim as anaerobic microbial cell factories.

Whenever farming of anaerobic microorganism in biotechnological way is accomplished, it might

be important to monitor microbial growth, viability, and substrate uptake and product formation
kinetics. Under anaerobic conditions, cultivation, sampling procedures, and the determination of
physiological characteristics of anaerobic microbial population have to be adapted. Those
physiological characteristics are essential biotechnological variables and can be used to improve
yield or productivity of an anaerobic culture. The determination of those characteristics in
anaerobic cultivation systems may be addressed by using different techniques for sampling,
measuring growth, viability, and substrate uptake and product formation kinetics.

However, determining the appropriate method or combination of methods respecting farming

conditions and the desired yield of information about the cultured microorganisms is still
sometimes puzzling. This topic gives a thorough guidance to be able to make a watchful decision
on which methods are suitable for the measurement of microbial growth. All presented advantages
and disadvantages of the summarized methods should assist the reader to choose a proper
measuring technique for their specific purpose for laboratory. Assigning a method to laboratory,
pilot plant, or industrial plant is more difficult as it seems and must be purposefully chosen for
careful process analytical technology.

Before ascribing a technique to a biological process in a cultivation vessel, whether laboratory,

experimental plant, or industrial plant scale, the operation mode has to be specified since not every
technique can be performed under each operation mode.

All discussed methods are graded in four groups: connection to the cultivation vessel, costs,
complexity, and how time-consuming the quantification is. This grading could support and
improve the decision-making process, and which method under which conditions and bioreactor
settings should be applied. This listing should give support to find the right method for the applied
scale: laboratory, experimental/pilot plant, or industrial plant.

30
Biomass quantification approaches

Biomass quantification can be performed by applying several different methods, all possessing
some advantages or disadvantages. Especially when working with offline biomass quantification
approaches, washing and purification steps have sometimes to be encountered to be able to
quantify the amount of produced biomass. Here we will categorize offline techniques into five
different subsections:

1. Direct cell counting

2. Colony counting

3. Most Probable Number (MPN)

4. Biomass measurement

5. Light scattering

1. Direct cell counting:

Direct cell counting methods based on the enumeration of detectable cells within a liquid medium
and it comprises of-

[A] Microscopic enumeration

[B] Electronic enumeration

[C] Fluorescence Activated Cell Sorting (FACS)

[A] Microscopic enumeration:

Microscopic enumeration is another term for cell counting. Counting of single cells can be
performed by using different approaches. One of the most common approaches is microscopic
enumeration that can either rely on using a membrane filter sampling technique (Brock 1983),
followed by a cell or nucleus staining procedure (Koch 2007), or by using a counting chamber.
Counting chambers are a well applied microbiological tool to directly count cells. Depending on
the microorganism, different counting chambers and microscope settings can be applied. For
counting bacteria, commonly counting chambers with counting chambers depth of 0.02 mm are
used, whereas for counting larger microbes like yeast or algae, a counting chamber depth of 0.1
mm should be preferably applied (Bast 2001b). The two main disadvantages of direct cell counting
are the reproducible filling of the counting chamber and the adherence of cells on the glassware
surfaces and pipette tip. The market offers a great variety of counting chambers which usually
differs in the applicable volume, design of the counting grids and compatibility with different
objectives. Besides that, every counting chamber is calibrated for specific objective types. For
instance, Neubauer counting cambers are suited for high-dry objectives (Talking et al. 2014),

31
whereas Hawksley counting chambers can be used under oil-immersion objectives (Koch 2007)
which are, e.g. more suited for counting small-sized cells. However, without using a cell staining
method, distinction between viable, dormant, and dead cells is not possible (Talking et al. 2014).
The use of a counting chamber is eased when applying auto fluorescent strains. This approach
enhances the visibility of cells by excitation of cellular compounds at a specific wavelength, e.g.
the UV-inducible blue-green autofluorescence of microorganisms. Many H2-utilizing
methanogens can be counted by exposing them to an UV light, subsequently strain originated
autofluorescence is induced by special cofactors. Coenzyme F420 absorbs light at a wavelength of
420 nm and emits blue-green light, which can be detected by a fluorescence microscope (Solera et
al. 2001; Kumar et al. 2011). Deazaflavin F420 functions as an essential coenzyme within the
methanogenesis pathway. The reduced form of F420 (F420H2) functions as an electron donor for
methylenetetrahydromethanopterin dehydrogenase (Mtd), cysteine-containing F420-reducing
hydrogenase (Frc), and for selenocysteine-containing F420-reducing hydrogenase (Fru)
(Hendrickson and Leigh 2008). However, due to their low coenzyme F420 content, counting of
acetoclasic methanogens is rather difficult (Kamagata and Mikami 1991; Solera et al. 2001).
Another aspect that needs to be considered when applying these enumeration methods is the
aggregation state of biomass. For example, Methanosarcina spp. may form aggregates under
certain environmental conditions, which complicates counting of single cells by autofluorescence
cell enumeration (Solera et al. 2001). Counting autofluorescent methanogens during cultivation in
bioreactors is frequently used (Ahn et al. 2000; Solera et al. 2001). The autofluorescence of
methanogens could also be used to distinguish methanogens in co-cultures from other microbes
which do not express the coenzyme F420.

[B] Electronic enumeration:

Electronic enumeration of cells is another approach for determining the cell number. The Coulter
counter is routinely used in clinical hematology and for the enumeration of non-filamentous yeast
and protozoa. However, this technique is hard to apply to bacteria and other microbes with similar
morphological characteristics, like small cell size and elongated shape (Kubitschek 1969).

[C] Fluorescence Activated Cell Sorting (FACS):

FACS allows the measurement of scattered light and fluorescence emissions produced by
illuminated single cells that are passing through a capillary that is intersected by a laser beam.
Once a cell passes through a beam of light a signal is produced. The scattered light and
fluorescence emissions of each cell are collected by detectors and are further processed in silico.
The in silico process allows the distribution a population with respect to different parameters
measured by a given equipment. Forward scattered light, collected in the same direction as the
incident light, is related to cell size. Collected side scattered light (angle of 90°) provides
information of cell surface properties and internal structure of the cell. Information concerning the
cell is obtained by staining the sample with different fluorochromes (Álvarez-Barrientos et al.

32
2000; Lehtinen 2007). Most FACS has been limited to aerobic microbial systems due to the
oxygenated atmosphere of the sort stream and the cell deposition.

[2] Colony counting:

The amount of viable microorganisms can be elucidated by colony counting (Hungate 1969). This
technique can be performed by Spreading the diluted sample over a solid agar (spread plate
method) Pipetting the culture into a sterile Petri plate and mixing it with molten agar medium
(pour plate method) (Postgate 1969). Pipetting a sample into a small amount of molten but cool
agar medium (bearable temperature for the microbe), followed by pouring the mixture onto a
sterile agar plate, allowing it to harden (thin layer plates). Using the thin layer technique, but
adding another agar layer on top of it (layered plates). Filtering the diluted sample with a pre-
sterilized filter and placing it onto the sterile agar medium plate (membrane filter method).

In anaerobic microbiology, all these techniques are utilized, but compared to aerobic conditions
they require some additional precautions. For solid media, the execution of the colony counting
methods has to be carried out under anaerobic conditions. This can be realized by making the
media anoxic, counting in a glove box or by using an anoxic chamber for inoculation. In most
cases, the samples have to be diluted before plating to obtain an adequate quantity of colony-
forming units (CFUs). This number generally lies between 30 and 300 colonies per plate (Sutton
2011). Dilution of samples is a sensitive step since it needs to be compatible with the physiological
requirements of the microbe in respect to pH and osmolality (Koch 2007). After preparing and
incubating the agar plates, CFUs may be determined by using an appropriate period. However,
CFUs mostly consists out of more than one initial starting cell, which must be considered as well
(Li et al. 1996; Lehtinen 2007; Madigan et al. 2012). The techniques in this section can only detect
viable and culturable microorganisms. Dormant, non-culturable microbes and microorganisms
with very low μ are not detected with the previously described methods (Barer and Harwood 1999;
Oliver 2005).

[3] Most Probable Number (MPN):

The concentration of viable cells in culture can be estimated by applying the MPN method. The
amount of proliferating microbes is determined with MPN by the amount of dilutions, where
growth is observable (Kott 1966). This method is based on statistics. MPN has already been
applied for anaerobes, especially for estimating the methanogenic population in an anaerobic
thermophilic digester and a mesophilic soil sample (Wagner et al. 2012).

[4] Biomass measurement methods:

Sometimes it might be preferred to assess the cell mass instead of the real number of cells. Biomass
can be measured by determining wet weight or dry weight of a culture sample (Tisa et al. 1982;
Guerrero et al. 1985). Cell dry weight is determined by drying pelleted biomass for a defined
period of time with approximately 105 °C in glass eprouvettes (Koch 2007), subsequently followed

33
by cooling in a desiccator and weighing. As dry mass corresponds to 10–20% (m/v) of the wet
mass (Madigan et al. 2012), also the wet mass can be determined. Wet mass can simply be obtained
after centrifugation of the sample and removing of the supernatant (Tisa et al. 1982; Troller 1989).
After this process, a packed cell pellet remains, which should be weighed to determine the wet
mass (Tisa et al. 1982). The quantification of biomass dry or wet weight can be correlated to other
biomass quantification approaches such as spectrophotometry. Furthermore, for improving
bioprocess quantification, the elementary composition of biomass can be determined (Mauerhofer
et al. 2018) to balance growth stoichiometry on an elemental molar basis.

[5] Light scattering:

Light scattering methods are mostly used to monitor the growth of pure cultures (Gunther and
Bergter 1971). However, methods based on light scattering give mainly information corresponding
molecular content/ dry weight and not about the number of cells (Koch 1970). The cell biomass
can be estimated through the turbidity of a culture, which is measured with a photometer (fix
wavelength) or spectrophotometer (whole wavelength spectrum). The principle of this
measurement is based on the absorption of light by cells in the suspension at a certain wavelength;
but only unscattered light is detected. The amount of cells in the light path decreases the intensity
of the incident light beam and gives an indirect correlation of the amount of biomass in the sample.
The method of turbidity measurements is better known as determination of the optical density
(OD), (Koch 1970; Koch 2007). The more cells are in the suspension the more light is scattered or
absorbed and less light can be detected (Madigan et al. 2012). This correlation is described by the
Beer–Lambert law). The Beer–Lambert law is empirically valid only for OD values < 0.5 (Locher
et al. 1992) because of light scattering effects increase with higher cell density. The incoming light
beam gets initially scattered by the cells (primary scattered light). If the amount of cells is too high,
the possibility for scattering already scattered light (secondary scattered light) is increased, which
results in measuring lower OD values than the real extinction value. However, with the preparation
of standard curves and appropriate dilution series measuring up to higher OD values is possible
(Bast 2001). A relation between the cell dry weight and the absorbance was found to be directly
proportional and shows a linear correlation (Koch 1961).

Φex=Φin⋅e−sn⋅c⋅d2

In Equation Φex (W m−2) is the intensity of the incident light, Φin (W m−2) is the intensity of
outgoing light, s(m2 mol−1) is described as the scattering coefficient, c(mol L−1) is the
concentration of the cell suspension, and d(m) is the layer thickness. Offline turbidity
measurements are being executed by an external photometer. Therefore, a small amount of
biomass (up to 1 mL) has to be harvested, further transferred into a dedicated cuvette, and
measured at a proper wavelength. Microplate systems in contrary to cuvette spectrophotometers
34
allow measurements even with 100 μL of harvested suspension (Stieber et al. 1994; Turcotte et al.
2004). Investigations on different spectrophotometers showed a high dependency in the OD
measurements in respect to geometry and the optical design resulting in different OD values for
the same cell suspension. This has to be taken into account when performing measurements with
different systems. OD measurements can only be compared when measuring with one specific
spectrophotometer. Then OD-based biomass quantification can be correlated to other offline
biomass quantification methods. However, the correlation of biomass concentration to light
scattering must be individually determined for each organism and growth media. Moreover, the
correlation is only valid in a specific range as discussed above. When performing OD
measurements, medium characteristics have to be taken in account, since quantification of
microbes within the medium could be affected. Some medium components could impede the
quantification of microbes via light scattering, especially when working with dark samples from a
digester or manure plant. To overcome darkness, samples including blank could be diluted, which
have to be considered later when elucidating the amount of cells. If a dilution is not realizable, due
to immense microbial biomass loss, other biomass determination techniques have to be
investigated.

[6] At-line biomass measurement:

At-line measurements represent an improvement over traditional offline methods and are close to
real-time analysis; of course the ideal approach is monitoring online, preferably in situ. However,
the installation of online measuring devices is not feasible at each bioprocess condition.
Commonly anaerobic digestion plants are regulated based on at-line or offline analytical results
(Madsen et al. 2011). By applying an at-line attenuated total reflectance-mid-infrared (ATR-MIR)
spectroscopy, ammonium, glucose, methyl oleate, and biomass were investigated in a complex
antibiotic fermentation process using Streptomyces clavuligerus (Roy Choudhury et al. 2006). At-
line information gathered from flow cytometry can also be used to change the biofuel production
control strategy to enhance the process yield (da Silva et al. 2012). In principle, almost every
measuring deceive can be installed at-line.

Online biomass measurement:

The most common in situ measurement devices (Vojinović et al. 2006; Kiviharju et al. 2008;
Hopfner et al. 2010) are as follows:

[1] Optical sensors

[2] Fluorescence optical sensors

[3] Other spectroscopic sensors

35
[1]Optical sensors:

Measurements of biomass with optical sensors are either based on transmission or backscattering.
Probes based on the backscattering principle do not show any limitation in case of increasing
biomass concentration compared to transmission probes. Visible optical sensors can produce
erroneous responses caused by cell morphology, or interfering gas bubbles (Ulber et al. 2003;
Vojinović et al. 2006). Other suspended effects, and the necessity for cleaning of optical sensors
are common problems of these probes (Locher et al. 1992). Individual calibration for optical
sensors is recommended since the signals depend strongly on the cell morphology. Measurements
of cell dry weight and optical online methods showed different correlations according to the
investigated strains (Ude et al. 2014).

[2] Fluorescence optical sensors:

Fluorescence optical sensors can be employed to measure lifetime fluorescence emitted by

microbes in a culture. When applying this method, only viable cells in the population can be
detected. In active and living cells NADPH plays an important role for the electron transfer from
electron donor to electron acceptor. The signal and amount of NADPH in a biological system was
found to correlate with the biomass concentration (Coppella and Rao 1990; Farabegoli et al. 2003).
This technique is limited respectively to inferences from medium compounds that emit or absorb
between 360 and 450 nm. Therefore, only well-defined medium compositions can be used when
applying optical sensors (Marose et al. 1999). Possible interferences by several fluorophores (e.g.
FAD, NAD and NADH) can be circumvented with 2D absorption/emission fluorescent spectra
measurements or multi-wavelength fluorometry (Morel et al. 2004; Vojinović et al. 2006;
Kiviharju et al. 2008). The robustness and the capability of measuring intracellular effects as well
as their rapidity in measuring of fluorescent samples are the main advantages of these systems
(Locher et al. 1992).

[3] Other spectroscopic sensors:

Infrared spectroscopy:

Spectroscopic sensors are commonly used to detect infrared light within a range of 0.74–1.00 nm
(Landgrebe et al. 2010). Infrared spectroscopy is an analytical technique which is used to analyze
a wide variety of organic compounds, substrates, products, metabolites, and biomass. This method
is based on molecular vibrations of organic compounds, which have spectral signatures that belong
to the infrared domain (Landgrebe et al. 2010). The infrared light is subdivided into three regions:
far infrared (FIR), mid-infrared (MIR) and near-infrared (NIR) region. To monitor bioprocesses,
two spectroscopic sensor types are available, MIR and NIR probes (Olsson and Nielsen 1997;
Landgrebe et al. 2010). Microbial growth can be either measured via light absorption (turbidity)
or light scattering (nephelometry) in the visible and NIR ranges (Marose et al. 1999). NIR shows
the best correlation between wavelength and biomass at 2300 nm. The majority of media do not
absorb light in this NIR region (2300 nm) (Olsson and Nielsen 1997; Marose et al. 1999).

36
Electrochemical impedance spectroscopy:

Low frequency electrochemical impedance spectroscopy (EIS) can be used as an online process
tool to monitor viable cell concentrations during cultivations. Via EIS, the relative permittivity
between two electrodes affected by cells with an integer cell membrane is detected. This signal is
in turn correlated to cell dry weight measurement of the organism of interest. Thus, estimation of
viable cell concentration can be conducted. The proposed technique has a high dynamic range
from low to high cell densities beyond 40 g/L-1 cell dry weight with low background interferences
(Slouka et al. 2016).

Modeling of growth kinetics:

Modeling is a powerful tool to get insight into a biological bioprocess. Modeling concepts are
mentioned below:

➢ State estimation
➢ Estimation of volumetric mass bio-density
➢ AMD1 model
State estimation:

Real-time monitoring of physiological characteristics such as biomass, product, substrate, and

precursor concentrations during cultivation is of great importance during biotechnological
processes. Particle filter algorithm could be applied for estimating these difficult-to-measure
process states. The particle filter represents a new algorithmic framework, combining several
already existing methods and techniques (online and offline) for state estimation (Kager et al.
2018).

Estimation of volumetric mass bio-density:

The biological biomass density (biomass/bio-volume) referred as bio-density is a physiological

variable that can be estimated by using dielectric spectroscopy and a soft sensor based on first
principle elemental balances. The combination of both signals allows a real-time estimation of the
bio-density during cultivation. Dielectric spectroscopy measures the permittivity of the
fermentation broth in dual frequency mode, a high frequency accounting for non-cellular
background and a low frequency accounting for the permittivity attributed to living cells.
Dielectric spectroscopy estimates the biomass via correlating the permittivity signal, which reflects
the encapsulated volume fraction of cells. Soft sensors are software algorithms that calculate non-
measured process parameters from readily available process signals. Accurate estimation of the
biomass concentration via elemental balancing can be performed. The application of this sensor
allows a real-time calculation of specific rates and yield coefficients, which provides insight to
physiological changes. The combination of both signals, dielectric spectroscopy and soft sensor,
provides a possibility to estimate the volumetric mass (Ehgartner et al. 2014, 2017).

37
ADM1 model:

The anaerobic digestion model No. 1 (ADM1) reflects the major processes steps during digestion
and product formation, conversion of complex organic substrates into CH4 and CO2 and inert by-
products (Batstone et al. 2002; Jimenez et al. 2015). The kinetic equations consider microbial
growth and biomass decay. Therefore, the model incorporates seven microbial trophic groups.
Growth of these groups is related to degradation rates of organic matter and is described by Monod-
like dependencies. Also, inhibitive effects of pH, H2, ammonium, and fatty acids are considered
by equations.

The model includes the degradation of complex solids into carbohydrates, proteins, and fats, which
get further hydrolyzed to sugars, amino acids, and VFAs. Carbohydrates and proteins are
fermented to VFA (acidogenesis) and H2. Fatty acids are converted into acetate and H2. CH4 is
produced by acetoclastic and autotrophic, hydrogenotrophic methanogenesis. The
physicochemical equations describe ion association and dissociation, and gas–liquid transfer
during the digestion process. This differential and algebraic equation set enables the determination
of 26 dynamic state concentration variables, and 8 implicit algebraic variables per bioreactor vessel
or element. For monitoring of the process, there are further 32 dynamic concentration state
variables provided, based on differential equations (Batstone et al. 2002; Jimenez et al. 2015). The
ADM1d model is an extension of the ADM1 model and describes biomass distribution within a
one-compartment model (Mu et al. 2008).

Analytical approaches for quantification of biomass:

Microbial growth during cultivation should to be monitored. Biomass quantification can be

targeted by using offline, at-line, and/or online approaches. The usage of offline direct cell
counting, including microscopic enumeration, electronic enumeration, and FACS implies the
possibility to count microbes in liquid media, although only a representative sample volume is
used to determine the number of cells. Direct cell counting techniques facilitate the determination
of microbes in liquid media without the requirement of turbidity compared to light scattering
technique. Under ideal conditions, medium characteristics should not affect the quantification of
microbes within the medium, although medium components could impede the quantification of
microbes, e.g. digester or manure samples, because they are mostly dark and of high viscosity. To
overcome darkness or viscosity, samples can be diluted, which have to be considered later when
elucidating the amount of cells. If a dilution is not applicable, indirect biomass determination
techniques that are based on substrate consumption, product formation, or biomass viability
investigations can be employed. Moreover, complex media compounds or polymeric substances
can also impede proper quantification (Reischl et al. 2018b). Microscopic enumeration is more
cost-efficient than electronic enumeration and FACS, although susceptibility of errors is increased.
Determination of growth through most probable number technique is easy to perform. Although it
has some disadvantages over direct cell counting, as they are, contaminations are not detectable,
cells are not counted, and only the amount of viable cell is being estimated. Growth determination

38
on solid media could be performed via colony counting. Colony counting does not allow the
elucidation of the actual cell number. Instead, growth is indicated by colonies which have to be
counted. Instead of counting colonies, wet and dry weight determination can be elucidated. This
method gives only an insight in weight increase or decrease and no accurate determination of cell
number. Additionally, OD measurements should be performed. When performing OD
measurements, the medium absorption have to be considered too. Depending on the purpose and
the available budget, different applications are possible.

Online biomass measurements provide the possibility to monitor microbial growth in real time.
Optical sensors detect cells directly, thus signals generated by optical sensors are strongly
dependent on the cell morphology, which could also produce erroneous responses. Whereas
fluorescence optical sensors measure lifetime fluorescence emitted by microbes, here only viable
microbes can be detected (Coppella and Rao 1990; Farabegoli et al. 2003). Low-frequency
electrochemical impedance spectroscopy (EIS) can be used as an online process tool to monitor
viable cell concentrations during cultivations. Microbial growth could also be quantified by using
a modeling strategy to estimate biomass increase during the cultivation process. This strategy is
cost-efficient since not every parameter (e.g. biomass, substrate, and product) has to be detected
by a single device. As space for measurement devices in cultivation vessels is limited, this
modeling strategy could improve the monitoring of the cultivation process. The ADM1 model has
been specifically developed for modeling of anaerobic digestion bioprocesses. This modeling
strategy is well applied and further advancements have been intended.

Quantification of live and dead biomass:

“What is life?” Life is a biochemical process or an energy flux in a biological system. The trial to
answer this question leads to the reverse questioning “What is death?” The philosophical
distinction between life and dead is problematic (Davey 2011), which is also true in microbiology.
According to Martin et al. (2014), the core of the living process of all organisms is based on energy-
releasing chemical reactions or metabolic energy (adenosine triphosphate (ATP)). Therefore, life
could be seen as a generation of metabolic energy within a defined compartment, envelope, or
membrane. On the other hand, death could be interpreted as lacking ATP production in the
organism. Generally, the determination of microbial viability under certain conditions is essential
to be able to control and monitor their productivity. The monitoring of viability has a great
importance in many fields of microbiology and even beyond such as in food production (Ercolini
2004), health care sector (hospital) (Galvin et al. 2012), ground water sustenance (Clinton Ezekwe
and Nwabuko Chima 2013), production of pharmaceuticals (Jimenez 2004), and biological product
generation (Gaylarde et al. 1999). To determine the physiological status of an anaerobic
population, knowledge of the amount of alive and dead cells in the population is relevant.
Therefore, some methods have been implemented to study the viability of anaerobes. Those
methods can be divided into the following groups:

39
Quantification of viable biomass by using physiochemical parameters, staining and quantification
of biomass by microscope and FACS.

There are several staining methods available to investigate the viability status of microorganisms.

• LDS-FISH
• BONCAT
• BONCAT-FISH
• BONCAT-FACS
• Micro autoradiography (MAR)
The detection of live and of dead cells can be either performed by microscopy or by using cell
sorting. Both detection techniques are discussed in the sections below.

The Backlight® bacterial viability kit staining can be used for the application of microscopy and
FACS. This kit was initially developed to investigate the viability of bacteria. The usage for
archaea has already been confirmed by some research groups, which are mentioned below.
LIVE/DEAD BacLight® bacterial viability kit is offered for instance by the company
MOLECULAR PROBES EUROPE BV Leiden (Netherlands, www.probes.com). The two-color
fluorescence assay can be used for the distinction between live and dead microbes. It provides a
mixture of the green (SYTO 9) and red (propidium iodide (PI)) fluorescent nucleic acid stains.
Both stains differ in their spectral characteristics and in their ability to penetrate viable cells. When
SYTO 9 stain is used separately, all microbes with intact and damaged membranes get labeled. In
contrast, propidium iodide penetrates only microbes with damaged membranes. Subsequently, a
reduction of SYTO 9 stain fluorescence is induced. Through appropriate mixture of both stains,
microbes with intact cell membranes stain fluorescent green, while microbes with defective
membranes stain fluorescent red. The excitation/emission maxima for these dyes are about 480–
500 nm for SYTO 9 stain and 490–635 nm for PI. The kit is well suited for fluorescence
microscopy or for the application in quantitative analysis with a fluorimeter, fluorescence
microplate reader, flow cytometer, or other instrumentation. The LIVE/DEAD BacLight kit® was
initially developed for investigations of vital and dead bacteria, but it is already used in a broad
range of application in Microbiology. The intolerance of halo archaea species, except halococci,
to distilled water (Garrity et al. 2001) was used for investigating the reliability of the BacLight
kit® to detect extremophilic archaea (Leuko et al. 2004). Halobacterium sp. strain NRC-1 was
chosen as a reference strain to detect dead haloarchaea (reduction of SYTO 9 by propidium iodide
“red fluorescence”) as it lyses in presence of distilled water easily, and cells of Halococcus
dombroskii H4 were used as reference to detect vital haloarchaea (SYTO 9 “green fluorescence”)
(Leuko et al. 2004). Also, the incubation with LIVE/DEAD BacLight kit® reagents SYTO 9 and
PI for up to 24 h did not noticeably reduce the growth of the two haloarchaeal species. To
summarize, the LIVE/DEAD BacLight kit® could be used to assess the viability of haloarchaea
(Leuko et al. 2004). Further, the cultivability is not affected upon usage of the kit up to 24 h.
LIVE/DEAD® BacLight™ kit can be used to study the viability of psychrophilic archaea (Moissl

40
et al. 2003). By applying this kit the physiological status of the SM1 euryarchaeal cells at 10 °C in
sterilized marsh water (pH 6.5) was evaluated. The staining indicated a cell viability of 90%. The
applicability of the LIVE/DEAD®BacLight™ kit was also tested for methanogenic archaea
(Methanobacterium lacus, Methanobacterium movilense, Methanosarcina soligelidi,
Methanosarcina barkeri) (Heise et al. 2016). The strains were stained before and after isopropanol
killing procedure. SYTO 9 stained all archaeal cells, whereas PI only penetrates cells with
damaged membranes. After isopropanol killing, both Methanosarcina spp. formed defense
aggregates of cells and medium components. The cell wall structure of single cell Methanosarcina
spp. consists of a fairly porous surface layer called S-layer, aggregated cells are encapsulated in a
methanochondroitin sheath (Sowers et al. 1993). Possibly, this method is not suitable for
aggregated Methanosarcina spp. to allow distinguishing between live and dead cells.

LDS-FISH:

LDS-FISH is another visualization method to differentiate alive and dead cells (Savichtcheva et
al. 2005). This method combines fluorescence-based live/dead staining and FISH; it is applicable
for microscopy and FACS (Álvarez-Barrientos et al. 2000; Lehtinen 2007). By applying LDS-
FISH, the viability and survival ability of fecal Bacteroides spp. in environmental waters was
tested (Savichtcheva et al. 2005). The authors successfully demonstrated that LDS-FISH method
is a powerful tool to monitor the viability of anaerobic fecal Bacteroides spp. in drinking water.
The combination of both methods, allows the detection of single microbes (FISH) and determining
their viability status.

BONCAT:

BONCAT is used for visualizing transcriptional active cell of either archaeal or bacterial pure
cultures inside of complex samples, for instance, biofilms, freshwater, and anoxic sediments. This
method is based on in vivo incorporation of the non-canonical amino acid l-azidohomoalanine
(AHA). AHA-containing cellular proteins get further fluorescently labeled by azide-alkyne click
chemistry (Hatzenpichler et al. 2014).

BONCAT-FISH:

The advantage of combining BONCAT and FISH (BONCAT-FISH) is based on the specific
labeling of transcriptional active cells within complex samples like biofilms. Through this method,
newly synthesized proteins can be detected via BONCAT, in combination specific strains can be
identified via rRNA-targeted FISH. As a control, 4′,6-diamidino-2-phenylindole (DAPI) can be
applied to stain all cells. For quantification of transcriptional active cells, overlay programs can be
used (Hatzenpichler et al. 2014).

41
BONCAT-FACS:

A novel approach combining BONCAT with fluorescence-activated cell sorting, referred to as

BONCAT-FACS, is to separate translationally active cells by tracking the incorporation of
synthetic amino acids into newly synthesized proteins from complex samples. By applying this
technique the authors were able to directly link the identities of anaerobic CH4-oxidizing archaea
with their partner bacteria and detect transcriptional active cells (Hatzenpichler et al. 2016).

MAR:

MAR is a well-applied method in the aquatic and terrestrial microbiology field to measure single-
cell activity. This method enables a direct visualization of active cells and their metabolic
capabilities without prior enrichment or cultivation (Nielsen and Nielsen 2010). The method is
based on a short-term incubation of radioactive-labeled substrate. Those substrates get up-taken
by individual cells, which can be visualized by an auto radiographic emulsion. This emulsion is
placed on top of the radioactive-labeled organisms and subsequently processed by standard
photographic procedures. Excited silver ions will precipitate as metallic silver, resulting in silver
grain formation adjacent to or on top of the active microbial cells. Those cells can be visualized
under the bright-field or phase-contrast microscope (Nielsen et al. 2003).

Viable biomass quantification by molecular methods

Before investigating the viability of a microbial population via a molecular based method is
performed, DNA and/or has to be extracted. Special medium characteristics or environmental
conditions can interfere with the extraction method (Rittmann and Holubar 2014). Those features
have to elucidate before starting the extraction or quantification and the applied methods have to
be adapted. When applying this technique for a strain that has not been investigated yet with this
method, adjustments have to be initially performed. The viability of microbes can also be studied
by using molecular based methods, like:

• PMA-qPCR
• DNase I/Proteinase K
• RNA analysis
• Genomics
PMA-qPCR:

The analysis of viable and dead cells in a population could be investigated by applying quantitative
PCR with prior propidium monoazide (PMA) treatment (Heise et al. 2016). PMA is a DNA-
intercalating dye. Due to its positive charge, PMA is incapable of penetrating cells with intact cell
membranes, but it selectively interfuses membrane-compromised cells. The photo-inducible azide
group of PMA can be converted into a highly reactive nitrene radical which binds covalently to
free DNA upon exposure to bright light (Nocker et al. 2006). Respectively to the masking nature
of PMA toward free DNA, qPCR amplification results only in amplicons from intact cells (Nocker

42
and Camper 2009). It was shown that PMA-qPCR technique is suitable for the differentiation
between live and dead methanogens (Heise et al. 2016). Further findings indicate that unscathed
membranes of methanogens have a natural barrier for PMA (50–130 μM, < 20 min). Thus, PMA
can be used for detecting a lack of membrane integrity. The company Biotium
(https://biotium.com) commercially distributes a LED photolysis device (PMA-Lite™ LED
Photolysis Device), specifically designed for photoactivation of PMA, ethidium bromide
monoazide (EMA), or other similar azido dyes.

DNase I/Proteinase K:

An alternative method for the discrimination between live and dead cells is the DNase I/Proteinase
K treatment. Before performing qPCR, extracellular DNA has to be removed to determine the
amount of vital cells. Through the activity of DNase I, extracellular DNA is digested. When using
this method, one has to directly focus their attention to reaction conditions, DNase I concentration,
exposure time of DNase, and inactivation of DNase I, which can be properly inactivated by
Proteinase K. Through DNase I/Proteinase K pretreatment, followed by qPCR, exclusively living
cells were detected in the reference sample as well as in the natural drinking water biofilms
(Villarreal et al. 2013). DNase I/Proteinase K treatment could be a promising alternative to PMA-
qPCR technique.

RNA analysis:

RNA (mRNA, pre-rRNA, and rRNA) can be used to quantify viable or recently active microbes
(Cangelosi et al. 2010; Blazewicz et al. 2013). To quantify viable microbes via RNA analysis,
RNA of high quality has to be extracted from the sample, which can be challenging (Rittmann and
Holubar 2014). The short half-life of mRNA of minutes (Passow et al. 2018) in active cells can be
seen as an advantage and a disadvantage at the same time. Specific metabolic responses of
microbes can immediately be detected. On the other hand, extraction has to be performed fast or
special sample preparation have to be considered, such as flash freezing in liquid nitrogen
(Rittmann and Holubar 2014), or the application of stabilizing components like RNAlater (Passow
et al. 2018). Compared to mRNA, rRNA has a half-life of hours (Karnahl and Wasternack 1992).
Thus, when aiming for rRNA instead of mRNA to quantify active cells, extraction or stabilization
of RNA can be performed slower compared to mRNA. Another advantage of using rRNA is that
rRNAs are part of the ribosomes and thereby more protected as mRNAs. Besides rRNAs,
ribosomes consist of ribosomal proteins, which among other tasks stabilize the protein
synthesizing complex (Smith et al. 2008). As ribosomes (103 to 105 ribosomes per cell among
different species) transcribe mRNAs and thereby synthesize new proteins, direct correlations with
growth rate can be drawn (Kemp et al. 1993; Amann et al. 1995). However, rRNA is also present
in dormant cell as well (Blazewicz et al. 2013). To circumvent this bias, precursor rRNA (pre-
rRNA) can be targeted and quantified via qPCR (Cangelosi et al. 2010). RNA can be detected via
microarrays, qPCR, 16S ribosomal RNA (rRNA) sequencing, and metatranscriptomics (Ozsolak
and Milos 2011; DeAngelis et al. 2011; Geisen et al. 2015).

43
Genomics:

If only the metagenomics data of a sample is available, iRep could be used to estimate genome
replication rates from single-sample metagenomic data (Brown et al. 2016).

Quantification of viable biomass by using physiochemical parameters

Physiochemical parameters can also be employed to estimate the viable amount of microbes in a
population, such as:

• Adenosine triphosphate (ATP)

• Heat flows
• Foam formation
ATP

Investigating biomass by measuring ATP is dependent on the fact that all viable cells contain ATP,
whereas non-living particulate matter do not. The ratio of ATP to carbon in cells is fairly constant
for living organisms even though it slightly varies from species to species. The energy-storing
macromolecule ATP is only present in viable cells and disappears right after cell death (Helm-
Hansen and Booth 1966). It was shown that the ATP content reflects the activity of anaerobic
digestion (Chung and Neethling 1988). The ATP content of the biomass was determined through
a luciferin-luciferase-mediated reaction. The generated luminescence intensity from the reaction
was found to be proportional to ATP concentration in the assay solution and consistent results with
10% accuracy were achieved (Chang et al. 1981). ATP might be used also as a total activity
indicator for anaerobic digesters. Adverse aspects are the limitation of distinguishing between the
various population groups in a digester, but it could be used when working with pure cultures.
Their results showed that the activity in a digester, measured as ATP concentration, responded
quickly to changes in digester operation. Those changes have to be included when interpreting the
results. Further, the ATP content of living cells is dependent on environmental conditions and
reflects the activity of the cellular metabolism (Graça et al. 2005). The distinction between various
species within a population cannot be performed via ATP measurements.

Heat flow

Another physiochemical marker for vital microbes is heat flow. Heat flow is an outstanding
indicator for microbial activity, for the quantity of substrate consumption or metabolic product
release. This can be measured by using isothermal calorimetry (IMC), which has already been
proven to be an accurate method for monitoring microbial activity for in situ samples with very
low detection limits. IMC provides a rapid real-time detection and monitoring of microbial growth
and metabolism. Measurements of heat flow less than a microwatt, produced by 1 · 104–1 · 105
active bacterial cells, are possible to be detected with this non-destructive method (Braissant et al.
2010). The generated signal can be related to the number of present cells and their activity
(Braissant et al. 2010). Investigations of lake and marine sediments have shown a linear relation

44
between dehydrogenase activity assayed by using triphenyltetrazolium chloride (TTC) or
iodonitrotetrazolium chloride (INT) and sediment heat production (Pamatmat and Bhagwat 1973;
Pamatmat et al. 1981). Furthermore, a strong correlation between the ATP concentration and the
heat production in the sediment was observed (Pamatmat et al. 1981). In 2003, a more recent study
on lake sediments containing mixed communities of anaerobic, fermentative aerobic strains was
performed (Haglund et al. 2003). They concluded that heat production followed the same trend as
radiolabeled leucine and thymidine incorporation. Calorimetric chips are a promising area of IMC
instrumentation (Van Herwaarden 2005). These chips have already been used to monitor bacterial
growth correlated to heat (Higuera-Guisset et al. 2005; Maskow et al. 2006). Auspicious
calorimetric techniques are enthalpy arrays (Torres et al. 2004), which detect molecular
interactions including protein–ligand binding, enzymatic turnover, and mitochondrial respiration
that reflect viable cells.

Foam formation

Foaming cultures indicate an augmented cell lysis, generated by an overload of lipids, proteins,
and carbohydrates in the liquid phase (Kougias et al. 2014). Foam is a dispersion of gas bubbles
in a liquid (Walstra 1989), where the biggest volume consists of gas surrounded by a thin liquid
film (Mollet and Grubenmann 1999). In bioprocesses, foaming can be caused by surface-active
compounds, VFLs, lipids, and proteins. Two groups of surface-active substances are closely
related to foam formation: surfactants and biosurfactants (Ganidi et al. 2009). VFAs, oil, grease,
detergents, and proteins are examples of surfactants (Moeller et al. 2012). Biosurfactants are
naturally produced substances through microbial activity in the bioreactor (Ganidi et al. 2009),
such as hydroxylated and cross-linked fatty acids, glycolipids, lipopolysaccharides, lipoproteins–
lipopeptides, phospholipids, and the complete cell surface (Saharan et al. 2012). Volatile fatty
acids like formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and
3-methylbutanoic acid are potential intermediates of biogas production (Moeller et al. 2012). Due
to their hydrophobic character, lipids have the tendency to diffuse to the surface (Berg et al. 2013),
and as a result, lipids incline foam formation. Not only lipids can enhance foam formation but all
cell lysis and cell metabolism-related compounds can contribute to foam formation. Further, gases
can promote foam formation (Subramanian and Pagilla 2015). Foaming caused by CO2 seems to
be spontaneous (Devereux and Lee 2011), whereas bubble nucleation in presence of CH4 requires
an initiator (mixing) for foam (Blatteau et al. 2006; Subramanian and Pagilla 2015). Changes in
microbial population of anaerobic digestors, fed with agro-industrial wastes, before and after foam
formation were studied. Interestingly, no archaea was found to be associated to the foaming event,
but some archaeal species increased their abundance corresponding to foam formation (Kougias
et al. 2014). Foaming is an indicator for cell lysis during fermentation, but due to complexity of
foam formation, it is not possible yet to correlate foaming intensity to the amount of dead cells.
However, foaming could in some cases be indirectly measured via quantification of the specific
lysing rate via the quantification of specific process parameters such as specific amino acid ratios

45
(Bernacchi and Herwig 2017). Correlating foaming intensity to the amount of dead cells would be
a useful tool in biotechnology.

Chromatography

Chromatography is a chemical technique that is primary used for the separation of components of
a mixture. The principal of separation is based on the interaction between the analyte and the
mobile and the stationary phase. The separation method and the downstream detector depend on
the investigated component. Liquid chromatography (LC) can be divided into thin-layer
chromatography and column liquid chromatography (high-performance liquid chromatography
(HPLC) and ultra-performance liquid chromatography (UPLC)). HPLC allows a faster separation
of the investigated analysts as LC (Gey 2015a). Compared to HPLC, the separation process in case
of UPLC is performed with approximately 1000 bar. This leads to an improved resolution and
sensitivity, as the peaks in the chromatogram became thinner. Further, the operation speed is
increased (Nováková et al. 2017). Commonly used techniques to quantify metabolized or produced
components of anaerobic microbes are LC and HPLC. HPLC allows the separation of amino acids,
peptides, proteins, lipids, vitamins, organic acids, or bases, e.g. within the sample. The
combination of HPLC and MS admits an accurate determination of the analyte (Nollet and Toldrá
2012). Using HPLC equipped with an autosampler, a quaternary pump, a UV detector, and an
Aminex HPX-87H (300 × 7, 8 mm) column, short-chain fatty acids (formic, acetic, butyric,
propionic acid) could be measured during anaerobic digestion processes and their effect toward
CH4 production (Wagner et al. 2011). Further, the concentrations of dissolved free taurine and
dissolved free amino acids could be determined via HPLC fitted with an autosampler, a quaternary
pump, a column oven and a fluorescence detector (Clifford et al. 2017). After performing a
supercritical fluid extraction (SFE), bacterial respiratory quinone (RQ), bacterial phospholipid
fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested
sludge can be analyzed. Bacterial RQ were determined using UPLC (Hanif et al. 2012). To identify
and quantify liquid components in microbial cultures, mass spectrometry (MS) could be coupled
to LC or HPLC.

Mass spectrometry

MS enables the measurement of atoms or molecules within a sample. A mass spectrometer consists
of a sample inlet, ion source, mass analyzer, detector, control unit, and evaluation module. MS is
mainly used to elucidate structures of organic molecules. The sample gets converted into a
positively or negatively charged gaseous ion by using an ion source followed by ion separation
and detection in the mass analyzer unit based on their mass-to-charge ratio (m/z). To avoid
collusions of ionized particles, high-pressure vacuum (10−4 mbar) is applied in the device (Gey
2015b). Depending on the sample different ion sources, mass analyzer and detectors can be
combined. In principle, ionization can be divided into gaseous (electrospray ionization (ESI),
chemical ionization (CI), field ionization (FI)) and desorption (field desorption (FD), fast atom
bombardment (FAB), matrix-assisted laser desorption/ionization (MALDI)) techniques and soft

46
and hard ionization. Hard ionization methods (electron ionization (EI)) cause several ion
fragmentations, whereas soft ionization methods (CI, FI, ESI, FD, FAB, atmospheric pressure
chemical ionization (APCI), atmospheric pressure photoionization (APPI), and MALDI) induce
no or hardly any fragmentation of molecules. The most commonly used mass analyzers are
magnetic sector mass spectrometers (MS/MS), quadrupole mass spectrometers (QMS), time-of-
flight mass analyzers (TOF), trapped-ion mass analyzers (IT), and quadrupole ion traps (QIT).
MS/MS provides high reproducibility, resolution, and sensitivity. Organic MS analysis, accurate
mass measurements, and isotope measurements can be performed with this set-up. Although this
mass analyzer is commonly used, it is more expensive than other mass analyzers; also, it is not
well suited for MALDI. QMS has a good reproducibility and is relatively small and low cost,
although the resolution is limited and the combination with pulsed ionization (MALDI) is not
recommended. This analyzer is compatible with MS/MS, GC/MS, and LC/MS. TOF is known to
be a fast MS analyzer and well suited for MALDI, pulsed ionization methods in general, and fast
GC/MS systems. IT has the highest recorded mass resolution. However, this device requires strict
low-pressure conditions. Compatible ionization techniques are MALDI and ESI with high mass
analytes. QIT has a high sensitivity but poor quantitation. Applications are ion chemistry and target
compound screening. Compatibility is ensured with GC/MS, LC/MS, and MS/MS. Toward
separation, ion detection is executed. Established detectors are photomultiplier tube (PMT),
electron multiplier tube (EMT), and Faraday cup (FC) (Brunnée 1987). Faraday cup detectors are
mostly used in IRMS devices (Evershed et al. 2006; Chartrand et al. 2007; Schulze-Makuch et al.
2011). Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)
coupled to time-of-flight (TOF) analyzer are the most appropriate ionization methods for
biomolecules like peptides, proteins, nucleic acids, oligosaccharides, and lipids (De Hoffmann and
Stroobant 2007). For the ionization of steroids, amino acids, vitamin D, fatty acids, and fullerenes,
ESI can be used (Wilson and Wu 1993). MALDI is used for the ionization of following
biomolecules (Duncan et al. 2016), lipids (Wang et al. 2015), carbohydrates (Harvey 2003), drugs
including drug metabolites (Buck and Walch 2014), hormones (Gao et al. 2015; Yi et al. 2015),
and nucleotides and nucleosides (Gao et al. 2012). Further, MS (ESI, MALDI) could function as
a tool to study enzymatic reactions (Liesener and Karst 2005). The combination of
chromatography and mass spectrometry enables a threshold of investigated compounds within
nanogram and femtogram range (Gey 2015b). Some application areas are listed below.

LC/ESI-QMS

Mass spectrometric analysis of large biomolecules is preferentially investigated by using ESI-MS,

which is predominantly coupled with LC. Since a QMS detector was used, the method is named
LC/ESI-QMS. ESI-MS has a broad applicability such as analyte quantification, structure
determination of biomolecules and protein–ligand interaction studies. Also, the competitive
consumption of two substrates was investigated of an archaeal glycogen synthase by using ESI-
MS (Zea et al. 2003).

47
LC/MALDI-TOF-MS

LC/MALDI-TOF-MS is commonly used in detection and verification of carbapenemase

production in anaerobic bacterium Bacteroides fragilis, which belong to the beta-lactamase protein
family and inhibits most beta-lactam-based antibiotics (Johansson et al. 2014).

HPLC/APCI-MS

HPLC combined with MS with positive ion atmospheric pressure chemical ionization mass
spectrometry (APCI-MS) could be used to investigate of intact glycerol dialkyl glycerol tetraethers
(GDGTs) in archaeal cell. Molecules could function as biomarkers to detect archaeal cells
(Hopmans et al. 2000).

UPLC-UV-ESI-MS/MS

The relative abundance of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and PhIP-

M1 in cultures of the strict anaerobic gut microbe Eubacterium hallii were analyzed with UPLC-
UV-ESI-MS/MS (Fekry et al. 2015). The separation was performed with UPLC, the ionization
with ESI, and the mass analysis with MS/MS.

Spectroscopy

Spectroscopy, particularly infrared and Raman spectroscopy, can be applied to monitor various
metabolites during cultivation (Kornmann et al. 2003). Infrared sensors are commonly used in
biotechnology. When monitoring the consumption of a substrate or production of a product in the
liquid phase, NIR, MIR, and FIR spectroscopy methods could be applied. Near-infrared
spectroscopy (NIRS) was used for simultaneous prediction of exopolysaccharide (EPS; 0–3 g/L)
and lactic acid productions (0–59 g/L), and lactose (0–68 g/L) concentration in supernatant
samples from pH-controlled batch cultures of Lactobacillus rhamnosus RW-9595 M (Acedo et al.
2002). Linoleic acid, oleic acid, and ammonia were measured in fermentation broth via an inline
NIR of following microbes: E. coli, Pichia pastoris, Streptomyces toxitricini, and Aspergillus niger
(Tiwari et al. 2013). Methanol concentrations were tracked by applying an online MIR sensor
while performing a fermentation with P. pastoris (Schenk et al. 2007). Online Raman application
could be used to determine starch, dextrins, maltotriose, maltose (Gray et al. 2013), glucose, and
ethanol (Shaw et al. 1999) concentrations in the fermentation broth of S. cerevisiae. During
fermentations of E. coli, online Raman was used to determine glucose, lactate, formate, acetate,
and phenylalanine (Lee et al. 2004). Online spectroscopy to track substrate uptake is a useful tool
to monitor various metabolites during fermentation.

Assay kits

Assay kits could be used to determine the uptake of the employed substrate. For instance, uptake
of starch, mono-, di-polysaccharides, alcohols, and organic acids could be tracked by using

48
designated assay kits (Megazyme Inc., USA; www.megazyme.com). For quantifying the amount
of residual substrate or produced product, ELISA could be examined (Neuhaus et al. 2015).

Stable isotope probing

SIP techniques could be used to illustrate and track substrate uptake and metabolic processes
through labeling of specific biomarkers (Musat et al. 2012). SIP approaches mainly use stable
isotopes, such as 13C, 15N, or 18O. 13C-tracers are widely used to asess the quantity of carbon
flux. SIP techniques are predicated upon the incorporation of labeled substrates into DNA (DNA-
SIP; Radajewski et al. 2000), RNA (RNA-SIP; Manefield et al. 2002), proteins, or phospholipid
fatty acids (PLFA-SIP; Middelburg et al. 2000).

DNA-SIP and RNA-SIP

The incorporation of labeled substrate with DNA-SIP and RNA-SIP approach could be visualized
via isotope ratio mass spectroscopy (IRMS) or at single cell level by FISH-MAR, FISH-SIMS
(Biddle et al. 2006), FISH-Raman (Haider et al. 2010), and NanoSIMS (Lechene et al. 2006). Also,
unstable isotopes (14C, 3H, 35S, 33P, 32P) are commonly used in research to study the metabolism
of microbes. FISH-MAR can be used for the specific detection of the microorganism (FISH) and
monitor the incorporation of labeled substrate, such as14C, 3H, 32/33P (Lee et al. 1999), and 35S
(Vila et al. 2004) into intracellular storage compartments. This technique is limited by the
availability and affordability of radioactive-labeled substrates (Nielsen and Nielsen 2010). Further,
microbes that assimilate radioactive-labeled substance cannot be discriminated from active ones
via the application of MAR (Musat et al. 2012). FISH-SIMS was applied to identify the
metabolism of two uncharacterized archaea, which naturally present in the subsurface of marine
sediments by studying their isotopic carbon (Biddle et al. 2006; Musat et al. 2012). FISH-Raman
is applicable to investigate the metabolic function of microbial cells (Haider et al. 2010).
NanoSIMS could be used as a sole approach or in combination with others, like FISH, SIMSISH,
EL-FISH/HISH-SIMS. The N2-fixation of a bacterial symbiont of a shipworm was intensively
studied with NanoSIMS (Lechene et al. 2006). Microbial cells could be identified by using FISH
or halogens (bromine, fluorine, or iodine) bonded directly to oligonucleotide probes that bind
specifically to rRNA genes of the targeted organism (Musat et al. 2012). The usage of SIMSISH
(iodine-labeled oligonucleotide probe) is favored, when the permeabilization of cell wall is barely
realizable (Amann and Fuchs 2008). EL-FISH (Behrens et al. 2008)/HISH-SIMS (Musat et al.
2012) was based on bromine- and fluorine-labeled tyramines in oligonucleotide probes. This
technique was used to study and identify rare microbes involved in N2 fixation in anoxic layers of
lake sediments (Halm et al. 2009).

49
50
Abbreviations
VFA volatile fatty acid

H2 molecular hydrogen

CO2 carbon dioxide

CH4 methane

CO carbon monoxide

O2 molecular oxygen

N2 molecular nitrogen

CO2-BMP CO2-based biological methane production

s substrate

t time

q specific substrate consumption rate

Y( x/ s) biomass and substrate yield coefficient

X biomass

μ specific growth rate

ORP oxidation-reduction potential

MPN most probable number

FACS fluorescence activated cell sorting

OD optical density

NIR near-infrared region

MIR mid-infrared region

FIR far infrared region

EIS electrochemical impedance spectroscopy

ADM1 anaerobic digestion model No. 1

ATP adenosine triphosphate

MAR microautoradiography

BONCAT bio-orthogonal non-canonical amino acid tagging

DNA deoxyribonucleic acid

PMA propidium monoazide

RNA ribonucleic acid

mRNA messenger ribonucleic acid

rRNA ribosomal ribonucleic acid

IMC isothermal calorimetry

51
LC liquid chromatography

HPLC high performance liquid chromatography

UPLC ultra performance liquid chromatography

MS mass spectrometry

IRMS isotope ratio mass spectrometry

ESI electrospray ionization

CI chemical ionization

FI field ionization

FD field desorption

FAB fast atom bombardment ionization

MALDI matrix-assisted laser desorption/ionization

EI electron ionization

APCI atmospheric pressure chemical ionization

APPI atmospheric pressure photoionization

MS/MS magnetic sector mass analyzer

QMS quadrupole mass analyzer

TOF time-of-flight mass analyzer

IT trapped-ion mass analyzers

QIT quadrupole ion trap mass analyzer

PMT photomultiplier tube detector

EMT electron multiplier tube detector

FC Faraday cup detector

SIP stable isotope probing

PLFA-SIP phospholipid fatty acids -SIP

Bq Becquerel (radioactive decay s−1)

Sv Sieverts (J kg−1)

GC gas chromatograph

WCOT wall coated open tubular column

PLOT porous layer open tubular column

SCOTT support coated open tubular column

FID flame ionization detector

TCD thermal conductivity detector

ECD electron capture detector

52
 CHAPTER: 5
MAINTENANCE AND PRESERVATION OF PURE
CULTURE

Once a microorganism has been isolated and grown in pure culture, it becomes necessary to
maintain the viability and purity of the microorganism by keeping the pure culture free from
contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into
a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The
transfer is always subject to aseptic conditions to avoid contamination. To maintain pure culture
for extended periods in viable conditions, without any genetic change (Mutations) as well as
avoiding its contamination is referred as Preservation. During preservation most important factor
is to stop microbial growth or at least lower the growth rate. Due to this toxic chemicals are not
accumulated and hence viability of microorganism is not affected.

Application of Preservation:

1. Academic Use

2. Research Purpose

3. Fermentation Industry

4. Biotechnological Field

Techniques for the Preservation of microbes broadly divided into two:

1. Methods where organisms are in Continuous metabolic active state

2. Methods where organisms are in Suspended metabolic state

3. Low temperature storage (Refrigeration methods)

1. Continuous metabolic active state preservation technique: In this technique, organisms

preserved on nutrient medium by repeated sub-culturing. In this technique, any organisms are
stored by using general nutrient medium. Here repeated subculturing is required due to depletion
or drying of nutrient medium. This technique includes preservation by following methods.

a. Periodic transfer to fresh media (Subculturing): Organisms grown in general media on slant,
incubated for particular period at particular temperature depending on the characteristics of the
selected organisms, then it is stored in refrigerator. These cultures can be stored for certain interval
of time depending on the organism and its growth conditions. After that time interval, again these
organisms transferred to new fresh medium and stored in refrigerator.

53
Advantages:

1. It is a simple method; any special apparatus are not required.

2. Easy to recover the culture

Disadvantages:

1. Since repeated subculturing is time consuming, it becomes difficult to maintain a large number
of pure cultures successfully for a long time.

2. Risk of contamination is more

3. Possibility of genetic changes due to the development of variants and mutants and changes in
biochemical characteristics Therefore, it is now being replaced by some modern methods that do
not need frequent subculturing

Example: All microbiology laboratories preserve microorganisms on agar slant. The slants are
incubated for 24hr or more and are then stored in a refrigerator. These cultures are periodically
transferred to fresh media.Time intervals at which the transfers are made which varies with the
origin and condition of growth.

b. Overlaying culture with mineral oil (Paraffin method): Organisms are grown on agar slant
then they are covered with sterile mineral oil to a depth of 1 cm. above the tip of the surface. This
method is simple; one can remove some organisms in aseptic condition with the help of sterile
wire loop and still preserving the initial culture. Some species preserved satisfactorily for 15 – 20
years by this method.

Advantages:

a. One can remove some of the growth under the oil with a transfer needle and inoculate a fresh
medium and still preserve the original culture.

b. It is simple and less expensive method.

Disadvantages:

a. It fails to prevent changes in the characteristics of a strain due to the development of variants
and mutants

c. Storage in sterile soil: This method is widely used for preserving spore forming bacteria and
fungi. In this method, organisms will remain in dormant stage in sterile soil. Soil sterilized then
spore suspension added to it aseptically, this mixture dried at room temperature and stored in
refrigerator. Viability of organisms found around 70 – 80 years.

54
d. Saline suspension: Normal Saline used to provide proper osmotic pressure to organism’s
otherwise high salt concentration is inhibitory for organisms. Organisms kept in screw cap bottles
in normal saline, stored at room temperature, wherever required transfer made on agar slats, and
incubated.

2. Methods where organisms are in Suspended metabolic state: Organisms preserved in

suspended metabolic state by either drying or storing at low temperature. Microbes are dried or
kept at low temperature carefully so that their revival is possible.

a. Drying in vacuum: In this technique, organisms dried over chemical instead of air dry. Cells
passed over CaCl2 in a vacuum and then stored in refrigerator. Organisms survive for longer
period.

b. Lyophilization (Freeze-drying): Lyophilization is vacuum sublimation technique. Cells grown

in nutritive media and then this culture distributed in small vials. These vials culture then immersed
in a mixture of dry ice and alcohol at -78 °C. These vials immediately connected to a high- vacuum
line, and when they are completely dried, each vial sealed under vacuum. This is most effective
and widely accepted method.

Advantage of Lyophilization:

1. Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small
area.

2. Small vials can be sent conveniently through the mail to other microbiology laboratories when
packaged in special sealed mailing containers.

3. Lyophilized cultures can be revived by opening the vials, adding liquid medium, and transferring
the rehydrated culture to a suitable growth medium.

Disadvantages:

a. It is expensive.

3- Low temperature storage: Refrigeration:

a. Refrigeration:

Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold rooms. This
method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the
metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their
growth continues slowly, nutrients are utilized and waste products released in medium. This results
in, finally, the death of the microbes after sometime.

55
b. Cryopreservation:

Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the liquid
nitrogen at -150°C) helps survival of pure cultures for long storage times. In this method, the
microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of
stabilizing agents such as glycerol or Dimethyl Sulfoxide (DMSO) that prevent the cell damage
due to formation of ice crystals and promote cell survival. This liquid nitrogen method has been
successful with many species that cannot be preserved by lyophilization and most species can
remain viable under these conditions for 10 to 30 years without undergoing change in their
characteristics, however this method is expensive.

Advantages:

a. The cultures can remain viable and with unchanged characteristics for 10 to 30 years or more.

b. Many species which can’t be preserved by lyophilization are successfully preserved with liquid
nitrogen method.

Disadvantage:

a. It is relatively expensive method as liquid nitrogen in the refrigerator has to be replenished at

regular intervals to replace the loss due to evaporation.

Culture collection centers:

Many countries have microbial culture collection centre whose main function is to acquire,
preserve and distribute authentic cultures of living microorganisms. For examples:

1. MTCC: Microbial Type Culture Collection-Chandigarh, India

2. ATCC: American Type Culture Collection-Maryland, USA

3. National Collection of Type Cultures -London, UK

4. Institute Pasteur-Paris, France

5. Institute for fermentation-Osaka, Japan

56
 CHAPTER: 6
INTRODUCTION TO THE CELL AND TISSUE
CULTURE

Chronological Developments:
An early attempt at tissue culture was made in 1885 by German zoologist Wilhelm Roux, who
cultivated tissue from a chick embryo in a warm salt solution. The first real success came in 1907,
however, when American zoologist Ross G. Harrison demonstrated the growth of frog nerve cell
processes in a medium of clotted lymph. French surgeon Alexis Carrel and his assistant Montrose
Burrows subsequently improved upon Harrison’s technique, reporting their initial advances in a
series of papers published in 1910–11. Carrel and Burrows coined the term tissue culture and
defined the concept. Thereafter, a number of experimenters succeeded in cultivating animal cells,
using as culture media a variety of biological fluids, such as lymph, blood serum, plasma, and
tissue extracts. In the 1980s and ’90s, methods were developed that enabled researchers to
successfully grow mammalian embryonic stem cells under artificial conditions. Those
breakthroughs ultimately enabled the establishment and maintenance of human embryonic stem
cell lines, which advanced researchers’ understanding of human biology and greatly facilitated
progress in therapeutics and regenerative medicine.

Culture Environments:

Distributed on or in the medium, and the flask, tube, or plate containing the culture is then
incubated, usually at a temperature close to that of the tissue’s normal environment. Sterile
conditions are maintained to prevent contamination with microorganisms. Cultures are sometimes
started from single cells, resulting in the production of uniform biological populations called
clones. Single cells typically give rise to colonies within 10 to 14 days of being placed under
culture conditions. Plant tissue culture techniques are the most frequently used biotechnological
tools for basic and applied purposes ranging from investigation on plant developmental processes,
functional gene studies, commercial plant micropropagation, generation of transgenic plants with
specific industrial and agronomical traits, plant breeding and crop improvement, virus elimination
from infected materials to render high-quality healthy plant material, preservation and
conservation of germplasm of vegetative propagated plant crops, and rescue of threatened or
endangered plant species. Additionally, plant cell and organ cultures are of interest for the
production of secondary metabolites of industrial and pharmaceutical interest. New technologies,
such as the genome editing ones combined with tissue culture and Agrobacterium tumefaciens
infection, are currently promising alternatives for the highly specific genetic manipulation of
interesting agronomical or industrial traits in crop plants. Application of omics (genomics,
transcriptomics, and proteomics) to plant tissue culture will certainly help to unravel complex

57
developmental processes such as organogenesis and somatic embryogenesis, which will probably
enable to improve the efficiency of regeneration protocols for recalcitrant species. Additionally,
metabolomics applied to tissue culture will facilitate the extraction and characterization of
complex mixtures of natural plant products of industrial interest. General and specific aspects and
applications of plant tissue culture and the advances and perspectives are described in this edition.
Introduction

Plant tissue culture is a broad term that refers to the culture of any part of a plant (cells, tissues, or
organs) in artificial media, in aseptic conditions, and under controlled environments. This set of
techniques emerged as an experimental approach to demonstrate the cell theory, which establishes
that all living organisms are constituted of cells, the basic units of structure and reproduction, and
also the totipotency concept, which is defined as the genetic potential of a cell to generate an entire
multicellular organism. Different attempts were conducted by several researchers to investigate
the conditions to initially achieve the growth of organs or tissues in an artificial nutrient culture
medium rather than isolated cells because of the complex nutritional and hormonal requirements
they need. Nutrient solutions alone or supplemented with natural extracts were used as starting
culture media, and some important results were reported ; however, the discovery of plant growth
regulators was determinant for the successful establishment of in vitro plant tissue cultures. A key
advance in plant tissue culture was the control of morphogenesis by using different levels and
combinations of growth regulators, because this allowed the regeneration of entire plants, opening
the possibility of using in vitro systems to study fundamental aspects of cell differentiation and
development, and also for the application of tissue culture for different purposes. Some other
relevant advances in plant tissue culture were the culture of meristems as a tool for getting virus-
free plants ; the demonstration of totipotency in haploid or gametophyte cells, which made possible
the faster generation of isogenic lines important for plant breeding programs ; the rescue of hybrid
embryos to overcome sexual incompatibility between plant species; the enzymatic degradation of
cell walls of plant cells to produce protoplasts and the fusion of these naked cells to eliminate
sexual barriers between different plant species to render intraspecific or interspecific somatic
hybrids ; and the production of secondary compounds using cell or organ cultures , and perhaps
the most relevant advance in plant tissue culture was the development and establishment of genetic
transformation systems by Agrobacterium tumefaciens infection and through particle
bombardment to allow the genetic manipulation of plant species .

Primary Cultures and Established Cell Lines

There are two main types of cultures: primary (mortal) cultures and cultures of established
(immortal) cell lines. Primary cultures consist of normal cells, tissues, or organs that are excised
directly from tissue collected by biopsy from a living organism. Primary cultures are advantageous
in that they essentially model the natural function of the cell, tissue, or organ under study.
However, the longer the samples are maintained in culture, the more mutations they accumulate,
which can lead to changes in chromosome structure and cell function. In addition, primary cultures
generally are mortal. Cells undergo an aging process whereby they multiply for only 50 to 100
58
generations, after which the rate decreases markedly. The point at which a cell in primary cultures
stops growing, or undergo replicative senescence, marks the so-called Hayflick limit (named for
its discoverer, American microbiologist Leonard Hayflick). By contrast, established cell lines can
be perpetuated indefinitely. Such cell lines generally are derived from tumor biopsies from
patients, or they may be generated from primary cells that have undergone mutations that enabled
them to overcome the Hayflick limit and continue replicating. Similar to cells in primary cultures,
cells in established lines accumulate mutations over time that can change their character. Thus, in
order for researchers from different laboratories to be able to compare results from experiments
using the same cell lines, they must confirm the identity of the cells that they are working with.
Cell identity is verified through a process known as authentication, in which the DNA profile of
the cultured cells is compared against the known or standard profile for that cell line.

Basic Principles of Cell, Tissue, and Organ Culture:

Anyone who wishes to start plant tissue cultures should have in mind the following basic
principles:

(1) Select an appropriate explant from a healthy and vigorous plant.

(2) Eliminate microbial contamination from the surface of the explant.

(3) Inoculate the explant in an adequate culture medium.

(4) Provide the explant in culture with the proper controlled environmental conditions. In the case
of in vitro regenerated plants, they are subjected to an adaptation process (acclimatization) in the
greenhouse before the transference to ex vitro conditions.

Depending on the part of the plant that is cultured, we can refer them as cell culture (gametic cells,
cell suspension, and protoplast culture), tissue culture (callus and differentiated tissues), and organ
culture (any organ such as zygotic embryos, roots, shoots, and anthers, among others). Each type
of culture is used for different basic and biotechnological applications.

Micropropagation:

Micropropagation or in vitro clonal propagation is one of the most current extended commercial
applications of tissue culture. Plant tissue culture is an excellent tool for the asexual multiplication
of those species that are naturally reproduced asexually, but it is also used to overcome some
problems of germination of seeds in different plant species; for example, recalcitrant species are
particularly characterized for their short-seed viability (recalcitrant seeds), and therefore, asexual
multiplication is a good alternative. Although tissue culture can be applied for the
micropropagation of almost any plant species, it is recommended only for those that are
economically profitable.

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Among the plant species that are currently micropropagated at the commercial level, the
ornamentals occupy the first place. Micropropagation of plants may be carried out through three
different ways:

(1) By promoting the proliferation of apical or axillary buds and then rooting them,

(2) By inducing adventitious bud formation and its further rooting by somatic embryo formation,
maturation, and germination.

Each alternative can be applied to several plant species at different efficiencies depending on the
genetic background or regeneration capacity, the culture media, and the incubation conditions.

Plant Tissue Culture:

Genomics (the study of gene structure, function and regulation, and related techniques),
transcriptomics (the study of the transcriptome or the set of genes that are transcribed in an
organism), proteomics (the study of the set of proteins translated in an organism), and
metabolomics (the study of all metabolites present in an organism) have become essential for the
study of biological processes in plants. The knowledge on plant genomes, transcriptomes,
proteomes, and metabolomes has impacted favorably in the comprehension of complex
developmental processes, such as in vitro organogenesis, embryogenesis, or dedifferentiation, and
the genetic changes induced during in vitro conditions.

Additionally, metabolomics can be very useful to investigate secondary metabolism not only
during morphogenetic processes but mainly in cell, tissue, and organ cultures of plant species
producing secondary metabolites of industrial and .pharmaceutical interest.

Future Perspectives:

Plant cell, tissue, and organ cultures have been applied to a range of different purposes including
micropropagation, which is the most extended and successful application at commercial level and
surely will continue in the future, and genetic engineering of important crops to confer tolerance
mainly to pests and herbicides enabling the increase in production and yield with less applications
of toxic insecticides and herbicides in millions of hectares worldwide. A significant impact is
predicted in the production of different transgenic crops resistant or tolerant to drought, salinity,
or cold under these stress conditions in the near future. Additionally, genetic transformation will
be certainly a strategic tool for facing the global warming and its consequences by generating
transgenic plants resistant to abiotic factors.

Genetic engineering is still expected to contribute to the development of transgenic crops with
increased nutritional or nutraceutical value or resistant to diseases caused by fungi, bacteria, or
viruses. Plant metabolic engineering contribution to the development of more metabolically
efficient crops or with modified biochemical pathway leading to the production of commercial
secondary metabolites has been slow and modest, but it should have great promise to regulate the

60
biosynthesis of diverse secondary metabolites of industrial and pharmaceutical interest. Much
more difficult is to evaluate quantitatively the impact that tissue culture has had or will have on
plant breeding and crop improvement using embryo rescue, double-haploid generation, or somatic
hybridization, but of course they will be contributing to get improved hybrid crops to increase
productivity. Somaclonal variation in tissue cultures has been employed to rescue or recover
interesting materials that have led to the generation of new varieties and undoubtedly will continue
to be applied in the future for the isolation of somaclones bearing polygenic novel traits in which
the mechanisms underlying complex agronomical characteristics are unknown.

Genome editing techniques have opened a new and wide avenue for the second green revolution
and certainly will allow the creation of new and novel plant varieties with useful agronomic traits
through the fine manipulation of specific genetic changes in important crop species. The
development of high-throughput genome and transcriptome sequencing techniques, the application
of protein separation and sequencing, and the improvement of extraction, separation, and
identification of metabolites, as well as the availability of data in public databases, have helped to
decipher genome organization, gene function and regulation, and prediction of protein function
and to know the set of metabolites produced in different plant species. Omics have therefore
become fundamental tools for the study of basic biological processes in plants. Integration of omics
is desirable for a better understanding of whole biological phenomena. It is evident that omics will
be of great benefit to investigate in vitro morphogenetic processes and should facilitate the
establishment of more efficient in vitro plant regeneration protocols if master control genes of
differentiation and development are identified and characterized. On the other hand, the
combination of different omics should enable the metabolic engineering of interesting biochemical
pathways in order to manipulate specific characteristics for the optimization and production of
secondary metabolites of industrial and pharmaceutical importance.

61
 CHAPTER: 7
Tissue culture Lab design and lab setup

Plant tissue culture is not a distinct branch of plant science like ecology, taxonomy, cytology, plant
physiology etc. Reasonably it is a collection of experimental techniques of growing large number
of isolated cells or tissues under sterile and organized conditions. The cells or tissues are obtained
from any part of the plant like stem, root, leaf etc. which are stimulated to produce more cells in
culture and to express their totipotency (i.e. their genetic ability to produce more plants). Cells or
tissues are grown in different types of glass vials containing a medium with mineral nutrients,
vitamins and phytohormones. Therefore, to carry out the experiments using tissue culture
techniques, a well-equipped laboratory is first required. In recent years there has been a large
in-crease in the number of research laboratories us-ing tissue culture techniques to investigate
many fundamental and applied aspects of higher plants. However, the use of these techniques is
not confined to research alone.

Tissue culture techniques are being exploited by many commercial laboratories. Even many
horticultural companies are setting up small units to multiply plants which are difficult to
propagate by conventional means. The general organization of a tissue culture laboratory and the
basic techniques will be discussed here

Tissue Culture Laboratory setup:

An ideal tissue culture laboratory should have at least two large rooms and a small room. Large
room is for general laboratory work such as preparation of media, autoclaving, distillation of water
etc. The other large room is for keeping cultures under controlled light, temperature and humidity.
The small room is for aseptic work and for keeping autoclaved articles. The general laboratory
for tissue culture should be provided with the following articles and arrangements:

Washing area: It should be provided with a large sink, running hot and cold water, brushes of
various sizes, detergent and distilled water for final washing of the glassware’s. A number of
plastic buckets are required for soaking the glassware’s to be washed. Another separate bucket
with lid is also required for disposing off the used or infected media before cleaning. Only this
bucket should be kept outside the room or cleaning area and should be cleaned thrice or twice in a
week.

Hot Air Oven: It is necessary for drying the washed glass goods. For this purpose, a number of
enameled trays of different sizes are required for keeping wet glass goods inside the oven.

Refrigerator: It is essential for storing various thermo- labile chemicals like vitamins, hormones,
amino acids, casein-hydrolysate, yeast extract, coconut milk, etc. Stock solutions of salts are also
kept to prevent contamination.

62
Distillation unit: A single distillation and a double distilla-tion water plant are indispensable. Two
big plas-tic containers are required for storing the dis-tilled water.

Weighing Balance:Three types of weighing balances viz. pan balance; chemical balance and
electric balance are required for weighing chemicals, sugars, agar- agar and other weighing
materials.

pH Meter: It is necessary for the measurement and ad-justment of pH of the nutrient medium
(Fig.).

Vacuum Pump: It is required for filtering liquid media, sugar solution etc. through filter apparatus
us-ing air suction.

Autoclave: It is very important for sterilization of nu-trient media, glass goods, instruments, etc.

Working Tables: These are necessary for preparation of medium.

Heater: It is needed for heating or warming the medium to dissolve agar or to melt the agarified
medium.

Microscope: Simple, compound, inverted binocular dis-section microscopes are essential for
various pur-poses. Microscopes should be fitted with a camera for taking photomicrograph.

63
Microtome: It is needed for sectioning the cultured tis-sue.

Wooden Rocks: These are required for keeping the various chemicals.

Laboratory for Aseptic Inoculation:

This room should be without any window or ventilator in order to make it dust-free. The room
should be provided with double doors. The doors should have a automatic door closer. In-side
floor should be fitted with a rubber mat to facilitate cleaning. For entering into the room, shoes
should be left outside. For aseptic work, a large wooden chamber (Ca, 4′ x 4′ x 7′) is made for
short term work. Upper half of the side walls of the chamber is made of large glass sheets. The
chamber should also have double doors provided with a door closer. The chamber is provided with
two UV (one small, one big) sterilizing lamps and a fluorescent lamp. The switches to operate
them are present outside the chamber so that the lamps can be safely switched on and off. Inside
the chamber the working table and shelves are made of thick glass sheets. For simple routine work
such as aseptic seed germination, harvesting of cultured tissue from the aseptic stock for
cytological work or for mi-crotome preparations, a small inoculating hood may be used. This can
be placed on a small table at the convenient corner of the room. The figure of an ideal chamber is
given here.

64
Laminar air flow cabinet (Fig) is the most suitable, convenient and reliable instru-ment for aseptic
work. It allows one to work for a longer period which is not possible inside the inoculation
chamber. Long hours of work inside the inoculation chamber may also cause suffocation and needs
the interruption of work. One can work openly and easily for a longer pe-riod on the table of
laminar air flow.

Laminar air flow has a number of small blower motors to blow air which passes through a number
of HEPA (high efficiency particulate air) filters. Such filters remove particles larger than 0.3 µm.
The ultraclean air which is free from fungal and bacterial contaminants, flows at velocity of about
27 ±3m/minute through the working area. All contaminants are blown away by the ultraclean air
and thereby an aseptic en-vironment is maintained over the working area. Before starting work,
laminar air flow is put on for 10-15 minutes. The flow of air does not put out the flame of a spirit
lamp. Therefore, a spirit lamp can be used conveniently during the work.

Culture Room:

The culture room means the room for keep-ing or incubating the culture under controlled
temperature, light and humidity. The culture room is also fitted with double doors in order to make

65
it dust free and to maintain a constant room temperature. One should enter the culture room
keeping the shoes outside the door. To maintain the temperature around 25 ±2 ° C in-sides the
culture room, air coolers are used.

This room is also provided with specially designed shelves to keep culture vessels. The shelves
are made of glass or plywood. Flask, bottles, jars; Petri plates can be placed directly on the shelves.
Culture tubes can be kept on a support such as empty paper cover of fluores-cent lamps.

Cultures can be grown in light or in dark. For light arrangement, each culture rack is provided with
fluorescent lamps which are photo periodically controlled by an automatic timer. Racks covered
with black curtains are suitable for dark incubation of culture. A thermome-ter and a hygrometer
are fixed on the wall at the safety corner of the room to check tempera-ture and relative humidity
respectively. The relative humidity of the culture room is main-tained above 50%. Some small
shelves are placed in the culture room for temporarily keeping the autoclaved articles and the
culture vials contain-ing the medium. The culture room should also have a shaker for suspension
culture or single cell culture in moving liquid medium. The speed of revolution of the shaker can
be controlled. The shaker is also provided with light. The platform of the shaker is fitted with clips
for holding conical flasks (150 ml to 200 ml).

Techniques Used in Plant Tissue Culture:

The below mentioned article provides an outline on the techniques used in plant tissue culture.

Some of the techniques are:

(1) Preparation of Culture Medium

(2) Sterilization Procedure

(3) Preparation of Aseptic Plants

(4) Aseptic Techniques

(5) Incubation of Culture.

Several techniques have been adopted for in vitro plant cell, tissue and organ culture. Among them
some are general techniques that are essentially followed in all experiments.

General Techniques:

Preparations of nutrient medium, sterilization, aseptic manipulation, maintenance of culture are

the general techniques.

66
Preparation of Culture Medium:

In vivo plant cells, tissues and organs get their appropriate nutrient and growth requirements from
the intact plant body for their organised growth and development. Isolated cell, tissues and organs
also need nutrients for their in vitro growth and development. So, nutrients are supplied artificially
according to the medium formulated by several workers. The main objective of medium
preparation is to culture the cell, tissue and organ in vitro.

Procedure: Media should be prepared with care and the following procedure is recommended.

To make 1litre of MS medium:

• Dissolve 30gms cane sugar in 200 ml DDH2O. Mix 1-2gms activated charcoal and filter
through filter paper, set inside the Buchner funnel fitted on a special conical flask with
small side arm attachment. Filtering is done by using a suction pump.
• Take DDH2O in another flask and add in sequence the appropriate amount of stock solution
as follows—

Constituents Volume (In ml.)

Stock solution of micronutrients 1
Stock solution of macronutrients 50
Stock solution of KI 1
Stock solution of Fe-EDTA 5
Stock solution of Glycine 1
Stock solution of meso-inositol 2
Stock solution of MS-3vits 1

Desired concentrations of auxin and/or cytokinin are added from stock solution according to the
formula:

Desired concentration / Stock concentration = amount (ml) of stock solution to be taken for
one lire medium.

If the quantity of the medium is less than one liter, then hormones are added using another
formula—

Required concentration X Volume of medium/Stock concentration X1, 000 = amount (ml) of

stock solution to be added.

• Pour filtered sucrose solution and salt, vitamins, amino acid, hormone solution mixture into
a one litre measuring cylinder. Make the final volume to one litre with DDH2O. Shake well
to mix up uniformly.
• Adjust the pH of the liquid medium 5.6-5.8 with the aid of 0.1(N) HCl or 0.1(N) NaOH.
This operation is done using the pH metre.

67
 • Add 5% to 8% agar to the liquid medium to make solid medium. Heat to 60°C to dissolve
the agar completely. Otherwise, without adding agar, liquid medium can be used for
culture.
• Dispense the culture medium into culture tube (20 ml/tube) or wide mouth conical flask
(25-40 ml/flask). Insert non-absorbent cotton plug wrapped with gauge cloth. Cover the
plug with the help of brown paper and rubber band.
• Medium is finally sterilized by autoclaving.
Sterilization Procedure:

The culture medium, especially when it contains sugar, will also support the growth of micro-
organisms like bacteria, fungi etc. So if they come in contact with medium either in cellular form
or in spore form, the micro-organisms grow faster than the higher plant tissue due to their brief life
cycle and will kill the tissue. The micro-organisms may come from glass vials, instruments,
nutrient medium used for culture and even from plant material itself. Therefore, the surface of
plant tissue and all non-living articles including nutrient medium should be sterilized.

Procedure:

• Sterilization of non-living Articles:

The routine sterilization procedure of non-living articles such as nutrient medium, glass goods,
distilled water, instruments (wrapped with brown paper) is by autoclaving under steam at a
pressure of 15 lb/in2 and a temperature of 120°C for 15 minutes. Thermolabile compounds are
often added in the medium and such medium is sterilized either at room temperature or in cold by
passing through bacterial filter. An alternative method of sterilizing glass goods and instruments
is by heating in an oven at 150°C for 3-4 hrs. It should be noted that when autoclaving screw
capped glass vials, care should be taken to ensure that the caps are not closed too tightly so that
gases can expand without the risk of explosion.

Sterilization of Plant Material:

Plant material which is to be cultured, should be surface sterilized to remove the surface borne
microorganisms. This procedure is done in front of a laminar air flow or inside the inoculation
chamber before the plant material is inoculated onto the culture medium (Fig.)

68
(1) Thoroughly washed plant material or ex- plant in tap water is immersed in 5% v/v solution of
liquid detergent such as ‘Teepol’ for 10-15 minutes. Then wash the material thoroughly in tap
water and finally in distilled water. This step can be done in the general laboratory. Subsequent
steps are done in front of a laminar air flow or the pre-sterilized inoculation chamber.

(2) Dip the explants in 70% ethyl alcohol for 60 seconds.

(3) Immediately transfer the material into an autoclaved jaw bottle and pour 0.1% mercuric
chloride (HgCl2) 5-10% Sodium hypochlorite (v/v) solution. Keep them for 10- 15 minutes. During
that period, the bottle is frequently swirled for shaking so that all surfaces of plant material come
equally in contact with sterilant.

(4) After 10-15 minutes, decant the sterilant and wash the explants thoroughly with several changes
of autoclaved distilled water to remove all traces of sterilant.

(5) Then the explants are ready for culture.

Preparation of Aseptic Plants:

Surface sterilization of plant tissue may cause some deleterious effect because most of the sterilant
is toxic chemicals. Seeds can more or less resist such deleterious effect due to the presence of its
seed coat. So to avoid the surface sterilization of plant tissue, seeds are surface sterilized and are
69
cultured on simple basal nutrient medium. Seeds in culture germinate and give rise to an aseptic
seedling. Explants from such seedlings grown under aseptic and controlled conditions are the most
suitable material for culture and need no further surface sterilization.

Procedure:

(1) Wash the dry seeds thoroughly with tap water (Fig.).

(2) dip the seeds in 5% Teepol solution (v/v) for 10-15 minutes. Decant the Teepol solution and
wash the seeds again with tap water and finally with distilled water.

(3) Rinse the seeds with 70% ethyl alcohol for 1 minute.

(4) In front of laminar air flow, transfer the seeds into an autoclaved bottle and pour 0.1%
HgCl2 solution (w/v) so that seeds are immersed. Leave for 10-15 minutes. Stir the bottle
frequently.

(5) Decant the sterilant and wash 3-4 times with autoclaved distilled water.

(6) Transfer the seeds from bottle to autoclaved petridish with the aid of sterile forceps.

(7) Open the closure of the culture vial containing the basal nutrient medium. Flame the neck of
the culture vial and in quick succession transfers a few seeds on to the medium. Replace the
closure.

(8) Incubate the seeds in continuous dark either at room temperature or at 25-28° C.

Aseptic Techniques:

Precautions must be taken to prevent the entry of any microorganism at the time of transferring
the surface sterilized explants on the nutrient medium (inoculation) using the sterilized
instruments. For this reason, manipulation and transfer should ideally be carried out under aseptic
condition. Starting from surface sterilization to inoculation, all operations should be done
aseptically.

Procedure:

70
A typical procedure of aseptic technique is given below:

(1) Put all the sterilized articles (media, instruments, glass goods etc.) for inoculation on the glass
racks of the inoculation chamber. Alternatively, if laminar air flow is available, keep all articles
on the table of air flow cabinet. Laminar air flow blows bacteria- free air over the working surface.

(2) Put on the switch of UV lamps of inoculation chamber for one hour before work. In case of
laminar air flow, the power switch is put on and allows the air flow to blow air for at least 15
minutes before work.

(3) Put off the UV lamp before entering inside the inoculation chamber. Do not put off laminar air
flow. The working glass table top of the inoculation chamber or the table of laminar air flow is
swabbed with alcohol before starting work.

(4) Wear a clean apron and use a mask. Clean the hands with alcohol and dry it.

(5) Pour alcohol in a clean coupling jar and dip all instruments into it. Light the spirit lamp. Take
the surface sterilized or aseptic plant material in a, sterile petri dish.

(6) Flame the neck of culture tube or flask and in quick succession remove the plug of glass vials.
Transfer the tissue onto the medium and replace the closure. Each time, the instruments are passed
through the flame of the spirit lamp.

Precautions:

(1) Always keep away the hands moistened with alcohol from the spirit lamp. So dry the alcohol
first.

(2) Exposure to UV light builds up a high concentration of Ozone gas (toxic) inside the closed
chamber. It is, therefore, healthy to enter the chamber only 15-30 minutes after switching off the
UV lamp.

(3) Do not dip hot instruments in alcohol and don’t use hot instrument for cutting or holding the
plant material.

(4) Work carefully and try to ensure that media and plant tissues are exposed for the plant material.

(5) Don’t heat the neck of the glass vials excessively.

Incubation of Culture:

High temperatures are likely to lead to dissociation of the culture medium and tissue damage while
at very low temperatures tissue growth is slow. Again some tissues grow well in dark while others
need both light and dark conditions. Low humidity causes the quick desiccation of culture medium
and high humidity is favorable for the contamination of culture medium. Therefore, cultures are
incubated in a culture room where light, temperature and humidity are controlled.

71
Procedure:

(1) After inoculating the tissue onto the culture medium, cultures are incubated on culture rack at
25-28°C constant temperature.

(2) Culture tubes are placed at 30-45° inclined position. For this purpose a long wooden stick or
an empty paper cover of fluorescence lamp is placed on the middle of culture rack and lay the
plugged end of the culture tube on the support.

(3) Illumination is provided by cool-white fluorescent light placed about 18 inches above the
culture to give a light intensity of 4 – 10 x103 Lux for 16 hours.

(4) If light is not necessary, then put off the light and cover the whole rack with a black cloth.

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 CHAPTER: 8
Tissue culture media composition and preparation

Nutrient supplies for optimal growth of a tissue in vitro vary with the species. An optimal nutrient
balance in the culture medium is a precondition for any response of explants to plant growth
regulators, and optimization of inorganic nutrients may reduce or eliminate the use of plant growth
regulators. The media constituents include:

(1) Inorganic nutrients,

(2) Organic nutrients,

(3) Growth hormones, and

(4) Gelling agents.

The pH of the media changes at various stages of preparation and culture. The pH changes then
influence the availability of various mineral ions in the medium and their uptake by the plant tissue.
The chapter also discusses the media selection. The simplest method of preparing media is to use
commercially available dry powdered media, containing inorganic salts, vitamins, and amino
acids. The chapter outlines the sequence of steps involved in preparing a medium.

Preparation of Culture Medium:

In vivo plant cells, tissues and organs get their appropriate nutrient and growth requirements from
the intact plant body for their organised growth and development. Isolated cell, tissues and organs
also need nutrients for their in vitro growth and development. So, nutrients are supplied artificially
according to the medium formulated by several workers. The main objective of medium
preparation is to culture the cell, tissue and organ in vitro.

Media Composition:

Two distinct parameters determine the culture media’s composition. These are:

✓ The plant species

✓ The part or type of plant material you will use (tissues, protoplasts, cells, etc.)
Therefore, it would be safe for you to guesswork that a medium’s composition will be based on
the culture system’s specific protocols or requirements. The medium will either be liquid or solid,
again, depending on how the culture will respond. Agar is one of the most effective, and therefore
one of the most popular, gelling agents to use for most plant media. When considering your plan
of action, you should take note that preparing a culture medium should be based on the
requirements that your chosen plants need. Each species should have a unique approach. However,

73
the general stock solution consists of organic elements, micronutrients, and macronutrients. An
aseptic technique should also be used for all culture medium preparations.

The following steps outline the proper preparation of media for tissue culture:

• Mix a powdered medium with the appropriate amount of water.

• If you are mixing for a 1-liter medium, then fill a beaker with 800ml distilled water. Slowly
begin adding the powered medium into the beaker.
• Add 30g sucrose.
• Set the PH at 5.8.
• Add agar to the beaker (8g).
• Add hormone (if using).
• Add 200 mL distilled water.
• Autoclave media.
• Dispense the melting media into sterile tubes and make sure that each tube is labeled.
• The media should fill up approximately a third of the tubes.
• The media should be left in a sterile environment, where it is monitored until it can be used
to culture cells seven days later.
Why is a sterile environment so important?

The plant tissue cultures must be maintained in a sterile environment to eliminate contaminating
microbes, fungi, and pathogens. These contaminants could interfere with cell reproduction and
minimize your chance of successful tissue culture. Ensure that your tissue culture equipment,
including your tissue culture flask and beaker, is sterilized if you want to avoid culture or lab
contamination.

Dealing with Contamination:

In tissue culture, preventing contamination is a much smarter tactic than scrambling to treat it.
Plant preservative mixture (PPM™) can be used as both prevention and treatment. Adding PPM™
to your medium will help prevent fungi growth and will minimize the microbial contamination

Why is Preventing Contamination Important?

Contamination of your cell or tissue culture can ruin your plants or kill them entirely and will
impair the reproducibility of your results. Despite the use of sterile techniques and aseptic
conditions, the contamination of plant cell and plant tissue cultures remains a persistent problem
(most studies estimate between 10% and 20% of all cell cultures to be contaminated).

How to Make PPM™:

Add your PPM™ to your plant growth media before the sterilization step. PPM™ can be
autoclaved for 20 minutes at a temperature of 121C at 1.05kg/cm2. Alternatively, you can add the

74
PPM™ to media containing proteins that have already undergone sterilization before dispensation.
Once PPM™ is diluted in the medium, it is at its final concentration and can be left (at a stable
ambient temperature) for no longer than one month.

What is inside PPM™?

PPM™ is a proprietary chemical product and is used as a biocide and a culture preservative.
PPM™ is a broad `spectrum biocide, formulated specifically for use in plant tissue culture
procedures. By targeting fungi, contaminated cells and bacteria, PPM™ both prevents and
eliminates contamination in your cultures.

How does PPM™ do this?

PPM™ interacts with primary enzymes in the Electron Transport Chain and the Krebs cycle and
can be used as both a biocidal component in plant culture mediums and a biostatic compound for
preventing contamination. PPM™ can be diluted with your plant media to be an effective fungicide
and bactericide. By using Plant Preservative Mixture with your plant growth media, you will be
giving your cultures a chance to grow free from harmful bacteria and fungi.

Media should be prepared with care and the following procedure is recommended.

To make 1litre of MS medium:

• Dissolve 30gms cane sugar in 200 ml DDH2O. Mix 1-2gms activated charcoal and filter
through filter paper, set inside the Buchner funnel fitted on a special conical flask with
small side arm attachment. Filtering is done by using a suction pump.
• Take DDH2O in another flask and add in sequence the appropriate amount of stock
solution as follows—
[1]. Desired concentrations of auxin and/or cytokinin are added from stock solution
according to the formula:
Desired concentration/Stock concentration = amount (ml) of stock solution to be taken for
one litre medium.

If the quantity of the medium is less than one litre, then hormones are added using another
formula—

Required concentration X Volume of medium/Stock concentration X1, 000 = amount (ml)

of stock solution to be added.

[2]. Pour filtered sucrose solution and salt, vitamins, amino acid, hormone solution mixture into a
one litre measuring cylinder. Make the final volume to one litre with DDH2O. Shake well to mix
up uniformly.

75
[3]. Adjust the pH of the liquid medium 5.6-5.8 with the aid of 0.1(N) HCl or 0.1(N) NaOH. This
operation is done using the pH metre.

[4]. Add 5% to 8% agar to the liquid medium to make solid medium. Heat to 60°C to dissolve the
agar completely, otherwise, without adding agar, liquid medium can be used for culture.

[5]. Dispense the culture medium into culture tube (20 ml/tube) or wide mouth conical flask (25-
40 ml/flask). Insert non-absorbent cotton plug wrapped with gauge cloth. Cover the plug with the
help of brown paper and rubber band.

[6]. Medium is finally sterilized by autoclaving.

Major Types of Media:

The composition of the most commonly used tissue culture media is given in Table, and briefly
described below.

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White’s medium:

This is one of the earliest plant tissue culture media developed for root culture.

MS medium:

Murashige and Skoog (MS) originally formulated a medium to induce organogenesis, and
regeneration of plants in cultured tissues. These days, MS medium is widely used for many types
of culture systems.

B5 medium:

Developed by Gamborg, B5 medium was originally designed for cell suspension and callus
cultures. At present with certain modifications, this medium is used for protoplast culture.

77
N6 medium:

Chu formulated this medium and it is used for cereal anther culture, besides other tissue cultures.

Nitsch’s medium:

This medium was developed by Nitsch and Nitsch and frequently used for anther cultures. Among
the media referred above, MS medium is most frequently used in plant tissue culture work due to
its success with several plant species and culture systems.

Synthetic and natural media:

When a medium is composed of chemically defined components, it is referred to as a synthetic

medium. On the other hand, if a medium contains chemically undefined compounds (e.g.,
vegetable extract, fruit juice, plant extract), it is regarded as a natural medium. Synthetic media
have almost replaced the natural media for tissue culture.

Expression of concentrations in media:

The concentrations of inorganic and organic constituents in culture media are usually expressed as
mass values (mg/l or ppm or mg I/1). However, as per the recommendations of the International
Association of Plant Physiology, the concentrations of macronutrients should be expressed as
mmol/l and micronutrients as µmol/l.

Constituents of Media:

Many elements are needed for plant nutrition and their physiological functions. Thus, these
elements have to be supplied in the culture medium to support adequate growth of cultures in vitro.
A selected list of the elements and their functions in plants is given in Table.

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The culture media usually contain the following constituents:

• Inorganic nutrients
• Carbon and energy sources
• Organic supplements
• Growth regulators
• Solidifying agents
• pH of medium
Inorganic Nutrients:

The inorganic nutrients consist of macronutrients (concentration >0.5 mmol/l) and micronutrients
(concentration <0.5 mmol/l). A wide range of mineral salts (elements) supplies the macro and
micronutrients. The inorganic salts in water undergo dissociation and ionization. Consequently,
one type of ion may be contributed by more than one salt. For instance, in MS medium, K+ ions
are contributed by KNO3 and KH2PO4 while NO3 ions come from KNO3 and NH4NO3.

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Macronutrient elements:

The six elements namely nitrogen, phosphorus, potassium, calcium, magnesium and sulfur are the
essential macronutrients for tissue culture. The ideal concentration of nitrogen and potassium is
around 25 mmol I/1 while for calcium, phosphorus, sulfur and magnesium; it is in the range of 1-
3 mmol/ I. For the supply of nitrogen in the medium, nitrates and ammonium salts are together
used.

Micronutrients:

Although their requirement is in minute quantities, micronutrients are essential for plant cells and
tissues. These include iron, manganese, zinc, boron, copper and molybdenum. Among the
microelements, iron requirement is very critical. Chelated forms of iron and copper are commonly
used in culture media.

Carbon and Energy Sources:

Plant cells and tissues in the culture medium are heterotrophic and therefore, are dependent on the
external carbon for energy. Among the energy sources, sucrose is the most preferred. During the
course of sterilization (by autoclaving) of the medium, sucrose gets hydrolyzed to glucose and
fructose. The plant cells in culture first utilize glucose and then fructose. In fact, glucose or fructose
can be directly used in the culture media. It may be noted that for energy supply, glucose is as
efficient as sucrose while fructose is less efficient. It is a common observation that cultures grow
better on a medium with autoclaved sucrose than on a medium with filter-sterilized sucrose. This
clearly indicates that the hydrolyzed products of sucrose (particularly glucose) are efficient sources
of energy. Direct use of fructose in the medium subjected to autoclaving, is found to be detrimental
to the growth of plant cells. Besides sucrose and glucose, other carbohydrates such as lactose,
maltose, galactose, raffinose, trehalose and cellobiose have been used in culture media but with a
very limited success.

Organic Supplements:

The organic supplements include vitamins, amino acids, organic acids, organic extracts, activated
charcoal and antibiotics.

Vitamins:

Plant cells and tissues in culture (like the natural plants) are capable of synthesizing vitamins but
in suboptimal quantities, inadequate to support growth. Therefore the medium should be
supplemented with vitamins to achieve good growth of cells. The vitamins added to the media
include thiamine, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin, ascorbic acid,
myo-inositol, Para amino benzoic acid and vitamin E.

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Amino acids:

Although the cultured plant cells can synthesize amino acids to a certain extent, media
supplemented with amino acids stimulate cell growth and help in establishment of cells lines.
Further, organic nitrogen (in the form of amino acids such as L-glutamine, L-asparagine, L-
arginine, L-cysteine) is more readily taken up than inorganic nitrogen by the plant cells.

Organic acids:

Addition of Krebs cycle intermediates such as citrate, malate, succinate or fumarate allow the
growth of plant cells. Pyruvate also enhances the growth of cultured cells.

Organic extracts:

It has been a practice to supplement culture media with organic extracts such as yeast, casein
hydrolysate, coconut milk, orange juice, tomato juice and potato extract. It is however, preferable
to avoid the use of natural extracts due to high variations in the quality and quantity of growth
promoting factors in them. In recent years, natural extracts have been replaced by specific organic
compounds e.g., replacement of yeast extract by L-asparagine; replacement of fruit extracts by L-
glutamine.

Activated charcoal:

Supplementation of the medium with activated charcoal stimulates the growth and differentiation
of certain plant cells (carrot, tomato, orchids). Some toxic/inhibitory compounds (e.g. phenols)
produced by cultured plants are removed (by adsorption) by activated charcoal and this facilitates
efficient cell growth in cultures. Addition of activated charcoal to certain cultures (tobacco,
soybean) is found to be inhibitory, probably due to adsorption of growth stimulants such as
phytohormones.

Antibiotics:

It is sometimes necessary to add antibiotics to the medium to prevent the growth of

microorganisms. For this purpose, low concentrations of streptomycin or kanamycin are used. As
far as possible, addition of antibiotics to the medium is avoided as they have an inhibitory influence
on the cell growth.

Growth Regulators:

Plant hormones or phytohormones are a group of natural organic compounds that promote growth,
development and differentiation of plants. Four broad classes of growth regulators or hormones
are used for culture of plant cells-auxins, cytokinin, gibberellins (Fig.) and abscisic acid. They
promote growth, differentiation and organogenesis of plant tissues in cultures.

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Auxins:

Auxins induce cell division, cell elongation, and formation of callus in cultures. At a low
concentration, auxins promote root formation while at a high concentration callus formation
occurs. A selected list of auxins used in tissue cultures is given in Table.

Among the auxins, 2, 4-dichlorophenoxy acetic acid is most effective and is widely used in culture
media.

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Cytokinin:

Chemically, cytokinins are derivatives of a purine namely adenine. These adenine derivatives are
involved in cell division, shoot differentiation and somatic embryo formation. Cytokinins promote
RNA synthesis and thus stimulate protein and enzyme activities in tissues. The most commonly
used cytokinins are given in Table. Among the cytokinins, kinetin and benzyl-amino purine are
frequently used in culture media.

Ratio of auxins and cytokinins:

The relative concentrations of the growth factors namely auxins and cytokinins are crucial for the
morphogenesis of culture systems. When the ratio of auxins to cytokinins is high, embryogenesis,
callus initiation and root initiation occur. On the other hand, for axillary and shoot proliferation,
the ratio of auxins to cytokinins is low. For all practical purposes, it is considered that the formation
and maintenance of callus cultures require both auxin and cytokinin, while auxin is needed for root
culture and cytokinin for shoot culture. The actual concentrations of the growth regulators in
culture media are variable depending on the type of tissue explant and the plant species.

Gibberellins:

About 20 different gibberellins have been identified as growth regulators. Of these, gibberellin A3
(GA3) is the most commonly used for tissue culture. GA3 promotes growth of cultured cells,
enhances callus growth and induces dwarf plantlets to elongate. Gibberellins are capable of
promoting or inhibiting tissue cultures, depending on the plant species. They usually inhibit
adventitious root and shoot formation.

Abscisic acid (ABA):

The callus growth of cultures may be stimulated or inhibited by ABA. This largely depends on the
nature of the plant species. Abscisic acid is an important growth regulation for induction of
embryogenesis.

Solidifying Agents:

For the preparation of semisolid or solid tissue culture media, solidifying or gelling agents are
required. In fact, solidifying agents extend support to tissues growing in the static conditions.

Agar:

Agar, a polysaccharide obtained from seaweeds, is most commonly used as a gelling agent for the
following reasons.

1. It does not react with media constituents.

2. It is not digested by plant enzymes and is stable at culture temperature.

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Agar at a concentration of 0.5 to 1% in the medium can form a gel.

Gelatin:

It is used at a high concentration (10%) with a limited success. This is mainly because gelatin melts
at low temperature (25°C), and consequently the gelling property is lost.

Other gelling agents:

Bio-gel (polyacrylamide pellets), phytagel, gelrite and purified agarose are other solidifying
agents, although less frequently used. It is in fact advantageous to use synthetic gelling compounds,
since they can form gels at a relatively low concentration (1.0 to 2.5 g l/1).

PH of medium:

The optimal pH for most tissue cultures is in the range of 5.0-6.0. The pH generally falls by 0.3-
0.5 units after autoclaving. Before sterilization, pH can be adjusted to the required optimal level
while preparing the medium. It is usually not necessary to use buffers for the pH maintenance of
culture media. At a pH higher than 7.0 and lower than 4.5, the plant cells stop growing in cultures.
If the pH falls during the plant tissue culture, then fresh medium should be prepared. In general,
pH above 6.0 gives the medium hard appearance, while pH below 5.0 does not allow gelling of
the medium.

Preparation of Media:

The general methodology for a medium preparation involves preparation of stock solutions (in the
range of 10x to 100x concentrations) using high purity chemicals and demineralized water. The
stock solutions can be stored (in glass or plastic containers) frozen and used as and when required.
Most of the growth regulators are not soluble in water. They have to be dissolved in NaOH or
alcohol.

Dry powders in Media Preparation:

The conventional procedure for media preparation is tedious and time consuming. Now days, plant
tissue culture media are commercially prepared, and are available in the market as dry powders.
The requisite medium can be prepared by dissolving the powder in a glass distilled or
demineralized water. Sugar, organic supplements and agar (melted) are added, pH adjusted and
the medium diluted to a final volume (usually 1 litre).

Sterilization of Media:

The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for 20 minutes.
Hormones and other heat sensitive organic compounds are filter sterilized, and added to the
autoclaved medium.

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Selection of a Suitable Medium:

In order to select a suitable medium for a particular plant culture system, it is customary to start
with a known medium (e.g. MS medium, B5 medium) and then develop a new medium with the
desired characteristics. Among the constituents of a medium, growth regulators (auxins,
cytokinins) are highly variable depending on the culture system. In practice, 3-5 different
concentrations of growth regulators in different combinations are used and the best among them
are selected. For the selection of appropriate concentrations of minerals and organic constituents
in the medium, similar approach referred above, can be employed.

Medium utmost Important for Culture:

For tissue culture techniques, it is absolutely essential that the medium preparation and
composition are carefully followed. Any mistake in the preparation of the medium is likely to do
a great harm to the culture system as a whole.

What is the Meaning of Flower Culture?

Flower culture can be defined as the aseptic culture of excised floral bud on a chemically defined
nutrient medium where they continue their development to produce a full bloom in a culture vessel.
Young and complete flower culture can also be described as flower culture. In culture medium,
the flowers remain healthy and they develop normally to mature seeds.

Flowers can be cultured at the different stages of development, such as primordial stage, bud stage,
pre-pollination stage and post-pollination stage. Flower primordia and the young flo-wer bud
require a complex medium containing inorganic salts, B-vitamins, amino acids, coconut milk,
auxins and cytokinins. The mature flowers at pre or post-pollination stage need comparatively
simple media containing inorganic salts, sucrose and a small quantity of hormones.

Protocol:

(1) Flower buds or mature flowers are collected from the healthy plants.

(2) Wash them thoroughly and dip them in 5% Teepol solution for 10 minutes and wash.

(3) Transfer them to laminar airflow cabinet. Surface sterilizes them by immersing in 5% Sodium
hypochlorite, wash with autoclaved distilled water.

(4) Using flamed forceps, transfer the flower bud or mature flower to culture tubes con-taining 20
ml solid medium.

(5) Incubate the culture in 16 hrs. light at 25° C.

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Importance:

(1) The main application of floral primordia or flower bud culture is in fundamental studies of
flower development.

(2) Flowers put into the culture before pollina-tion do not usually produce fruits. In some cases,
parthenocarpic fruit development has been observed, particularly in presence of auxins.

(3) The culture of pollinated flowers is very im-portant to study the fruit development. Of-ten the
in vitro fruits are smaller than their natural counterparts, but the size can be in-creased by
supplementing the medium with an appropriate combination of growth hor-mones such as auxins,
gibberellins and cytokinins.

(4) Flower culture has been used to study the sex expression in flower. In the cucumber (Cucumis
sativus), there exist different genetic lines that are monoecious (with unisexual male or female
flowers on the same plant), gynoecious (purely female) or hermaphrodite. Under suitable natural
conditions, the monoicous types will produce only staminate male flower and the gynoecious types
only pistilated female flower.

It has been observed that in culture the potentially male buds tend to form ovaries and this tendency
is promoted when IAA is added to the culture medium. In contrast, addition of gibberellic acid
counteracts the effect of auxin. Isolated potentially female or bisexual flower buds in culture
remain unchanged even in presence of IAA or gibberellic acid or cytokinins. Such culture
techniques are also important for experimental studies on floral morphogenesis.

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 CHAPTER: 9
General Design and setup of Laboratory

Plant tissue culture labs have design requirements that differentiate them from other types of
laboratories and some needs are unique for a particular type that would be setting up a laboratory.
Therefore, careful initial planning is required in order to avoid problems associated to special
requirements. Unlike other facilities, e.g. microbiology the plant tissue culture laboratory is still in
a mid-phase and little specialized tools has been developed. As a result, some designers have come
across particular requirements of the laboratory by drawing from non-laboratory sources such as
food processing and office supply manufacturing units.

The building infrastructure:

While some tissue culture companies have the means to construct a facility from the ground up,
most laboratories have been set up by adapting existing structures. In either case, certain important
details should be considered. Adequate insulation is necessary to protect against temperature
extremes. In some instances, a smaller building has been constructed entirely inside a larger
existing one with the dead air space between them helping to insulate the interior structure against
the exterior environment. Of course, the air layer between the two would need to be vented to
provide an advantage during hot periods.

The selection of interior building materials should be made relative to durability, availability and
cost. Special considerations will be discussed in the descriptions of the different areas. A single
level structure that provides easy access to the different work areas is preferable for safety reasons
and because typical tissue culture activities require frequent movement of materials between areas.
The details of water access and disposal need thorough consideration in a location where public
water and sewer facilities are not available. A tissue culture laboratory needs a source of good
quality water, although some variation in quality of incoming water can be tolerated because it can
be purified to different degrees in the laboratory. A large quantity of waste water is generated,
particularly from glassware washing. Therefore, the permeability of the soil must be adequate to
accommodate all the liquid waste generated if disposed of through a septic tank and drainage field.
Furthermore, local and state health and environment codes may regulate waste disposal. Even
where a reliable source of electricity is available, power outages can occur, particularly in areas
prone to thunderstorms, ice storms and high winds. A power failure can be critical for temperature
control of cultures in the growth room and cold storage. Therefore, it is advisable to have a
generator to provide electricity in an emergency. The electric wiring for these critical areas must
be planned initially in such a way that the heating/cooling and lights can be switched over readily
to generator power. Generators are available that start up automatically if the main power source
fails. These often have an automatic monthly start-up feature to keep the engine in running
condition. However, they need to be checked periodically to ensure that they operate correctly. A

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less expensive type of generator is one that requires manual start-up. Use of a manual generator
requires an alarm system that will notify the appropriate personnel in the event of a power failure
during off hours.

Air flow and temperature control needs differ among the rooms; however, positive pressure is
required in the entire facility. These needs must be considered during the planning stages. Where
a room has specific requirements, these will be described.

Layout:

The types of activities to be performed and their requirements in terms of cleanliness, equipment
and patterns of movement have led to the designation of areas that are common to most commercial
laboratories: medium preparation room, washing/sterilizing room, transfer room, one or more
growth rooms, and rooms for cold storage, office, general storage, employee center.

Trained laboratory personnel must understand how chemical laboratory facilities operate. Given
the chance, they should provide input to the laboratory designers to ensure that the facilities meet
the needs of the functions of the laboratory. Laboratory personnel need to understand the
capabilities and limitations of the ventilation systems, environmental controls, laboratory chemical
hoods, and other exhaust devices associated with such equipment and how to use them properly.
To ensure safety and efficiency, the experimental work should be viewed in the context of the
entire laboratory and its facilities.

GENERAL LABORATORY DESIGN CONSIDERATIONS

Relationship Between Wet Laboratory Spaces and Other Spaces. Modern laboratories, particularly
in academia, often have contiguous spaces that include wet laboratories, computer laboratories,
instruments, write-up spaces, office areas, and other spaces with varying degrees of chemical use
and hazards. Maintaining a positive safety culture and at the same time meeting the safety and
comfort needs of laboratory personnel are challenging under these circumstances.

Wherever possible, separate wet chemical areas or those with a higher degree of hazard from other
areas with a physical barrier, such as a wall, divider, or control device. The objective is to protect
the computer laboratory or otherwise low-hazard area from the risk of the higher hazard, and thus
eliminate the need to use protective equipment in the low hazard area.

When such areas cannot be physically separated, or where the risk cannot be eliminated
completely, individuals working at the computer or in the write-up area need to evaluate what level
of protection may be needed to control the risk of exposure to the hazards in the other areas. For
example, all individuals in a computer laboratory must wear eye protection if there is a risk of eye
injury from operations in a contiguous area.

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Relationship between Laboratory and Office Spaces

Almost all laboratory personnel require both laboratory and office support space. Their desire to
be aware of procedures and to have a constant presence in the laboratory usually demands that
office space be located near the laboratory. The need for personnel safety, evolutionary technology
allowing for computer-based research and data monitoring outside of the laboratory, as well as a
desire to foster better interaction between researchers has driven the offices outside the laboratory
proper. Locating all offices outside the laboratory environment allows for a safer workspace where
food can be consumed, quiet work can be done, and more paper and books can be stored. Locating
the office zone very close to or adjacent to the laboratory for easy access and communication is
desirable.

Some laboratories have office spaces within research areas. In this design, it is best to have an
obvious separation between the laboratory area and the office area using partitions or, at a
minimum, aisle space, but preferably using a wall and a door that can be closed. Occupants should
not have to walk through laboratory areas to exit from their office space. Visitors and students
should not have to walk through laboratories to get to researchers' offices, because those persons
do not have personal protective equipment (PPE).

Open Laboratory Design

Traditionally, laboratories were designed for individual research groups with walls separating the
laboratories and support spaces. Group sizes ranged from 2 to 10 people, and most groups were
completely self-contained, each with its own equipment and facilities. Since the 1990s, the trend
has been for researchers to collaborate in a cross-disciplinary nature; chemists, biologists,
physicists, engineers, and computer scientists work together on a common goal. At the same time,
laboratory designers have moved to open multiple-module laboratories that allow a wide variety
of configurations for casework and equipment setups. These laboratories often support large or
multiple teams and are configured with relocatable furnishings. Even when not using a
multidiscipline approach, many facilities have moved toward larger, more open laboratories with
the belief that working in teams raises overall productivity, promote open communication, and
facilitates resource sharing. Team sizes, in some disciplines, have risen and are frequently as high
as 12 to 20 individuals.

Open versus closed laboratory design

The top figure is an example of a typical closed laboratory design with four separate laboratories.
The three walls separate the space and extend from floor to ceiling, with no shared spaces. The
bottom figure is an example of an open laboratory in the same space. The wall extends from floor
to ceiling but not from wall to wall (although in some designs, it could). Smaller working rooms
with permanent or movable walls are set up for storage or activities that require closed spaces.

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Considerations for Open Laboratory Design

There are advantages and disadvantages to open laboratory design.

Advantages:

• visibility among researchers

• better communication and collaboration
• easy to share resources, including equipment, space, and support staff; flexibility for future
needs because of open floor plan with adaptable furnishings;
• Significant space savings compared with smaller, enclosed laboratories; and cost savings
(first building/renovation costs and ongoing operating costs) compared with smaller,
enclosed laboratories.
Disadvantages:

for large spaces, challenging to balance the ventilation system; limitations to the size or placement
of the laboratory (e.g., the floor of the building, the type of research) because of chemical storage
code limitations for flammable and other materials needed for isolated spaces because of specific
types of work being conducted, such as cell or tissue work where cross-contamination is an issue,
use of certain radioactive materials, lasers, materials requiring special security measures, glass-
washing facilities. challenge of storing chemicals and supplies when there is a lack of natural
spaces created by walls and other fixtures; noise from people and equipment may be higher than
in a closed laboratory; and inability of some researchers to work effectively in an open laboratory
environment.

Design teams should work with the research teams to find solutions that accommodate the needs
of the researchers as much as possible. A combination of open laboratory spaces with smaller areas
dedicated to special functions is often necessary.

Closed Laboratories and Access:

Closed or separate laboratory spaces are often necessary for certain functions because of the nature
of the operation, equipment needs, or security concerns. These areas may or may not be separated
with a door. The need for a door and access control should be examined carefully for code
requirements, safety protocol, and containment concerns. The following issues should be
considered:

• Do the exits require doors by code?

• Must the corridor walls, doors, and frames be fire-rated by code?
• Is containment of spills or smoke an issue that demands doors?
• Is noise an issue that demands separation and attenuation?
• Does the need for room air pressure control necessitate a door closing the laboratory space
off from other areas?

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 • Does the work present a hazard that requires that access by untrained personnel be
controlled?
• Do some materials or equipment present a security risk?
• Do the materials require compliance with biosafety guidelines?
Equivalent Linear Feet of Workspace

When designing new laboratory spaces, consider the equivalent linear feet (ELF) of work surface
within the laboratory. ELF can be divided into two categories: bench and equipment. Bench ELF
is the required length of benchtop on which instruments can be set and where preparatory work
takes place, as well as the length of laboratory chemical hoods. Equipment ELF includes the length
of floor space for equipment that does not fit on a bench. Typically, every two laboratory personnel
whose work mostly involves hazardous chemicals should have at least one chemical hood, and
these should be large enough to provide each person with a minimum of 3 linear ft., but it could
be 8 ft. or more depending on the planned activities and type of chemistry.

Typical chemistry laboratories are designed to provide from 28 to 30 ELF per person. Quality
control, biology, and analytical laboratories range from 20 to 28 ELF per person. Quality control
and production laboratories tend toward the low end of this range, whereas research laboratories
are at or above the high end of the range. This number includes the support space outside the
laboratory that is needed. These values can vary widely and must be addressed carefully for each
project.

Laboratory Layout and Furnishing

Compliances:

The frequency of change in laboratory use has made it desirable to provide furnishings and services
that can be moved and adapted quickly. Although some services and surfaces will be fixed
elements in any laboratory, such as sinks and chemical hoods, there are several options available
to meet the adaptable needs for various types of research. Current design practice is to locate fixed
elements such as laboratory chemical hoods and sinks at the perimeter of the laboratory, ensuring
maximum mobility of interior equipment and furniture. Although fixed casework is common at
the perimeters, moveable pieces are at the center to maximize flexibility. The central parts of the
laboratory are configured with sturdy mobile carts, adjustable tables, and equipment racks.

Another trend for new laboratory buildings is to design interstitial spaces between the floors and
to have all the utilities above the ceiling. The interstitial spaces are large enough to allow
maintenance workers to access these utilities from above the ceiling for both routine servicing and
to move plumbing and other utilities as research demands change. Here interstitial spaces are not
possible, overhead service carriers may be hung from the underside of the structural floor system.
These service carriers may have quick connects to various utilities, such as local exhaust
ventilation, computer cables, light fixtures, and electrical outlets.

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Casework, Furnishings, and Fixtures

Casework should be durable and designed and constructed in a way that provides for long-term
use, reuse, and relocation. Some materials may not hold up well to intensive chemistry or
laboratory reconfiguration. Materials should be easy to clean and repair. For clean rooms,
polypropylene or stainless steel may be preferable.

Work surfaces should be chemical resistant, smooth, and easy to clean. Bench work areas should
have knee space to allow for chairs near fixed instruments or for procedures requiring prolonged
operation. Work areas, including computers, should incorporate ergonomic features, such as
adjustability, task lighting, and convenient equipment layout. Allow adequate space for ventilation
and cooling of computers and other electronics. Handwashing sinks for particularly hazardous
materials may require elbow, foot, or electronic controls. Do not install more cup sinks than are
needed. Unused sinks may develop dry traps that result in odor complaints.

Shared Spaces:

Many facilities encourage sharing of some pieces of equipment.Locating the equipment in a space
that is not defined as part of an individual's work zone facilitates sharing. In an open laboratory
setting, duplication of much of this equipment can be avoided. Often, if the equipment is centrally
located near a laboratory, it can be walled off to reduce noise. The team needs to carefully address
the need for alarms on specific pieces of equipment such as freezers and incubators that contain
valuable samples. Care must be taken, however, not to assume that sharing is always effective.
There are certain pieces of equipment that must be dedicated to specific users.

Flooring:

Wet laboratories should have chemically resistant covered flooring. Sheet goods are usually
preferable to floor tiles, because floor tiles may loosen or degrade over time, particularly near
laboratory chemical hoods and sinks. Rubberized materials or flooring with a small amount of grit
may be more slip-resistant, which is desirable in chemical laboratories. Coved flooring that allows
4 to 8 in. of flooring material secured to the wall to form a wall base is also desirable. Floors above
areas with sensitive equipment, such as lasers, should be sealed to prevent leaks.

Doors, Windows, and Walls

Walls should be finished with material that is easy to clean and maintain. Fire code may require
certain doors, frames, and walls to be fire-rated. Doors should have view panels to prevent
accidents caused by opening the door into a person on the other side and to allow individuals to
see into the laboratory in case of an accident or injury. Doors should open in the direction of egress.
Laboratories should not have operable windows, particularly if there are chemical hoods or other
local ventilation systems in the lab.

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Noise and Vibration Issues

Many laboratories utilize equipment that may emit significant noise, require a stable structural
environment, or both. During early planning stages, all equipment should be discussed regarding
any unique noise or vibration sensitivity in order to locate the equipment properly. Large
equipment such as centrifuges, shakers, and water baths often work best in separate equipment
rooms. Pumps for older mass spectrometer units are both hot and noisy and are often located in
either a small room or a hall. If in a closet, the area must have extra exhaust to remove heat, or else
equipment may fail from overheating. With smaller and newer mass spectrometers, the pumps are
often small and can fit into cabinets specifically designed for them. These pumps work especially
well when water cooling is not required. Very few researchers need to hear their instrumentation
running, but many want to see the equipment.

Another consideration crucial to equipment-intensive areas is the allowable vibration tolerance.

Most analytical equipment such as NMRs, sensitive microscopes, mass spectrometers, and
equipment utilizing light amplification (laser) require either vibration isolation tables or an area
that is structurally designed to allow for very little vibration. Clarify the tolerance requirements
with the user and equipment manufacturer during the equipment-programming phase, or early
design process, so that the appropriate structure can be designed and the construction cost can be
estimated more accurately.

Safety Equipment and Utilities

Each laboratory should have an adequate number and placement of safety showers, eyewash units,
and fire extinguishers for its operations. (See Chapter 6, section 6.C.10, for more information.)
The American National Standards Institute (ANSI) Z358.1-2004 standard provides guidance for
safety shower and eyewash installation. The 2004 version recommends provision of tepid water,
which can be complicated from an engineering standpoint. Although this standard does not address
wastewater, most designers agree that emergency eyewash and shower units should be connected
to drain piping. It is prudent to have floor drains near the units, preferably sloped to the drain to
prevent excessive flooding and potential slip hazards. Consider choosing barrier-free safety
showers and eyewash units that can accommodate individuals with disabilities. The maximum
reach height for the activation control for safety showers is 48 in.

Sprinkler systems may be required by the building code and are almost always recommended. For
areas with water-sensitive equipment or materials, consider pre action systems. Most dry or
alternative systems do not function in a laboratory environment with chemical hoods and other
ventilation. There may be resistance to the idea of installing sprinkler systems in laboratories,
particularly laboratories that use water-sensitive chemicals or equipment. The following facts may
be helpful: Each sprinkler head is individually and directly activated by the heat of the fire, not by
smoke or an alarm system. Thus, small fires are not likely to activate the sprinkler and moderate-

93
size fires will likely activate only one or two heads. Indeed, more than 95% of fires are
extinguished by one or two sprinkler heads.

Statistics show that the sprinkler head failure rate is 1 in 16 million.

In the event that the water from the sprinkler system reacts with water-sensitive materials, ensuing
fires would be quenched once the reaction stopped. Damage is likely to be less severe than if a fire
was not suppressed and was allowed to reach other flammable or combustible materials in the
laboratory.

Laboratory equipment, including lasers, is just as likely to be harmed by the fire as by the water.
Without the sprinkler system, a fire that is large enough to activate the sprinkler system would
result in response by the fire department. The sprinkler heads are designed to release water at a
rate of 10–15 gallons per minute (gpm), whereas a firefighter's hose delivers 250–500 gpm.

Dry chemical systems can seriously damage electronic and other laboratory equipment and are
impractical in a building-wide system. Alternative agents are impractical because of the amount
of space required for the cylinders and are most effective in rooms or areas that are sealed, which
is not how laboratories are designed. These systems are most practical for an individual
application, such as a piece of equipment or a “sealed” room. Locate utility shutoff switches
outside or at the exit of the laboratory. The purpose of the switch is to shut down potentially
hazardous operations quickly in the event of an emergency.

Locate room purge buttons at the exits in laboratories with chemical hoods. For most laboratory
buildings, activating the room purge button shuts down or minimizes supply air while increasing
exhaust ventilation. In the event of a chemical spill, activating the purge system will help ventilate
the resulting chemical vapors more quickly. Laboratories should have abundant electrical supply
outlets to eliminate the need for extension cords and multiplug adapters. Place electrical panels in
an accessible area not likely to be obstructed. Install ground-fault circuit interrupters near sinks
and wet areas.

Assess and provide for emergency power needs.

Where possible, install chilled water loops for equipment requiring cooling. Chilled water loops
save energy, water, and sewer costs. The design team and the owner are responsible for identifying
what reasonable accommodations should and can be made to meet guidelines or requirements. In
addition, some school systems and municipalities require a minimum number or percentage of
accessible work areas in teaching laboratories. Accessible furniture, including laboratory chemical
hoods, is readily available from most suppliers. The American Chemical Society has an excellent
resource available online or in print, Teaching Chemistry to Students with Disabilities. It is prudent
to provide barrier-free safety showers and eyewash units for all laboratories.

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The other accommodations will likely need to be made individually, depending on the special
needs of the researcher, supervisor, and a laboratory safety professional will help determine the
extent of the accommodations. For wet laboratories, service animals should either have a place
outside the lab or an area within the laboratory that is accessible without the animal having to
traverse areas where chemicals or other hazardous materials could be present at floor level,
including spills.

Older Facilities:

Getting old facilities can present multiple challenges. As materials of construction begin to
degrade, the safety and environmental provisions of the facility often degrade as well. Although
some equipment and materials may continue to function well for many years, modern alternatives
may offer better safety and environmental sustainability features. For older facilities, it is important
to have a strong operations and maintenance program that monitors and maintains plumbing,
ventilation, and structural components. Nonetheless, as individual laboratories or spaces are
renovated for new uses or upgrades, there are opportunities for improving and modernizing
building systems.

Depending on the location of the laboratory building, there may be requirements for bringing the
entire building up to current building codes and standards once a certain percentage of the building
is under renovation. These code requirements may include fire protection systems, accessibility,
plumbing, ventilation, alarm systems, chemical storage restrictions, and egress issues. With rising
interest in energy conservation, there have been numerous studies and instances of retro-
commissioning of laboratories. The focus is generally on the laboratory ventilation system, with
the goal of managing airflow and temperature control to eliminate waste and reduce overall energy
use

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The retro-commissioning process proceeds in five major steps:

Organization: Bring facility and EHS staff, design engineers, and users together to discuss goals.
Gather information about the current system, including the original plans, as-built plans, major
alterations, and current function, including ventilation rates. Develop the retro-commissioning
plan.

Primary investigation: Verify all systems including the direct digital control or building
automation systems, evaluate all components that affect energy use, and verify monitoring
systems.

Investigation: Benchmark utility and energy use data, analyze trends, and test all equipment.
Testing should include functional testing of chemical hoods and related components, including
face velocity tests, containment tests, etc.

Implementation: Select which improvements will be made and arrange them. Implement the
improvements and test performance.

Handoff: Clearly document information and provide training to laboratory personnel and
maintenance personnel.

Common conditions that lead to energy waste include:

•overabundance of laboratory chemical hoods,

•laboratory chemical hoods with large bypass openings,
•dampers in fixed positions,
•over ventilated laboratory spaces,
•excessive duct pressure,
•Fans set to override position, fans that are no longer operating efficiently, constant volume
systems with no setback for temperature or airflow when unoccupied, and high face
velocities.
• Whether retro-commissioning for energy efficiency or for safety, ensure that all
stakeholders are involved in the process.
• Once the work is complete, continue to monitor efficiency and safety. It is important to
include trained laboratory personnel in the feedback process. If systems are not used
correctly or if they are bypassed, the retro-commissioning efficiency may deteriorate.
LABORATORY VENTILATION

The laboratory ventilation system, whether it is the general ventilation, a chemical hood, or a
specialized exhaust system, is a critical means to control airborne chemicals in the laboratory. At
a minimum, a well-designed laboratory ventilation system should include the following:

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 • Heating and cooling should be adequate for the comfort of laboratory occupants and
operation of laboratory equipment.
• A differential should exist between the amount of air exhausted from the laboratory and
the amount supplied to the laboratory to maintain a negative pressure between the
laboratory and adjacent non laboratory spaces. This pressure differential prevents
uncontrolled chemical vapors from leaving the laboratory.
• Clean rooms may require a slightly positive pressure differential. There should be
separation between common spaces and the clean room to prevent migration of airborne
contaminants.
• Exhaust ventilation devices should be appropriate to materials and operations in the
laboratory.
Many devices are used to control emissions of hazardous materials in the laboratory. A risk
assessment helps to determine the best choice for a particular operation or material.

Table: Laboratory Manufacturing Controls for Personal Protection

Typical Number of Air

Changes or Face Velocity
Type of in Linear Feet per Minute
Ventilation (fpm) as Appropriate Examples of Use

General 6–12 air changes/hour, • Nonvolatile chemicals

laboratory depending on laboratory
• Nonhazardous materials
ventilation design and system
operation

Environmental 0 air changes • Materials that require special

rooms environmental controls

• Nonhazardous amounts of flammable,

toxic, or reactive chemicals.

Laboratory 10–15 air changes/minute • Flammable, toxic, or reactive materials

chemical hoods or 60- 100 fpm depending
• Products or mixtures with
on hood type
uncharacterized hazards

Unventilated 0 air changes • Flammable liquids

storage cabinets
• Corrosives

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 Typical Number of Air
Changes or Face Velocity
Type of in Linear Feet per Minute
Ventilation (fpm) as Appropriate Examples of Use

• Moderately toxic chemicals

Ventilated 1–2 air changes/minute • Highly toxic, hazardous, or odiferous

storage cabinets chemicals (if equipped with flame
arrestors)

Recirculating A1: 75 fpm • Biological materials

biosafety A2: 100 fpm
• Nanoparticles, as of the date of
cabinets
B1: 100 fpm publication

• Biological materials

• Nanoparticles, as of the date of

publication

• Minute amounts of volatile chemicals

Total exhaust B2: 100 fpm • Biological materials

biosafety cabinet
• Nanoparticles, as of the date of
publication

• Minute amounts of volatile chemicals

Glovebox Varies from no change to • Positive pressure for specialty

very high rate of change, environments
depending on the glovebox
• Negative pressure for highly toxic
and the application
materials

Downdraft table 150–250 fpm depending on • Perfusions with paraformaldehyde,

design work with volatile, low to moderately
hazardous materials with higher vapor
density where access from more than
one side is necessary

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 Typical Number of Air
Changes or Face Velocity
Type of in Linear Feet per Minute
Ventilation (fpm) as Appropriate Examples of Use

Elephant trunk 150–200 fpm at opening • Local ventilation of a tabletop

• Discharge from equipment such as a gas

chromatograph

Canopy N/A • Ventilation of heat, steam, low or

nontoxic materials with low vapor
density

Ductless 10–15 air changes/minute • Materials that are compatible with the
laboratory filtration system, in controlled quantities
chemical hood and under controlled conditions

• Not suitable for particularly hazardous

substances

Slot hood Varies with application • Local ventilation of higher density

materials at the source, such as an acid
bath

Ventilated 5–10 air changes/minute • Weighing and initial dissolution of

balance highly toxic or potent materials
enclosure

Benchtop Variable per the needs of • Benchtop equipment, such as rotovaps

ventilated the materials
enclosures

NOTE: Clean benches are not designed for use with hazardous materials. These are appropriate
for use in work with materials that necessitate clean work conditions and should only be used for
materials or chemicals that one could safety use on a benchtop.

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Threat Calculation:

For all materials, the objective is to keep airborne concentrations below established exposure
limits. Where there is no established exposure limit, where mixtures are present, or where reactions
may result in products that are not completely characterized, prudent practice keeps exposures as
low as reasonably achievable. For chemicals, determine whether the material is flammable or
reactive or if it poses a health hazard from inhalation. If no significant risk exists, the work does
not likely require any special ventilation. If potential risk does exist, look at the physical properties
of the chemical, specifically its vapor pressure and vapor density. Vapor pressure is usually
measured in millimeters of mercury. A low vapor pressure (<10 mmHg) indicates that the chemical
does not readily form vapors at room temperature. General laboratory ventilation or an alternative
such as the elephant trunk or snorkel may be appropriate, unless the material is heated or in a
higher temperature room that might promote vapor formation. High vapor pressure indicates that
the material easily forms vapors and may require use of a ventilated enclosure, such as a chemical
hood.

Vapor density is compared to that of air, which is 1. A chemical having a vapor density greater
than 1 is heavier than air. If the vapors need to be controlled, a chemical hood or a ventilation
device that draws air from below, such as a downdraft table or a slot hood or elephant trunk with
the exhaust aimed low may be appropriate. Conversely, a chemical with a vapor density less than
1 is lighter than air. Besides a chemical hood, a ventilation device that draws air from above, such
as an elephant trunk or snorkel with the exhaust positioned above the source, may work best.

For radioactive or biological materials, consider whether the operations might cause the materials
to aerosolize or become airborne and whether inhalation poses a risk to health or the environment.
Determine whether filtration or trapping is required or recommended. For manipulating solid
particulates, a chemical hood and similar equipment with higher airflow may be too turbulent.
Weighing boxes or ventilated balance enclosures may be a better fit for such work. For
nanomaterials, a laboratory chemical hood might be too turbulent for manipulating the materials.
Also, consider whether the exhaust containing these tiny particles should be filtered. Studies have
shown that high-efficiency particulate air filters are very effective for Nano-size particles.
Containment tests for chemical hoods allow for a very minor amount of leakage into the breathing
zone of the user. For chemical vapors, such an amount may be insignificant, but in the same volume
of nanoparticles, the number of particles may be quite large, and biosafety cabinets, gloveboxes or
filtering hoods would be superior. More specialized ventilation systems, such as biosafety cabinets
and gloveboxes, may be necessary to control specific types of hazards.

Laboratory Chemical Hoods:

Laboratory chemical hoods are the most important components used to protect laboratory
personnel from exposure to hazardous chemicals and agents. Functionally, a standard chemical
hood is a fire- and chemical-resistant enclosure with one opening (face) in the front with a movable
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window (sash) to allow user access to the interior. Large volumes of air are drawn through the face
and out the top into an exhaust duct to contain and remove contaminants from the laboratory. Note
that because a substantial amount of energy is required to supply tempered supply air to even a
small hood, the use of hoods to store bottles of toxic or corrosive chemicals is a very wasteful
practice, which can seriously impair the effectiveness of the hood as a local ventilation device.
Thus, it is preferable to provide separate vented cabinets for the storage of toxic or corrosive
chemicals. The amount of air exhausted by such cabinets is much less than that exhausted by a
properly operating hood.

A well-designed hood, when properly installed and maintained, offers a substantial degree of
protection to the user if it is used appropriately and its limitations are understood. Chemical hoods
are the best choice, particularly when mixtures or uncharacterized products are present and any
time there is a need to manage chemicals using the ALARA principle.

Laboratory Chemical Hood Face Velocity:

The average velocity of air drawn through the face of the laboratory chemical hood is called the
face velocity. The face velocity greatly influences the ability to contain hazardous substances, that
is, its containment efficiency. Face velocities that are too low or too high reduce the containment
efficiency. Face velocity is only one indicator of hood performance and one should not rely on it
as a sole basis for determining the containment ability of the chemical hood. There are no
regulations that specify acceptable face velocity. Indeed, modern hood designs incorporate interior
configurations that affect the airflow patterns and are effective at different ranges of face velocity.

For traditional chemical hoods, several professional organizations have recommended that the
chemical hood maintain a face velocity between 80 and 100 feet per minute (fpm). Face velocities
between 100 and 120 fpm have been recommended in the past for substances of very high toxicity
or where outside influences adversely affect hood performance. However, energy costs to operate
the chemical hood are directly proportional to the face velocity and there is no consistent evidence
that the higher face velocity results in better containment. Face velocities approaching or
exceeding 150 fpm should not be used; they may cause turbulence around the periphery of the sash
opening and actually reduce the capture efficiency, and may reentrant settled particles into the air.

With the desire for more sustainable laboratory ventilation design, manufacturers are producing
high-performance hoods, also known as low-flow hoods, which achieve the same level of
containment as traditional ones, but at a lower face velocity. These chemical hoods are designed
to operate at 60 or 80 fpm and in some cases even lower. Average face velocity is determined by
measuring individual points across the plane of the sash opening and calculating their average. A
more robust measure of containment uses tracer gases to provide quantitative data and smoke
testing to visualize airflow patterns. ASHRAE/ANSI 110 testing is an example of this technique.
This type of testing should be conducted at the time the chemical hood is installed, when

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substantial changes are made to the ventilation system, including rebalancing and periodically as
part of a recommissioning or maintenance program.

Once a chemical hood is tested and determined to be acceptable via the ASHRAE/ANSI 110
method or an equivalent means, the face velocity should be noted and used as the reference point
for routine testing. Each chemical hood, laboratory, facility, or site must define the acceptable
average face velocity, minimum acceptable point velocity, and maximum standard deviation of
velocities, as well as when ASHRAE/ANSI 110 or visualization testing is required. These
requirements should be incorporated into the laboratory's Chemical Hygiene Plan and ventilation
system management plans.

When first installed and balanced, a laboratory chemical hood must be subjected to the
ASHRAE/ANSI 110 or equivalent test before it is commissioned. When multiple similar chemical
hoods are installed at the same time, at least half should be tested, provided the design is
standardized relative to location of doors and traffic, and to location and type of air supply
diffusers.

Elements That Affect Laboratory Chemical Hood Performance:

Tracer gas containment testing of chemical hoods reveals that air currents impinging on the face
at a velocity exceeding 30 to 50% of the face velocity reduce the containment efficiency by causing
turbulence and interfering with the laminar flow of the air entering the chemical hood. Thirty to
fifty percent of a face velocity of 100 fpm, for example, is 30 to 50 fpm, which represents a very
low velocity that can be produced in many ways. The rate of 20 fpm is considered to be still air
because that is the velocities at which most people first begin to sense air movement.

Proximity to Traffic:

Most people walk at approximately 250 fpm (approximately 3 mph [4.8 kph]) and as they walk,
vortices exceeding 250 fpm form behind them. If a person walks in front of an open chemical
hood, the vortices can overcome the face velocity and pull contaminants into the vortex, and into
the laboratory. Therefore, laboratory chemical hoods should not be located on heavily traveled
aisles, and those that are should be kept closed when not in use. Foot traffic near these chemical
hoods should be avoided when work is being performed.

Proximity to Supply Air Diffusers:

Air is supplied continuously to laboratories to replace the air exhausted through laboratory
chemical hoods and other exhaust sources and to provide ventilation and temperature/humidity
control. This air usually enters the laboratory through devices called supply air diffusers located
in the ceiling. Velocities that exceed 800 fpm are frequently encountered at the face of these
diffusers. If air currents from these diffusers reach the face of a chemical hood before they decay
to 30 to 50% of the face velocity, they cause the same effect as air currents produced by a person

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walking in front of the chemical hood. Normally, the effect is not as pronounced as the traffic
effect, but it occurs constantly, whereas the traffic effect is transient. Relocating the diffuser,
replacing it with another type, or rebalancing the diffuser air volumes in the laboratory can alleviate
this problem.

Proximity to Windows and Doors:

Exterior windows with movable sashes are not recommended in laboratories. Wind blowing
through the windows and high-velocity vortices caused when doors open can strip contaminants
out of the chemical hoods and interfere with laboratory static pressure controls. Place hoods away
from doors and heavy traffic aisles to reduce the chance of turbulence reducing the effectiveness
of the hood.

Prevention of Intentional Release of Hazardous Substances into Chemical Hoods:

Laboratory chemical hoods should be regarded as safety devices that can contain and exhaust toxic,
offensive, or flammable materials that form as a result of laboratory procedures. Just as you should
never flush laboratory waste down a drain, never intentionally send waste up the chemical hood.
Do not use the chemical hood as a means of treating or disposing of chemical waste, including
intentionally emptying hazardous gases from compressed gas cylinders or allowing waste solvent
to evaporate. For some operations, condensers, traps, and/or scrubbers are recommended or
necessary to contain and collect vapors or dusts to prevent the release of harmful concentrations
of hazardous materials from the chemical hood exhaust.

Laboratory Chemical Hood Performance Checks:

When checking if laboratory chemical hoods are performing properly, observe the following
guidelines: Evaluate each hood before initial use and on a regular basis (at least once a year) to
visualize airflow and to verify that the face velocity meets the criteria specified for it in the
laboratory's Chemical Hygiene Plan or laboratory ventilation plan.

Many factors can compromise the efficiency of chemical hood operation, and most are avoidable.
Be aware of all behavior that can, in some way, modify the chemical hood and its capabilities.
Always consider the following:

• Keep chemical fume hood exhaust fans on at all times.

• If possible, position the chemical hood sash so that work is performed by extending the
arms under or around the sash, placing the head in front of the sash, and keeping the sash
between the person and the chemical source. View the procedure through the sash, which
acts as a primary barrier if a spill, splash, or explosion should occur.

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 • Avoid opening and closing the sash rapidly, and avoid swift arm and body movements in
front of or inside the chemical hood. These actions may increase turbulence and reduce the
containment efficiency.

• Place chemical sources and apparatus at least 6 in. behind the face. Paint a colored stripe
or apply tape to the work surface 6 in. back from the face to serve as a reminder Quantitative
chemical hood containment tests reveal that the concentration of contaminant in the
breathing zone can be 300 times higher from a source located at the front of the face than
from a source placed at least 6 in. back. This concentration continues to decline as the
source is moved farther toward the back.

• Place equipment as far to the back of the chemical hood as practical without blocking the
bottom baffle.

• Separate and elevate each instrument by using blocks or racks; air should flow easily
around all apparatus.

• Do not use large pieces of equipment in a chemical hood, because they tend to cause dead
spaces in the airflow and reduce the efficiency.

• If a large piece of equipment emits fumes or heat outside a chemical hood, have a special-
purpose hood designed and installed to ventilate that particular device. This method of
ventilation is much more efficient than placing the equipment in a chemical fume hood,
and it will consume much less air.

• Do not modify chemical hoods in any way that adversely affects performance. This
includes adding, removing, or changing any of the components, such as baffles, sashes,
airfoils, liners, and exhaust connections.

• Make sure all highly toxic or offensive vapors are scrubbed or adsorbed before the exit
gases are released into the chemical hood exhaust system.

• Keep the sash closed whenever the chemical hood is not actively in use or is unattended.

Housekeeping:

Keep laboratory chemical hoods and adjacent work areas clean and free of debris at all times. Keep
solid objects and materials (such as paper) from entering the exhaust ducts, because they can lodge
in the ducts or fans and adversely affect their operation. The chemical hood will have better airflow
across its work surface if it contains a minimal number of bottles, beakers, and laboratory
apparatus; therefore, prudent practice keeps unnecessary equipment and glassware outside the
chemical hood at all times and stores all chemicals in approved storage cans, containers, or
cabinets. Furthermore, keep the workspace neat and clean in all laboratory operations, particularly

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those involving the use of chemical hoods, so that any procedure or experiment can be undertaken
without the possibility of disturbing, or even destroying, what is being done.

Sash Operation:

Except when adjustments to the apparatus are being made, keep the chemical hood closed, with
vertical sashes down and horizontal sashes closed, to help prevent the spread of a fire, spill, or
other hazard into the laboratory. Horizontal sliding sashes should not be removed. The face
opening should be kept small to improve the overall performance of the hood. If the face velocity
becomes excessive, the facility engineers should make adjustments or corrections.

The standard operating position for the vertical sash may be comfortable for the majority of users.
However, shorter laboratory personnel may find that this position does not provide an adequate
barrier from the materials within the chemical hood and may need to adjust downward. Taller
laboratory personnel may need to raise the sash more in order to comfortably work in the chemical
hood. For chemical hoods with horizontal sashes, the intended operating configuration is to open
the panes in such a way that at least one pane is between both arms, providing a barrier between
the user and the contents of the chemical hood. In addition,

Testing and Verification:

The OSHA lab standard includes a provision regarding laboratory chemical hoods, including a
requirement for some type of continuous monitoring device on each chemical hood to allow the
user to verify performance and routine testing of the hood. It does not specify a test protocol.
Laboratory chemical hoods should be tested at least as follows:

✓ containment test by manufacturer;

✓ containment test after installation and prior to initial use (commissioning);
✓ annual or more frequent face velocity and airflow visualization;
✓ performance test any time a potential problem is reported; and
✓ Containment test after significant changes to the ventilation system, including rebalancing
or recommissioning.
The total volume of air exhausted by a laboratory chemical hood is the sum of the face volume
(average face velocity times face area of the hood) plus air leakage, which averages about 5 to
15% of the face volume. If the laboratory chemical hood and the general ventilating system are
properly designed, face velocities in the range of the design criteria will provide a laminar flow of
air over the work surface and sides of the hood. Higher face velocities (150 fpm or more), which
exhaust the general laboratory air at a greater rate, waste energy and are likely to degrade hood
performance by creating air turbulence at the face and within the chemical hood, causing vapors
to spill out into the laboratory (Figure).

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 FIGURE: Laminar versus turbulent velocity profile

Velocity data are from a single traverse point on two separate hoods. The light line represents a
hood where supply air interference caused large variations in velocity, a “typical” turbulent profile.
Eddy currents and flow reversals caused by a turbulent airflow pattern may cause spillage and
leakage of contaminants from the hood into the laboratory environment. In contrast, the bold line
represents a hood having an almost ideal velocity profile, indicative of a laminar airflow pattern.
The coefficient of variation (COV) is used as a predictor of the level of turbulence experienced at
the face of a hood. A high COV indicates a turbulent air profile and most likely is a strong indicator
of poor containment; a low COV indicates a laminar flow profile and likely good
containment.[SOURCE: Maupins and Hitchings (1998) ]

An additional method for containment testing is the EN 14175, which is the standard adopted by
the European Union and replaces several other procedures that were in place for individual
countries

Routine Testing:

At least annually, the following test procedures should be conducted for all chemical hoods.

Instrumentation:

Anemometers and other instruments used to measure face velocity must be accurate in order to
supply meaningful data. Instruments should be calibrated at least once a year and the calibration
should be National Institute of Standards and Technology traceable.

Laboratory Chemical Hood Design and Construction

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When specifying a laboratory chemical hood for use in a particular activity, laboratory personnel
should be aware of the design features. Assistance from an industrial hygienist, ventilation
engineer, or laboratory consultant is recommended when deciding to purchase a chemical hood.

General Design Recommendations

Construct laboratory chemical hoods and the associated exhaust ducts of nonflammable materials.
Equip them with vertical, horizontal, or combination vertical/horizontal sashes that can be closed.
For the glass within the sash, use either laminated safety glass that is at least 7/32-in. thick or other
equally safe material that will not shatter if there is an explosion inside. Locate the utility control
valves, electrical receptacles, and other fixtures outside the chemical hood to minimize the need
to reach within the chemical hood proper. Other specifications regarding the construction
materials, plumbing requirements, and interior design vary, depending on the intended use.

Special Design Features

Since the invention of the chemical hood, two major improvements have been made in the
design—airfoils and baffles. Include both features on any new purchases. Airfoils built into the
bottom and sides of the sash opening significantly reduce boundary turbulence and improve
capture performance. Fit new chemical hoods with airfoils and retrofit any hoods without airfoils
When air is drawn through a laboratory chemical hood without a baffle, most of the air is drawn
through the upper part of the opening, producing an uneven velocity distribution across the face
opening. All chemical hoods should have baffles. When baffles are installed, the velocity
distribution is greatly improved. Adjustable baffles can improve hood performance and are
desirable if the adjustments are made by an experienced industrial hygienist, consultant, or
technician.

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Liquid Scrubbers

A laboratory chemical hood scrubber is a laboratory-scale version of a typical packed-bed liquid

scrubber used for industrial air pollution control. Figure 9.10 shows a schematic of a typical
chemical hood scrubber.

108
Contaminated air from the chemical hood enters the unit and passes through the packed-bed, liquid
spray section, and mist eliminator and into the exhaust system for release up the stack. The air and
the scrubbing liquor pass in a countercurrent fashion for efficient gas-liquid contact. The scrubbing
liquor is recirculated from the sump and back to the top of the system using a pump. Water-soluble
gases, vapors, and aerosols are dissolved into the scrubbing liquor. Particulates are also captured
quite effectively by this type of scrubber. Removal efficiencies for most water-soluble acid- and
base-laden airstreams are usually between 95 and 98%. Scrubber units are typically configured
vertically and are located next to the chemical hood as shown in Figure. They are also produced
in a top-mount version, in which the packing, spray manifold, and mist eliminator sections are
located on top of the chemical hood and the sump and liquid-handling portion are underneath for
a compact arrangement taking up no more floor area than the hood itself. Most hoods do not require
a scrubber unit, assuming the exhaust stack is designed properly and chemical quantities of volatile
materials are low.

ROOM PRESSURE CONTROL SYSTEMS

Laboratories and clean rooms usually require that a differential pressure be maintained between
them and adjoining no laboratory spaces. This requirement for a pressure differential may come
from code considerations or from the intended use of the space. For example, NFPA 45 states that
“air pressure in the laboratory work areas shall be negative with respect to corridors and non-
laboratory areas of the laboratory unit …” (NFPA, 2004). This rule helps prevent the migration of
fire, smoke, and chemical releases from the laboratory space. Laboratories containing radiation

109
hazards or biohazards may also be required by government agencies to maintain a negative
pressure to contain these hazards. Clean rooms, on the other hand, are normally operated at a
positive static pressure to prevent infiltration of particulates. (See sections 9.E.2 and 9.E.3, below,
for further information.)

SPECIAL SYSTEMS

Gloveboxes

Unlike a chemical hood, gloveboxes are fully enclosed and are under negative or positive pressure.
Gloveboxes are usually small units that have multiple openings in which arm-length rubber gloves
are mounted. The operator works inside the box by using these gloves. Construction materials vary
widely, depending on the intended use. Clear plastic is frequently used, because it allows visibility
of the work area and is easily cleaned.

Clean Rooms

Clean rooms are special laboratories or workspaces in which large volumes of air are supplied
through HEPA filters to reduce the particulates present in the room, in order to protect research
materials. As nanotechnology becomes more prevalent in scientific research across many
disciplines, clean rooms are becoming more and more in demand, not just in pharmaceutical,
microbiological, optical, and microelectronics laboratories. Special construction materials and
techniques, air-handling equipment, filters, garments, and procedures are required, depending on
the cleanliness level of the facility. Consult a laboratory expert in clean room operation before a
clean room is designed, built, or worked in.

Clean Room Protocols

The main objective of a clean room is to protect the materials and equipment from particulates.
Whereas most laboratories maintain negative airflow with respect to adjacent no laboratory areas,
clean rooms may be slightly positive. Thus, it is important to ensure that hazardous materials are
stored in ventilated cabinets and work with volatile hazardous materials is done with proper
ventilation.

• Use an air shower before entering the clean room.

• Keep personal items out of the clean room.
• Use only specially made notebooks and paper in the clean room; no felt-tip pens (except
permanent markers).
• Avoid bringing wood-pulp-based products into the clean room, such as magazines, books,
regular tissues, and regular paper.
• Do not bring Styrofoam or powders or any products that may produce dusts or aerosols
into the clean room.

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Laboratory Chemical Hoods and Laboratory Furniture in Clean Rooms

Laboratory chemical hoods and laboratory furniture in clean rooms must be easy to clean and not
subject to rust or chalking. Most prefer not to use materials with painted surfaces, which may chalk
or peel over time, or wood products that may form wood dusts. Stainless steel and thermoplastics
are the most common materials. Polypropylene chemical hoods are commonplace in clean rooms.
The main concern is that this material burns and melts very easily. In the event of a fire, a
polypropylene chemical hood may become fully involved. For this reason, it is prudent to choose
either a fire-retardant polypropylene or another thermoplastic or to install an automatic fire
extinguisher within the hood. For nanomaterials, consider whether a chemical hood might be too
turbulent for manipulating the materials. A biosafety cabinet, a ventilated enclosure with HEPA
filtration, or a glovebox may be better alternatives.

Biological Safety Cabinets and Biosafety Facilities

BSCs are common containment and protection devices used in laboratories working with
biological agents. BSCs and other facilities in which viable organisms are handled require special
construction and operating procedures to protect laboratory personnel and the environment.
Conventional chemical hoods should never be used to contain biological hazards. Biosafety in
Microbiological and Biomedical Laboratories (HHS/CDC/NIH, 2007a), Primary Containment for
Biohazards: Selection, Installation, and Use of Biological Safety Cabinets ((HHS/CDC/NIH,
2007b), and Biosafety in the Laboratory: Prudent Practices for the Handling and Disposal of
Infectious Materials (NRC, 1989) give detailed information on this subject.

Design Criteria:

The institution should determine the criteria to use for all new installations of chemical hoods and
other ventilation systems. This might include

• testing criteria as installed (e.g., all or a representative sampling of the hoods must pass
ANSI/ASHRAE 110-1995 containment testing as installed);
• chemical hood design criteria (e.g., face velocity criteria at specific sash height and sash
design);
• types of continuous monitoring systems preferred or required (e.g., face velocity reading,
magnehelic gauge);
• acceptable diversity factors;
• energy conservation strategies;
• alarm systems;
• type of duct work;
• noise criteria;
• preference for VAV systems (designing one extra fan into each system); and
• backup power.

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Many laboratories, particularly academic research laboratories, experience high turnover rates.
Good signage and postings complement training and act as constant reminders (Figure).

Inspection and Maintenance

Maintenance is key to a ventilation system management program. The program should describe
the elements of the inspection and maintenance program, including designation of who conducts
inspections and how often; how inspections are recorded; inspection criteria for laboratory
chemical hoods including face velocity testing—equipment used, history, how recorded, how
posted on the chemical hood, and will maximum sash height be marked and how; criteria for
working on roofs and around stacks.

Commissioning

When new components are installed, or any significant change to the ventilation system occurs,
consider hiring a commissioning agent with experience with laboratory facilities. An outside
commissioning agent will ensure that the system meets the criteria you have selected, note any
design errors, handle problems, and facilitate testing, installation, etc. In-house staff or hired
consultants will continue to maintain the equipment, but the startup issues can be overwhelming.
Ensure that those who will be using and maintaining the system receive training.

LABORATORY DECOMMISSIONING

A laboratory must be properly decommissioned prior to changing its use. Among other steps,
decommissioning entails decontamination and the removal of hazards to ensure the safety of future

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occupants and others who may enter the space. Decommissioning must be done prior to
renovation, even if the space is to be reused as a laboratory. Because laboratory operations differ,
it is appropriate to decommission a laboratory whenever there is a significant change in occupancy.
Areas outside of the laboratory, such as ventilation ductwork, coldrooms, hallway freezers and
common storage areas, should also be decommissioned if they are concurrently subject to a
significant change in use or occupancy. Decommissioning must also be done prior to the
demolition of a laboratory.

Removal, Cleaning, and Decontamination

The second step in decommissioning is to remove all hazards from the space. Be sure that all
chemicals, radioactive materials, and biologicals have been removed from use and storage areas,
including refrigerators and freezers. Movable equipment should be appropriately cleaned and/or
disinfected, and removed from the lab.

Clearance

Final tests or survey results can be used to verify decontamination. In some cases regulatory
authorities allow permanent marking of a porous floor or wall where a radioactive material or
chemical has penetrated deeply, and destructive removal is impractical prior to the building's
demolition. When removal, decontamination, and cleaning meet planned decommissioning
standards, a final area clearance statement can be issued, and renovation, demolition, or the new
occupancy can commence.

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 CHAPTER: 10
Culture types and Isolation of culture

In nature microbes occurs as mixed populations of number of widely different species. It is not
generally feasible to work with single microorganism. For this fact we study cultures that contain
numerous microorganisms. A culture that contains single species population or single kind of
microorganisms regardless of number of individuals, in an environment that is free from other
living beings is called Pure or Axenic culture, but technically pure culture is derived from single
cell (clone). If two or more than one kind of microorganism grow together as this type commonly
occurs in nature are referred as mixed culture or sometimes cultures accidentally contain more
than one type of species of microorganisms are sometimes called as contaminated culture. There
are two broad categories of culture-

1. Pure culture

2. Mixed culture

Types of culture (specially pure culture):

1. Stock culture
2. Streak culture
3. Starter culture
4. Stick or Stab culture
5. Enrichment culture
6. Batch culture
7. Continuous culture
8. Synchronous culture
1.Stock culture :

The stock culture are those type of cultures of known species of microbes which are maitained in
laboratory for longer period of time. This type of cultures generally used as stock of particular type
of microorganisms or their strains and researchers and students can take sample from them for
study or research work.

2. Streak culture :

Streak culture is developed by drawing a straight line with an inoculated needle on surface of the
culture media.

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3. Starter culture :

The starter culture consists of combination of species or particular species best suited for the
manufacture of the desired product. As a case study several commercial laboratories engaged in
production of starter culture for the dairy products. The desired microorganisms indulge are the
species of Streptococcus, Lactobacillus, and Leuconostoc which are liable for the desired changes
in fermented milk production.

4. Stick or Stab culture

These cultures are prepared in solidified media agar or gelatin or liquid media by inserting straight
inoculation needle for some distance. Here growth of microorganism occure along the line of
puncture and changes seen along these line.

5. Enrichment culture :

The enrichment culture is a type of culture in which the growth of particular microorganism is
favoured in mixed microorganisms by manupulating the nutritional ecosystem of culture. For
instance , if we desired to isolate Lactobacillus from mix population of Streptococcus, Leuconostoc
and Lactobacillus, the nutritional ecosystem is manupulated by enrichment method so as to the
growth of Lactobacillus is favoured. Now the Lactobacillus can be isolated and transferred to other
medium and desired pure culture can be obtained.

6. Batch culture :

Microbial growth in limited volume of liquid medium is referred as batch culture. This type of
culture is supportive in studying the growth characteristics chiefly of bacteria. When bacteria are
transferred to a known volume of batch culture, growth population of bacteria undergoes a
characteristic sequence in its rate of increase in cell number. four phases are seen when cell number
increases and is determined in relative to time as Lag phase, Log phase, Stationary phase and
Decline or death phase.

7. Continuous culture :

It is a type of culture which permits growth of microorganisms at a constant rate, more precisely
at steady rate is called as continuous culture. This type of cultures are extremely useful for
biochemical studies of microorganisms.

8. Synchronous culture :

Synchronous culture represents population of cells of specific microorganisms that are at the same
stage of life cycle. All the cells in culture behave similarily i.e. if they divide then divide at the
same time. Thus in such type of culture population kept uniform with respect to division and

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growth. It can be maintained by Helmstetter cummings technique however other technique also
used by researchers.

Isolation of Pure culture :

1. Streak Plate Method:

This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed
culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the
agar medium (Fig.). The successive streaks “thin out” the inoculum sufficiently and the micro-
organisms are separated from each other. It is usually advisable to streak out a second plate by the
same loop/needle without inoculation. These plates are incubated to allow the growth of colonies.
The key principle of this method is that, by streaking, a dilution gradient is established across the
face of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution
gradient, confluent growth does not take place on that part of the medium where few bacterial cells
are deposited. Presumably, each colony is the progeny of a single microbial cell thus representing
a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating
loop/needle and re-streaked onto fresh media to ensure purity.

Different streaking methods

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2. Pour Plate Method:

This method involves plating of diluted samples mixed with melted agar medium (Fig.). The main
principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to
permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of
bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state
at a temperature of 42-45°C (agar solidifies below 42°C). The bacteria and the melted medium are
mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify,
and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both
within the agar medium (subsurface colonies) and on the medium (surface colonies). These
isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to
insure purity.

Pour plate method has certain disadvantages as follows:

• The picking up of subsurface colonies needs digging them out of the agar medium thus
interfering with other colonies, and
• The microbes being isolated must be able to withstand temporary exposure to the 42-45°
temperature of the liquid agar medium; therefore this technique proves unsuitable for the
isolation of psychrophilic microorganisms.
However, the pour plate method, in addition to its use in isolating pure cultures, is also used for
determining the number of viable bacterial cells present in a culture.

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 Pour plate method

3. Spread Plate Method:

In this method (Fig.), the mixed culture or microorganisms is not diluted in the melted agar medium
(unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid,
usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the
center of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod.
The medium is now incubated. When the colonies develop on the agar medium plates, it is found
that there are some plates in which well-isolated colonies grow. This happens as a result of
separation of individual microorganisms by spreading over the drop of diluted liquid on the
medium of the plate. The isolated colonies are picked up and transferred onto fresh medium to
ensure purity. In contrast to pour plate method, only surface colonies develop in this method and
the microorganisms are not required to withstand the temperature of the melted agar medium.

Spread plate method

4. Serial Dilution Method:

As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms
that have not yet been successfully cultivated on solid media and grow only in liquid media.

A microorganism that predominates in a mixed culture can be isolated in pure form by a series of
dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large
number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.
The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that
there are some tubes showing growth of only one individual microbe. For convenience, suppose
we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms (Fig.),
i.e., 100 microorganisms/ml of the liquid medium. If we take out 1 ml of this medium and mix it
with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10
microorganisms/ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid
medium, each ml would now contain a single microorganism. If this tube shows any microbial

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growth, there is a very high probability that this growth has resulted from the introduction of a
single microorganism in the medium and represents the pure culture of that microorganism.

Serial dilution method

5. Single Cell Isolation Methods:

An individual cell of the required kind is picked out by this method from the mixed culture and is
permitted to grow. Following two methods are used-

[A] Capillary pipette method:

Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a
sterile pipette drawn to a capillary. One then examines each drop under the microscope until one
finds such a drop, which contains only one microorganism. This drop is removed with a sterile
capillary pipette to fresh medium. The individual microorganism present in the drop starts
multiplying to yield a pure culture.

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 Capillary method for isolation of single cell

[B] Micromanipulator method:

Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture.
This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial
cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by
means of which its micropipette can be moved right and left, forward, and backward, and up and
down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a
micropipette. Now a hanging drop is searched, which contains only a single microorganism cell.
This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of
sterile medium on another sterile coverslip. When the number of cells increases in that drop as a
result of multiplication, the drop is transferred to a culture tube having suitable medium. This
yields a pure culture of the required microorganism.

The advantages of this method are that one can be reasonably sure that the cultures come from a
single cell and one can obtain strains with in the species. The disadvantages are that the equipment
is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason
why this method is reserved for use in highly specialized studies.

6. Enrichment Culture Method:

Generally, it is used to isolate those microorganisms, which are present in relatively small numbers
or that have slow growth rates compared to the other species present in the mixed culture. The
enrichment culture strategy provides a specially designed cultural environment by incorporating a
specific nutrient in the medium and by modifying the physical conditions of the incubation. The
medium of known composition and, specific condition of incubation favors the growth of desired
microorganisms but, is unsuitable for the growth of other types of microorganisms.

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 CHAPTER: 11
Chromatography techniques and its Applications

Chromatography Technique:

Chromatography is a non-destructive process for resolving a multicomponent mixture of traces,

minor or major constituents in to its individual fractions. It is technically a separation tool based
on the partition or distribution coefficient which describes the way in which a compound
distributes itself between two immiscible phases. Chromatography technique has speeded up the
development of modern biochemistry and has made identification, separation, quantitative
estimation and study of many of the substances of biological origin possible. With the aid this
technology substances like amino acids, pigments, sugars, organic acid, phenolic compounds and
many others can be separated quantitatively and studied.

In year 1906, Michael Tswett, who coined the terms chromatogram and chromatography while
trying to separate the pigments in plants initiated to develop chromatography technique. In his
study when petroleum ether extracts of plant pigments were placed on the top of column of finely
powdered calcium carbonate (CaCo3) and run down with the help of added ether, definite green
and yellow zones of the pigments were separated due to different adsorption capacities of different
pigments.

Chromatography is based on the principle where molecules in mixture applied onto the surface or
into the solid, and fluid stationary phase (stable phase) is separating from each other while moving
with the aid of a mobile phase. The factors effective on this separation process include
molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and
affinity or differences among their molecular weights. Because of these differences, some
components of the mixture stay longer in the stationary phase, and they move slowly in the
chromatography system, while others pass rapidly into mobile phase, and leave the system faster.
Basically three components form the basis of the chromatography technique.

➢ Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface a solid support”.
➢ Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
➢ Separated molecules
A number of types of chromatography have been developed, during twenty century based on
simple differential separation principle of molecules. Basically all chromatography methods
consists of the stationary phase which may be gel, sol, liquid or a solid-liquid mixture and the
mobile phase which may be liquid or gaseous and which flows over or through the stationary
phase. The process of separately extracting a substance in relatively pure form was known as
“Elution” and the extract obtained as a result of the process as “Elute”.

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Stationary phase in chromatography is a solid phase or a liquid phase coated on the surface of a
solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile
phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas
chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids,
and solid material. Liquid chromatography is used especially for thermal unstable and non-volatile
samples. The type of interaction between stationary phase, mobile phase, and substances contained
in the mixture is the basic component effective on separation of molecules from each other.
Chromatography methods based on partition are very effective on separation, and identification of
small molecules as amino acids, carbohydrates, and fatty acids. However, affinity
chromatography’s (i.e. Ion-exchange chromatography) are more effective in the separation of
macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of
proteins, and in studies related to protein synthesis; gas-liquid chromatography is utilized in the
separation of alcohol, ether, lipid, and amino groups, and observation of enzymatic interactions,
while molecular-sieve chromatography is engaged especially for the determination of molecular
weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA
particles, and viruses.

Initially chromatographic techniques were used to separate substances based on their color as was
the case with herbal pigments. With time its application area was extended considerably.
Nowadays, chromatography is accepted as an extremely sensitive and effective separation method.
Column chromatography is one of the useful separation, and determination methods. Column
chromatography is a protein purification method realized especially based on one of the
characteristic features of proteins. Besides, these methods are used to control purity of a protein.
HPLC technique which has many superior features including especially its higher sensitivity, rapid
turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids,
hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.

The purpose of applying chromatography which is used as a method of quantitative analysis apart
from its separation is to achieve a satisfactory separation within a suitable time interval. Various
chromatography methods have been developed. Some of them include column chromatography,
thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange
chromatography, gel permeation chromatography, high-pressure liquid chromatography, and
affinity chromatography.

Types of chromatography

➢ Column chromatography
➢ Ion-exchange chromatography
➢ Gel Filtration/Gel-permeation (molecular sieve) chromatography
➢ Affinity chromatography
➢ Paper chromatography
➢ Thin-layer chromatography

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 ➢ Gas chromatography
➢ Dye-ligand chromatography
➢ Hydrophobic interaction chromatography
➢ Pseudo affinity chromatography
➢ High-pressure liquid chromatography (HPLC)
Column chromatography or Adsorption chromatography:

The addition of liquid, gas or a solid dissolved in a liquid to the surface of a solid body is known
as adsorption. In adsorption chromatography, the unknown substances are separated and
recognized making use of the phenomenon of adsorption. Many solids including charcoal, alumina
and other metallic acids, metallic salts, silica gels and special ones like agar (for plant pigments)
are used as adsorbing agents in adsorption chromatography. One of the main impurities is the water
which can be removed by heating the adsorbent. This process is known as activation.

As we know that proteins have different characteristic features as size, shape, net charge, stationary
phase used, and binding capacity, each one of these characteristic components can be purified
using chromatographic methods. Among these methods, most frequently column chromatography
is applied. This technique is used for the purification of biomolecules. On a column (stationary
phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied (Figure).
Their flow through inside column material placed on a fiberglass support is ensured. The samples
are accumulated at the bottom of the device in a time and volume-dependent manner.

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Ion- exchange chromatography:

Ion- exchange chromatography is based on electrostatic interactions between charged protein

groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein
to be separated, and the affinity of the protein to the column is achieved with ionic links. Proteins
are separated from the column either by changing pH, concentration of ion salts or ionic strength
of the buffer solution. Positively charged ion- exchange matrices are called anion-exchange
matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged
groups are known as cation-exchange matrices, and adsorb positively charged proteins (Figure).

Gel Filtration/Gel- permeation (molecular sieve) chromatography:

The basic principle is to use dextran containing materials to separate macromolecules based on
their differences in molecular sizes. This procedure is basically used to determine molecular
weights of proteins, and to decrease salt concentrations of protein solutions. In a gel- permeation
column stationary phase consists of inert molecules with small pores. The solution containing
molecules of different extents are passed continuously with a constant flow rate through the
column. Molecules larger than pores can not infiltrate into gel particles, and they are retained
between particles within a restricted area. Larger molecules pass through spaces between porous
particles, and move rapidly through inside the column. Molecules smaller than the pores are
diffused into pores, and as molecules get smaller, they leave the column with proportionally longer
retention times (Figure). Sephadex G type is the most frequently used column material. Besides,
dextran, agarose, polyacrylamide are also used as column materials.

Application of gel filtration:

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1. Purification or Fractionation

2. Group separation or Desalting

3. Determination of molecular weight

Affinity chromatography:

This chromatography technique is used for the purification of enzymes, hormones, antibodies,
nucleic acids, and specific proteins. A ligand which can make a complex with specific protein
(dextran, polyacrylamide, cellulose etc) binds the filling material of the column. The specific
protein which makes a complex with the ligand is attached to the solid support (matrix), and
retained in the column, while free proteins leave the column. Then the bound protein leaves the
column by means of changing its ionic strength through alteration of pH or addition of a salt
solution (Figure).

Paper chromatography:

In paper chromatography support material consists of a layer of cellulose highly saturated with
water. In this method a thick filter paper comprised the support, and water drops settled in its pores
made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a
developing tank. Paper chromatography is a “liquid-liquid” chromatography.

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Thin-layer chromatography:

Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method

stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material solid
substances are used. In column chromatography (alumina, silica gel, and cellulose) can be utilized.
In this method, the mobile phase travels upward through the stationary phase. The solvent travels
up the thin plate soaked with the solvent by means of capillary action. During this procedure, it
also drives the mixture priory dropped on the lower parts of the plate with a pipette upwards with
different flow rates. Thus the separation of analytes is achieved. This upward travelling rate
depends on the polarity of the material, solid phase, and of the solvent.

In cases where molecules of the sample are colorless, florescence, radioactive or a specific
chemical substance can be used to produce a visible coloured reactive product so as to identify
their positions on the chromatogram. Formation of a visible colour can be observed under room
light or UV light. The position of each molecule in the mixture can be measured by calculating the
ratio between the distances travelled by the molecule and the solvent. This measurement value is
called relative mobility, and expressed with a symbol Rf. Rf. value is used for qualitative
description of the molecules.

Gas chromatography:

In this method stationary phase is a column which is placed in the device, and contains a liquid
stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography is a
“gas-liquid” chromatography. Its carrier phase consists of gases as He or N2. Mobile phase which
is an inert gas is passed through a column under high pressure. The sample to be analyzed is
vaporized, and enters into a gaseous mobile phase. The components contained in the sample are
dispersed between mobile phase and stationary phase on the solid support. Gas chromatography is
a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent
separation of very minute molecules. It is used in the separation of very little amounts of analytes.

Dye- ligand chromatography:

Development of this technique was based on the demonstration of the ability of many enzymes to
bind purine nucleotides for Cibacron Blue F3GA dye. The planar ring structure with negatively
charged groups is analogous to the structure of NAD. This analogy has been evidenced by
demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD.
The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is
10–20-fold stronger that of the affinity of other adsorbents. Under appropriate pH conditions,
elution with high-ionic strength solutions and using ion-exchange property of adsorbent, the
adsorbed proteins are separated from the column.

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Hydrophobic interaction chromatography and Reverse phase liquid chromatography (HIC)

In this method the adsorbents prepared as column material for the ligand binding in affinity
chromatography are used. HIC technique is based on hydrophobic interactions between sides chain
bound to chromatography matrix. The variable hydrophobic nature of proteins allows their
separation through differential hydrophobic surface interactions. From these observations two
modes of protein chromatography have been developed, hydrophobic-interaction chromatography
(HIC) and reversed-phase chromatography (RPC). Selectivity of the HIC column can be easily
manipulated by changing mobile phase variables. Protein retention was increased by decreasing
the pH from neutrality or by using a salt with a greater "salting-out" ability. In addition, selectivity
can be altered through chemical modification of the matrix surface. Protein retention and
resolution decreased concomitantly with matrix ligand density.

There were several major differences in HIC and RPC selectivity. Hydrophilic proteins such as
cytochrome c and myoglobin were weakly retained on the HIC column but strongly retained on
the RPC column. In contrast, a hydrophobic protein such as beta-glucosidase was strongly retained
on the HIC column and only weakly retained on the RPC column. Other proteins were retained
equally by RPC and HIC columns. Load capacity on the HIC column was determined by plotting
resolution as a function of protein load. Resolution decreased significantly after 7.5 mg of total
protein had been loaded onto the column per cm3 of column material. Samples of lactic
dehydrogenase and alpha-chymotrypsin ranging in size from 10-200 micrograms were recovered
from an HIC column with greater than 86% enzymatic activity in all cases.

The recovery of enzymatic activity of alpha-chymotrypsin ranged from 55-91%, while none of the
activity of beta-glucosidase was recovered from the RPC column. Correct adjustment of the mobile
phase is equally important as the selection of the appropriate column for the separation of polar
compounds in LC. Both solvophobic and selective polar interactions control the retention in the
Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects
of the volume fraction of the strong eluent component in binary mobile phases on the sample
retention factors apply in a limited mobile phase composition range. We introduced a three-
parameter retention model, which provides improved prediction of retention over a broad mobile
phase range, under isocratic and gradient elution conditions.

The model does not imply any assumptions concerning either adsorption or partition distribution
mechanism, but allows estimating retention in pure strong and in pure weak mobile phase
components. The experimental retention data for phenolic acids and flavones on several core-shell
columns with different types of stationary phases agree with the theory.

Many polar columns with important structural hydrophobic moieties show dual retention
mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high
acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the
two modes for separations of samples containing compounds largely differing in polarity. The

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three-parameter model describes the retention in each mode, with separately determined best-fit
parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic
acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column.

Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins:

The variable hydrophobic nature of proteins allows their separation through differential
hydrophobic surface interactions. From these observations two modes of protein chromatography
have been developed, hydrophobic-interaction chromatography (HIC) and reversed-phase
chromatography (RPC). Selectivity of the HIC column can be easily manipulated by changing
mobile phase variables. Protein retention was increased by decreasing the pH from neutrality or
by using a salt with a greater "salting-out" ability. In addition, selectivity can be altered through
chemical modification of the matrix surface. Protein retention and resolution decreased
concomitantly with matrix ligand density. There were several major differences in HIC and RPC
selectivity.

Hydrophilic proteins such as cytochrome c and myoglobin were weakly retained on the HIC
column but strongly retained on the RPC column. In contrast, a hydrophobic protein such as beta-
glucosidase was strongly retained on the HIC column and only weakly retained on the RPC
column. Other proteins were retained equally by RPC and HIC columns. Load capacity on the HIC
column was determined by plotting resolution as a function of protein load. Resolution decreased
significantly after 7.5 mg of total protein had been loaded onto the column per cm3 of column
material. Samples of lactic dehydrogenase and alpha-chymotrypsin ranging in size from 10-200
micrograms were recovered from an HIC column with greater than 86% enzymatic activity in all
cases. The recovery of enzymatic activity of alpha-chymotrypsin ranged from 55-91%, while none
of the activity of beta-glucosidase was recovered from the RPC column.

Pseudo affinity chromatography:

Some compounds as anthraquinonoid dyes, and azo-dyes can be used as ligands because of their
affinity especially for dehydrogenases, kinases, transferases, and reductases the mostly known type
of this kind of chromatography is immobilized metal affinity chromatography (IMAC).

High-pressure liquid chromatography (HPLC):

HPLC chromatography technique is use to perform structural, and functional analysis, and
purification of many molecules within a short time, This technique yields perfect results in the
separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins,
steroids, and other biologically active molecules, In HPLC, mobile phase passes through columns
under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In this technique,
use of small particles, and application of high pressure on the rate of solvent flow increases
separation power, of HPLC and the analysis is completed within a short time. Essential
components of a HPLC device are solvent end, high- pressure pump, commercially prepared

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column, detector, and recorder. Duration of separation is controlled with the aid of a computerized
system, and material is accrued.

A new, weakly hydrophobic, high-performance liquid chromatography column has been

developed for the separation of native proteins based on their relative hydrophobicity. Starting
with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized
through acylation with anhydrides and acid chlorides of increasing chain length to obtain
increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind
hydrophobically to the columns by the use of high salt concentrations in the mobile phase.

Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture
of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving
power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was
achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer,
pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin
binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of
support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes
eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-
glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column
with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase
was recovered from the benzoate column with 92% retention of enzymatic activity.

Fast Protein liquid chromatography:

Fast protein liquid chromatography (FPLC) is a type of liquid chromatography that provides high
resolution by small-diameter stationary phases for protein characterization and separation. The
technique features high loading capacity, biocompatible buffer systems, fast flow rates, and
stationary phases for common chromatography modes (e.g., gel filtration, ion exchange, reversed
phase, and affinity chromatography). The system enables reproducible separation by incorporating
a high level of automation including samplers, gradient program control, and peak collection.
FPLC allows the users to monitor several parameters at a time such as UV level, pH, and
conductance and it allows for multiple columns to be run in tandem; therefore, minimizing the
time needed to isolate pure protein. The method is applicable to proteins as well as other kinds of
biological samples including oligonucleotides and plasmids.

Principle:

In fast protein liquid chromatography, the solvent velocity is controlled by a microprocessor

through a software interface to maintain the constant flow rate of the solvents. The mobile phase
is an aqueous buffer, and the flow rate is kept constant by a positive-displacement pump while the
buffer composition can be varied by drawing fluids from external reservoirs. The stationary phase
is composed of beads, usually of cross-linked agarose, packed into a cylindrical column. The eluent

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is passed through the detectors to measure the salt concentration (by conductivity) and protein
concentration (by absorbing the ultraviolet light at a wavelength of 280nm).

Apparatus:

The FPLC system consists of a control unit, high-precision pumps, a column, detection system,
and a fraction collector. The pump allows the fraction of each buffer entering the column to be
continuously varied. The injection loop, a segment of tubing of known volume, is filled with the
sample solution to be injected in the column. The sample loading is done by the injection valve
which links the mixer and sample loop to the column. The FPLC column is a glass or plastic
cylinder packed with beads of resin. It is mounted vertically with the buffer flowing downward
from top to bottom. The eluent from the column passes through the flow cells for protein
concentration measurements. The detector records the salt and protein concentration.

Protocol (Madadlou, Sullivan, & Sheehan, 2011)

Sample preparation:

✓ A spire Sephadex G-25 resin as slurry in water into the column before packing.
✓ Equilibrate the column in 3-4 column volumes of buffer a (10 mM Tris-HCl, pH 7.0).
✓ Load the sample (50 mL) under gravity flow.
✓ Pass buffer A through the column and monitor the protein by measuring A280, and collect
it as a single large peak.
✓ Measure the conductivity using the conductivity meter.
Ion-exchange FPLC:

✓ Prime the pumps A and B with filtered and degassed buffers A (10 mM Tris–HCl, pH 7.0.)
and B (10 mM Tris–HCl, pH 7.0, 1 M NaCl), respectively.
✓ Set the pressure limits on both the pumps below the maximum for the column in use.
✓ Equilibrate the Mono Q column (1 mL volume) with 5 volumes of buffer A and 10 volumes
of buffer B and then with 5 volumes of buffer A.
✓ Wash the sample loading loop with buffer A.
✓ Load the sample (0.5–10 mL) (approximately 1 mg/mL) and wash the column with buffer
A. Collect the flow-through and evaluate for the protein of interest.
Note: If protein not bound then replace the Mono Q column with Mono S column.

✓ Regenerate the column by washing it with 10 volumes of buffer B, then with 5 volumes of
buffer A.
Scouting FPLC methods:

Create gradients of shallowness by varying the concentration of buffer B at different time-points.

Identify the column to which the protein binds and perform chromatography at different pH values
using various buffer systems.

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Gel-filtration FPLC:

✓ Prime the pumps A and B with buffer C (50 mA/Tris-HCl, pH 7.0, 100 mM KCl) with no
column in the system.
✓ Set the pressure limits on both P-500 pumps.
✓ Connect Superose 12, and operate the system at 0.5 mL/min flow rate for 90 minutes for
column equilibration.
✓ Wash the sample loading loop with buffer C.
✓ Load 200 µL of the sample at the rate of 2 mg/mL.
✓ Monitor A280; collect peaks with the fraction collector and record the elution volume of
peak.
Applications:

Diagnosis of β-thalassemia (Tangvarasittichai, & Jermnim, 2009)-β-thalassemia is a genetic

blood disorder in which hemoglobin production is reduced. For its diagnosis, quantitative
hemoglobin A2 (HbA2) levels are used. High-performance liquid chromatography (HPLC),
electrophoresis, and microcolumn chromatography are the conventional techniques for beta-
thalassemia diagnosis. In the study, fast protein liquid chromatography (FPLC) method was
established to measure HbA2 levels in the samples. The FPLC method, using a DEAE Sepharose,
anion-exchange column chromatography technique was set up for diagnosis. The FPLC results
were highly correlated (r = 0.985, P<0.001) with those of HPLC, cellulose acetate electrophoresis
(r = 0.977), and microcolumn chromatography (r = 0.980). The method was found 100 percent
sensitive and specific for β-thalassemia diagnosis. In addition, the technique was simple, rapid,
cheaper, and reproducible.

Assessment of protein unfolding (Uversky, 1993)-Fast protein size-exclusion liquid

chromatography (SEC-FPLC) was used to explore the solvent-induced unfolding of proteins. Six
proteins were evaluated: two of them (sperm whale myoglobin and hen white lysozyme) and the
other four proteins (bovine and human a-lactalbumin, bovine carbonic anhydrase B (BCAB), and
8-lactamase from Staphylococcus aureus). It was shown that the permeation properties of the
Superose 12 columns are independent of temperature, pH, and denaturants. It was also indicated
that protein molecules could only be in one of the two unfolding states with different compactness
under denaturant effect. The results suggested that FPLC is one of the most direct approaches to
establish the “all-or-none” mechanism to study the solvent-induced denaturation of globular
proteins. The fast protein size-exclusion liquid chromatography (FPLC) does not shift the
equilibrium between the unfolding states and therefore; could be used for qualitative and
quantitative evaluation of protein denaturation.

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Precautions:

✓ Use neutral pH to allow the assessment of both anion- and cation-exchange columns at the
same ph.
✓ Avoid pH extremes to prevent protein denaturation.
✓ The FPLC procedure should be performed at 4°C for the purification of labile proteins.
✓ To avoid NaCl precipitation, prime pumps with water to remove 20% ethanol.
✓ Decreased flow rates should be used for improved resolution in gel-filtration
chromatography.
✓ Wash the system with water after the procedure.
Strengths and limitations:

The fast-protein liquid chromatography is a simple and reproducible separation technique with
efficient resolution. The chromatography columns have longer lifetime because of the inert
construction against the very high salt concentrations and corrosive liquids. The FPLC supports a
wide range of columns as the procedures are carried out under low pressure.

The wide flow range makes it a suitable technique for analytical and preparative chromatography.
The technique needs glass columns and cannot withstand high pressures. This method is not
sensitive to thermolabile proteins.

Application of chromatography in medicine:

Chromatography technique is a valuable tool for biochemists, besides it can be applied easily
during studies performed in clinical laboratories For instance; paper chromatography is used to
determine some types of sugar, and amino acids in bodily fluids which are associated with
hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids,
barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and
proteins.

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 CHAPTER: 12
Spectroscopy

The interaction of electromagnetic radiation with matter is a quantum phenomenon and is

dependent on the properties of the radiation and the appropriate structural parts of the material
involved. Interaction of electromagnetic radiation with matter leads to variety of spectrum types
which form the basis of the spectroscopic technique.

Spectroscopy is the study of light as a function of length of the wave that has been emitted,
reflected or shone through a solid, liquid, or gas. ... Spectroscopy separates and measures the
brightness of the different wavelengths. Spectroscopy is the technique of splitting light (or more
precisely electromagnetic radiation) into its constituent wavelengths (a spectrum), in much the
same way as a prism splits light into a rainbow of colours. However, in general, a spectrum is
generally more than a simple ‘rainbow’ of colours. The energy levels of electrons in atoms and
molecules are quantised, and the absorption and emission of electromagnetic radiation only occurs
at specific wavelengths. Consequently, spectra are not smooth but punctuated by ‘lines’ of
absorption or emission.

Old style spectroscopy was carried out using a prism and photographic plates. These days, modern
spectroscopy uses diffraction gratings to disperse the light, which is then projected onto CCDs
(Charge Coupled Devices) similar to those used in digital cameras. The 2-dimensional spectra are
easily extracted from this digital format and manipulated to produce 1-dimensional spectra like the
galaxy spectrum shown below.

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These 1-dimensional spectra contain an astonishing amount of useful data:

The spectrum of an S7 spiral galaxy showing both emission and absorption line features.
Wavelength is measured in angstroms while the flux is in arbitrary units.

Credit: VizieR catalogue III/219, Spectral Library of Galaxies, Clusters and Stars (Santos et al.
2002)

The precise position (wavelength) at which known emission and absorption lines are detected can
be used to measure the redshift of the observed object. For example, if the Hβ (486.2 nm) hydrogen
line is detected at 487.8 nm, we calculate that the object has a recession velocity of 1,000 km/sec.
Apart from measuring distances, this type of analysis can also be used to detect spectroscopic
binaries and extrasolar planets.

The widths of both the absorption and emission lines can be used to measure the internal velocity
dispersion of complex objects (e.g. the average velocity of stars within galaxies). This is achieved
through measurement of spectral line broadening from which the velocity broadening can be
calculated.

Many (indeed most) absorption and emission lines are produced by metals. The depths or heights
of these lines can be used to estimate the abundances of the metals responsible. In stars, these also
permit the measurement of the temperature and pressure of the stellar atmosphere. In objects such
as galaxies, they permit an estimation of age, though the spectra are somewhat susceptible to the
age-metallicity degeneracy making this a difficult procedure.

Finally, the overall shape of the spectrum contains clues to many other aspects of the observed
population including age and interstellar reddening (extinction). Spectroscopic techniques have
been applied in virtually all technical fields of science and technology. Radio-frequency

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spectroscopy of nuclei in a magnetic field has been employed in a medical technique called
magnetic resonance imaging (MRI) to visualize the internal soft tissue of the body with
unprecedented resolution. Microwave spectroscopy was used to discover the so-called three-
degree blackbody radiation, the remnant of the big bang (i.e., the primeval explosion) from which
the universe is thought to have originated (see below Survey of optical spectroscopy: General
principles: Applications). The internal structure of the proton and neutron and the state of the early
universe up to the first thousandth of a second of its existence are being unraveled with
spectroscopic techniques using high-energy particle accelerators. The constituents of distant stars,
intergalactic molecules, and even the primordial abundance of the elements before the formation
of the first stars can be determined by optical, radio, and X-ray spectroscopy. Optical spectroscopy
is used routinely to identify the chemical composition of matter and to determine its physical
structure.

Historical background:

The basis for analytical spectroscopy is the discovery, made in 1859 by the German physicist
Gustav R. Kirchhoff, that each pure substance has its own characteristic spectrum. Another
German physicist, Joseph von Fraunhofer, repeating more carefully an earlier experiment by a
British scientist, William Wollaston, had shown in 1814 that the spectrum of the Sun’s
electromagnetic radiation does not grade smoothly from one colour to the next but has many dark
lines, indicating that light is missing at certain wavelengths because of absorption. These dark
lines, sometimes called Fraunhofer lines, are also collectively referred to as an absorption
spectrum. The spectra of materials that were heated in flames or placed in electric-gas discharges
were studied by many scientists during the 18th and 19th centuries. These spectra were composed
of numerous bright discrete lines, indicating that only certain wavelengths were present in the
emitted light. They are called brightline, or emission, spectra.

Spectroscopic techniques are extremely sensitive. Single atoms and even different isotopes of the
same atom can be detected among 1020 or more atoms of a different species. (Isotopes are all
atoms of an element that have unequal mass but the same atomic number. Isotopes of the same
element are virtually identical chemically.) Trace amounts of pollutants or contaminants are often
detected most effectively by spectroscopic techniques. Certain types of microwave, optical, and
gamma-ray spectroscopy are capable of measuring infinitesimal frequency shifts in narrow
spectroscopic lines. Frequency shifts as small as one part in 1015 of the frequency being measured
can be observed with ultrahigh resolution laser techniques. Because of this sensitivity, the most
accurate physical measurements have been frequency measurements.

Spectroscopy now covers a sizable fraction of the electromagnetic spectrum. The table summarizes
the electromagnetic spectrum over a frequency range of 16 orders of magnitude. Spectroscopic
techniques are not confined to electromagnetic radiation, however. Because the energy E of a
photon (a quantum of light) is related to its frequency ν by the relation E = hν, where h is Planck’s

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constant, spectroscopy is actually the measure of the interaction of photons with matter as a
function of the photon energy. In instances where the probe particle is not a photon, spectroscopy
refers to the measurement of how the particle interacts with the test particle or material as a
function of the energy of the probe particle.

Electromagnetic phenomena approximate wavelength approximate frequency range

range (metres) (hertz)
radio waves 10–1,000 3 × 105–3 × 107
television waves 1–10 3 × 107–3 × 108
microwaves, radar 1 × 10−3–1 3 × 108–3 × 1011
infrared 8 × 10−7–1 × 10−3 3 × 1011–4 × 1014
visible light 4 × 10−7–7 × 10−7 4 × 1014–7 × 1014
ultraviolet 1 × 10−8–4 × 10−7 7 × 1014–3 × 1016
X-rays 5 × 10−12–1 × 10−8 3 × 1016–6 × 1019
gamma rays (γ rays) <5 × 10−12 >6 × 1019

An example of particle spectroscopy is a surface analysis technique known as electron energy loss
spectroscopy (EELS) that measures the energy lost when low-energy electrons (typically 5–10
electron volts) collide with a surface. Occasionally, the colliding electron loses energy by exciting
the surface; by measuring the electron’s energy loss, vibrational excitations associated with the
surface can be measured. On the other end of the energy spectrum, if an electron collides with
another particle at exceedingly high energies, a wealth of subatomic particles is produced. Most of
what is known in particle physics (the study of subatomic particles) has been gained by analyzing
the total particle production or the production of certain particles as a function of the incident
energies of electrons and protons. Atomic absorption spectroscopy

AAS is an instrumental analysis method for determining the concentration of the element in the
sample based on the absorption intensity of the ground state atom of the element. According to
different atomization techniques, AAS can be classified as flame AAS, graphite furnace AAS,
hydride AAS, and cold vapor AAS. For flame AAS, the sample should be liquid and the detection
limits are around ppm range (Wang et al., 2012). Different from the flame AAS, graphite furnace
AAS uses graphite tube, which can stand for 3000 oC atomization, to replace flame and its
detection limit (around ppb range) is higher than flame AAS (Gomez-Nieto et al., 2013). Hydride
AAS is suitable to detect the metalloid elements such as arsenic and lead that are introduced to
instrument in gas phase. This method can reduce the detection limit by 10–100 times (Maragou et
al., 2017). Cold vapor AAS is generally used to detect mercury because this element has enough
vapor pressure at room temperature. However, this technique cannot be used to detect organic
mercury compounds, as they cannot be reduced to the element by sodium tetrahydroborate.
Therefore, digestion is necessary before the detection using this technique. The detection limit of
cold vapor AAS is around ppb range (Adlnasab et al., 2014).

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AAS has the advantages of high sensitivity, strong selectivity, wide analysis range, strong
antiinterference ability, accurate and reliable results, simple and rapid operation, simple
instrument, and automation of the whole operation. Based on these advantages, AAS is
unparalleled in the field of heavy metal analysis and detection of water, and is even listed as an
arbitration method for multimetal analysis of water. However, the instrument of AAS is expensive
and its operating cost is high, which limits its application.

Cadorim et al. (2019) used disposable pipette extraction coupled with high-resolution continuum
source graphite furnace atomic absorption spectrometry (HR-CS GF AAS) to detect Pb and Cd in
the wastewater and the limit of detection was 0.2 ppb for Pb and 0.1 ppb for Cd, respectively.
Khayatian et al. (2018b) used FAAS to detect Cu(II) and Pb(II) in refinery wastewater and the
detection limit was 4 ppb for Cu(II) and 11 ppb for Pb(II). Islam et al. (2014b) developed a novel
solid-phase extractant for the preconcentration of lead in electroplating wastewater and detected
this metal using FAAS.

Principles of Spectroscopy:

Spectroscopy is the study of the interaction of electromagnetic radiation with matter. When matter
is energized (excited) by the application of thermal, electrical, nuclear or radiant energy,
electromagnetic radiation is often emitted as the matter relaxes back to its original (ground) state.
The spectrum of radiation emitted by a substance that has absorbed energy is called an emission
spectrum and the science is appropriately called emission spectroscopy. Another approach often
used to study the interaction of electromagnetic radiation with matter is one whereby a continuous
range of radiation (e.g., white light) is allowed to fall on a substance; then the frequencies absorbed
by the substance are examined. The resulting spectrum from the substance contains the original
range of radiation with dark spaces that correspond to missing, or absorbed frequencies. This type
of spectrum is called an absorption spectrum. In spectroscopy the emitted or absorbed radiation is
usually analyzed, i.e., separated into the various frequency components and the intensity is
measured by means of an instrument called spectrometer.

The resultant spectrum is mainly a graph of intensity of emitted or absorbed radiation versus
wavelength or frequency. There are in general three types of spectra: continuous, line, and band.
The sun and heated solids produce continuous spectra in which the emitted radiation contains all
frequencies within a region of the electromagnetic spectrum. A rainbow and light from a light bulb
are examples of continuous spectra. Line spectra are produced by excited atoms in the gas phase
and contain only certain frequencies, all other frequencies being absent. Each chemical element of
the periodic chart has a unique and, therefore, characteristic line spectrum. Band spectra are
produced by excited molecules emitting radiation in groups of closely spaced lines that merge to
form bands.

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These categories of emission and absorption spectra contain tremendous amounts of useful
information about the structure and composition of matter. Spectroscopy is a powerful and
sensitive form of chemical analysis, as well as a method of probing electronic and nuclear
structure and chemical bonding. The key to interpreting this spectral information is the
knowledge that certain atomic and molecular processes involve only certain energy ranges. Fig.
12.1 shows the regions of the electromagnetic spectrum and the associated energy transitions that
occur in atomic and molecular processes. Much of the scientific knowledge of the structure of
the universe, from stars to atoms, is derived from interpretations of the interaction of radiation
with matter. One example of the power of these techniques is the determination of the
composition, the velocities, and the evolutionary dynamics of stars.

The source of the incredible amount of energy produced by the sun is nuclear fusion reactions
going on within the hot interior (temperature 40 × 106 K). Two fusion cycles, the carbon cycle
and the proton cycle, convert hydrogen nuclei into helium nuclei via heavier nuclei, such as
carbon 12 and nitrogen 14. The enormous radiation of energy from the hot core seethes outwards
by convection.

This radiation consists of the entire electromagnetic spectrum as a continuous spectrum. Towards
the surface of the sun (the photosphere), the different elements all absorb at their characteristic
frequencies. The radiation that shoots into space toward the earth is a continuous emission
spectrum with about 22,000 dark absorption lines present in it (Fraunhofer lines), of which about
70% have been identified. These absorption lines, i.e., missing frequencies, prove that more than
60 terrestrial elements are certainly present in the sun.

Classification of Spectroscopic Methods:

Different spectroscopic techniques have been classified mainly on two parameters, first what type
of radiation is to be measured or by what measurement procedure is employed.

1. Nature of Radiation Measured:

This category of spectroscopy depends on the physical quantity measured. Normally, the quantity
that is measured is an amount or intensity of something.

i. Electromagnetic spectroscopy involves interactions with electromagnetic radiation, or light.

Ultraviolet-visible spectroscopy is an example.

ii. Electronic spectroscopy involves interactions with electron beams. Auger spectroscopy involves
inducing the Auger effect with an electron beam.

iii. Mechanical spectroscopy involves interactions with macroscopic vibrations, such as phonons.
An example is acoustic spectroscopy, involving sound waves.

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iv. Mass spectroscopy involves the interaction of charged species with a magnetic field, giving
rise to a mass spectrum. The term “mass spectroscopy” is deprecated in favour of mass
spectrometry, tor the technique is primarily a form of measurement, though it doe- produce a
spectrum for observation.

2. Measurement Process

Most spectroscopic methods are differentiated as either atomic or molecular based on whether or
not they apply to atoms or molecules. Along with that distinction, they can be classified on the
nature of their interaction:

i. Absorption spectroscopy uses the range of the electromagnetic spectra in which a sub stance
absorbs. This includes atomic absorption spectroscopy and various molecular techniques, such as
infrared spectroscopy in that region and nuclear magnetic resonance (NMR) spectroscopy in the
radio region.

ii. Emission spectroscopy uses the range of electromagnetic spectra in which a substance radiates
(emits). The substance first must absorb energy. This energy can be from a variety of sources,

139
which determines the name of the subsequent emission, like luminescence. Molecular
luminescence techniques include spectrofluorimetry.

iii. Scattering spectroscopy measures the amount of light that a substance scatters at certain
wavelengths, incident angles, and polarisation angles. The scattering process is much faster than
the absorption/emission process. One of the most useful applications of light scattering
spectroscopy is Raman spectroscopy.

Infrared (IR) Spectrophotometry:

IR-light was used in this spectrophotometric analysis, Infra-red spectrophotometry with Gas-
Liquid Chromatography or gas analysis techniques often used as a powerful technique for drug
metabolism study and also provides a convenient and sensitive means of detecting and measuring
differences in the concentration of gases such as carbon monoxide, carbon dioxide and acetylene
in biological samples. The light sources in different spectrophotometric techniques are:

Circular Dichromism (CD) Spectroscopy:

Information on the three-dimensional structure (con-formation) of macromolecules in solution

can be obtained by studying their absorption of polarized light, using Circu-lar dichromism (CD)
spectroscopy. CD spectroscopy mea-sures this differential absorption of right (R) and left (L)
cir-cularly polarized light as a function of wavelength. The main components of a CD
spectrometer are illus-trated in Fig. 1.2. Usually L and R circularly polarized radia-tion is
produced from a single monochromator by passing plane-polarized light through an electro-optic
modulator.

140
This is a crystal subject to alternating currents that transits either the R or L component of light
dependent on the polar-ity of the electric field to which it is exposed.

The photomultiplier detector produced a voltage in proportion to the ellipticity of polarization of

the combined beam falling on the photomultiplier. CD-spectroscopy is often done to get
information about the protein conformation (L, B and random coil) and also to study the
conformation of nucleic acids.

Spectrofluorimetry:

Spectrofluorimetry is a specialized spectroscopic method where two monochromators may be

used, one selecting the activating wavelength and the other the phorescent wavelength. No
reference cuvette is required, but a calibration curve must be constructed. This method is most
accurate at very low concentra-tions when absorption spectrophotometry is least accurate. The
sensitivity of the instrument is usu-ally easily adjusted over a large range by am-plification of the
current produced in the pho-tocell circuit. The basic components of a complete spectra-fluorimeter
are mentioned in Fig. 1.3. This includes a continuous spectrum source (mercury lamp or Xenon
arc) a monochromator (M) for irradiating the specimen with any chosen wavelength, a second
monochromator (M2) which, under conditions of constant irradiating wavelengths, enables the
determi-nation of I .v fluorescent spectrum of the speci-men; and a detector which is usually a
sensi-tive photocell.

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This technique is often used for the quan-titative study of Vitamin B,, NADH Cork set and other.
Group-specific hydrolases may be readily assayed by measuring the rate of appearance of
fluorescence of 450 nm of the amino of 4-methylumbellifore one when the enzyme acts upon an
ether of ester derivative of 4-methyl umbelliferone.

Luminometry:

This is a photometric technique in which light — enlisted as a result of a chemical reaction

(lumines-cence) in contrast to the result of a physical reaction (fluorescence or atomic emission)
— is measured in a luminometer.

Luminometers are relatively simple photometers, complicated only slightly by the need to amplify
and record the signal from the photocell. It has a photomultiplier tube with a well-stabilized high
voltage power supply to ensure sensitive, reproducible measurement of light emission, a direct
current amplifier with a wide range of sensitivity and linear response and a reaction chamber which
allows temperature control, adequate mixing of reactants and protection from extraneous light
(Fig. 1.4).

Atomic/Flame Spectrophotometry:

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Volatilisation of atoms — either in a flame or electro thermally — causes them to emit and absorb
light of specific wavelengths. Atomic/flame spectrophotometry takes advantage of the specificity
of line spectra to determine the amounts of a specific elements present. Emission flame
spectrophotometry measures the emission of light of a specific wavelength by atoms in a flame.

Atomic absorption spectro-photometry measures the absorption of a beam of monochromatic light

by atoms in a flame or alterna-tively by atoms heated electro thermally in a graphite furnace. The
energy absorbed is proportional to the number of atoms present in the official path. The amount of
radiation emitted is proportional to the number of excited atoms present, which depends on the
temperature and compositions of the flame. Standard additions must always be used to calibrate
the system.

The basic components of an atomic (flame) emission spectrophotometer are: nebulizer, burner,
monochromator, detector and read-out unit. Nebulizers are usually of the scent spray type, in which
a forced stream of air passes over a capillary tube dipping into the test solution. Large and small
drops of sample solution then passes along with air-stream into the burner. Then light thus emitted
is passed through the monochromator or filter. Finally, emitted light was detected and read out.

In order to produce a beam of radiation with a very narrow band, either a source of white light plus
a double monochromator, or a hollow cathode discharge lamp is used. The discharge lamps are
specific to the element being assayed.

These techniques are often used to detect the metallic elements present in the inorganic and organic
compounds — more than 20 elements in biological samples, particularly Ca8, Mg++, Mn++,
Zn++, Ca++, Pb++, Cr++, Ni++ etc. When assaying metal in biological samples it is usual to
degrade organic molecules by ashing.

The detection limits for various elements in emission and absorption flame spectrophotometry are
depicted below:

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Electron Spin Resonance (ESR) Spectrometry:

This is a technique used for detection of Paramagnetism, i.e. the magnetic movement associated
with an unpaired Electron Paramagnetic Resonance (EPR). The technique may be used for
detecting transition metal ions and their complexes, free radicals and excited states.

The basic components of an ESR spectrometer are Klystron source, Metal waveguide tube, Field
magnet, Sweep coils, Detector, Amplifier, Recorder and Oscillator. Electromagnets generating
fields of 50 to 100 millitesla with a uniformity of 1 in 106 are required for accurate work.

Most experiments are conducted at around 330 millitesla, in conjunction with an auxiliary sweep
of 10 to 100 millitesla. Klystron oscillator produces the monochromatic miss-wave radiation
usu-ally with a wavelength of about 3 × 102 m (9,000 MH2). Samples must be in the solid state,
so biological samples are usually frozen in liquid.

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Nuclear Magnetic Resonance (NMR) Spectrometry:

NMR-technique is used for detecting atoms which have nuclei that possess a magnetic movement.
These are usually atoms containing an odd number of protons in their nuclei. In the same way that
pairs of electrons in the same atomic orbital have opposite spin and no resultant magnetic
movement, pairs of protons in a nuclei do not have a magnetic moment.

However, an odd portion in a nudens imports a magnetic moment to the molecule which can
interest with an applied magnetic field. This interaction is major concern of nuclear magnetic
resonance Spectrometry. In a magnetic field of several hundred millitesla (several thousand gauss)
such nuclei absorb radia-tion in the resonance spectrum, giving rise to the phenomenon known as
nuclear magnetic resonance (NMR). Most studies are conducted using Hydrogen (H).

The basic components of an NMR Spectrometer are similar to those illustrated for an ESR-
Spectro- meter. A radio frequency transmitter in place of a Klystron source used to irradiate the
sample. For DMR the sample must be dissolved to a relatively high concentration in a solvent
which lacks protons, such as D20 or CDCl2. To minimize variations in the magnetic field, the
sample is contained in a tube of high precision diameter and is usually rotated at high speed by an
air turbine. The absorption signal, detected by a radio receiver, is amplified and recorded.

The major use of NMR is for the study of the molecular structure of relatively simple organic
molecules. This structural information relevant to the biological action of the antibiotics has also
been obtained from NMR studies. NMR has also been particularly useful in the study of phosphate
compounds viz., AMP, ADP, ATP, and phosphocreatine.

Mass Spectrometry:

J.J. Thomson for the first time in year 1900, introduced mass spectrometer which employed fixed
magnetic and electric field. It is based upon the principle that moving ion may be deflected by a
magnetic field to an extent that is dependent upon its mass and velocity. Thus ions of a larger
momentum are deflected less than ones of lower momentum, whilst a mixture of ions of different
mass but constant velocity will be deflected in proportion to their mass. So, in a Mass
Spectrometer, molecules of a compound are 145onized, either by ejection of an electron or capture
of a proton, to give the parent molecular ion the energy which is such that some fragmentation
occurs to give a series of fragment ions. Knowledge of the mass of the molecular ion and its major
fragment ions is frequently sufficient to enable the structure of the parent compound to be uniquely
deduced. This method is very sensitive and often used for as little as 10-6 to 10-9 g of material.
Mass spectra show a series of peaks or lines corresponding to the m/c values of the positive ions
produced from the compound. The height of the peaks corresponds to the relative abundance of
the ions. A reference ion of similar m/c value to that of the parent ion is used to calibrate the mass
axis (abscissa) of the intensity spectrum.

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The parent ion is the peak with the greatest mass, although it is not necessarily the most abundant
(base peak). Ion intensities in a mass spectrum are usually recorded as percentage of the intensity
of the base peak. The basic components of a Mass Spectrometer are Ionization chamber vacuum
pump unit, Electrostatic field, Ion trajectory, Magnetic field, Detector, Amplifier and Recorder.

During 1999-2000, mass spectrometry became an important tool for proteomics and genomics
leading to Nobel Prize (2002) conferred to J. B. Fenn and K. Tanaka. The earliest biochemical
uses of mass spectrometry were in the study of metabolic pathways — particularly the
determination of chemical structure and, hence, the identification of compounds. This technique
has also been used to determine the amino acid sequence of oligopeptides derived from protein
hydrolysate and other sources.

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The Mass spectroscopy requires that molecules examined should be in gaseous ionic form. There
are several methods for ionization of biomolecules but here ESI- MS and MALDI-TOF MS,
SELDI-TOF MS will be discussed.

Electrospray Ionization Mass Spectrometry (ESI-MS):

. The Electrospray Ionisation Process

ESI uses electrical energy to assist the transfer of ions from solution into the gaseous phase before
they are subjected to mass spectrometric analysis. Ionic species in solution can thus be analysed
by ESI-MS with increased sensitivity. Neutral compounds can also be converted to ionic form in
solution or in gaseous phase by protonation or cationisation (e.g. metal cationisation), and hence
can be studied by ESI-MS.

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The transfer of ionic species from solution into the gas phase by ESI involves three steps: (1)
dispersal of a fine spray of charge droplets, followed by (2) solvent evaporation and (3) ion ejection
from the highly charged droplets (Figure 1). tube, which is maintained at a high voltage (e.g. 2.5
– 6.0 kV) relative to the wall of the surrounding chamber. A mist of highly charged droplets with
the same polarity as the capillary voltage is generated. The application of a nebulising gas (e.g.
nitrogen), which shears around the eluted sample solution, enhances a higher sample flow rate.
The charged droplets, generated at the exit of the electrospray tip, pass down a pressure gradient
and potential gradient toward the analyser region of the mass spectrometer. With the aid of an
elevated ESI-source temperature and/or another stream of nitrogen drying gas, the charged droplets
are continuously reduced in size by evaporation of the solvent, leading to an increase of surface
charge density and a decrease of the droplet radius. Finally, the electric field strength within the
charged droplet reaches a critical point at which it is kinetically and energetically possible for ions
at the surface of the droplets to be ejected into the gaseous phase. The emitted ions are sampled by
a sampling skimmer cone and are then accelerated into the mass analyser for subsequent analysis
of molecular mass and measurement of ion intensity.

To obtain structural information, the precursor ions of interest can be mass selected and further
fragmented in a collision cell. The fragment ions can then be mass analysed by a second mass
analyser of a tandem mass spectrometer system to be described below.

2. Mass Analysers

I. The quadrupole mass analyser

When ions travel through a magnetic or electrical field, their movement is affected by their m/z
ratio and this is the main principle of separating ions in MS. In the clinical laboratory, a quadrupole
mass analyser is most commonly used. In such system, an assembly of 4 parallel metal rods is kept
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at equal distance (Figure 2). Each pair of opposite rods is connected electrically. An equal but
opposite DC voltage superimposed with a radio frequency (RF) AC voltage is applied to the
diagonally placed pair of rods. The resulting electrical field causes the ions to travel forward in the
z direction with oscillatory motion in the x-y plane. The amplitude of oscillation bears a unique
relationship with the m/z ratio and can be controlled by changing the DC and RF voltages
simultaneously in a pre-fixed ratio. These DC and RF voltages can be set so that amplitudes of
oscillation for desirable m/z ratios are “stable” with the ions travelling along the z-axis without
hitting the quadrupole rods, and finally reaching the detector. On the other hand, the oscillatory
amplitudes of undesirable ions are large and “unstable”; they hit the metal rods, get neutralised,
and fail to reach the detector. Quadrupole mass analysers are robust, economical, physically small,
and more readily interfaced with a wide variety of inlet systems when compared with other
conventional mass analysers like the magnetic sector.

II. Tandem quadrupole system

In a typical tandem quadrupole system there are three quadrupoles set up in a linear fashion, often
called “triple-quad” (Figure 3). The analyte ion of interest (usually called the precursor ion) is
mass-selected by the first quadrupole (Q1) and allowed to collide with a collision gas (usually
argon) in a second RF-only quadrupole collision cell (Q2), where the precursor ions are activated
by collision and undergo further fragmentation. This process is known as collision-induced
dissociation (CID). The daughter ions resulting from CID are related to the molecular structure of
the ions and can be monitored by a third quadrupole mass analyser (Q3) providing structural
information of the molecular ions. This tandem system is commonly denoted as MS/MS in the
literature. When Q1 is set to select only one specific m/z ratio, it filters out other molecular ions
having different m/z ratios. This is a “purification” step inside the MS system, eliminating
complicated and time-consuming sample purification procedures prior to MS analysis.

The following modes of data acquisition are commonly used in a tandem quadrupole system:

Product scan (daughter scan): Q1 is static allowing only one ion of specific m/z ratio to pass
through and Q3 scans the different CID product ions. This mode can be used for studying
molecular structure; for example, amino acids sequencing of a peptide molecule.

Precursor scan (parent scan): Q1 scans over a range of possible precursor ions and Q3 is static
focusing on one unique product ion resulting from CID of a class of precursor ions. For example,
m/z ratio of 85 is a common fragment ion from all the butylated acylcarnitines precursor ions (C2–
C18).2

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Neutral loss: Both Q1 and Q3 scan together at a constant difference in m/z ratio. This is used to
monitor the loss of a neutral fragment for a class of molecules from CID. For example, there is a
neutral loss of 102 from most of the butylated amino acids.2

Multiple reaction monitoring: Both Q1 and Q3 are static for a pre-determined pair of precursor
and product ions. This confers the highest specificity and sensitivity and is commonly used in ESI-
MS/MS quantification procedures.

III. The ion trap mass analyser

This system, which has become recently available, has the advantages of being smaller in size and
cheaper, and being capable of performing tandem CID monitoring. It consists of three hyperbolic
electrodes: the ring electrode, the entrance end cap electrode, and the exit end cap electrode (Figure
4). These electrodes form a cavity in which it is possible to trap (store) and analyse ions. Both end
cap electrodes have a small hole in their centres through which the ions can travel. The ring
electrode is located halfway between the two end cap electrodes. Ions produced from the source
enter the trap through the quadrupole and the entrance end cap electrode. Various voltages are
applied to the electrodes to trap and eject ions according to their m/z ratios. The ring electrode RF
potential,

an AC potential of constant frequency and variable amplitude, is applied to the ring electrode to
produce a 3-dimensional quadrupolar potential field within the trapping cavity. This will trap ions
in a stable oscillating trajectory confined within the trapping cell. The nature of the trajectory is
dependent on the trapping potential and the m/z ratio of the ions. During detection, the electrode
system potential is altered to produce instabilities in the ion trajectories and thus eject the ions in
the axial direction. The ions are ejected in order of increasing m/z ratio, focused by the exit lens
and detected by the ion detector system.

During tandem MS, the precursor ion is selected inside the trap where an inert gas is introduced
for CID. After that, the product ions are ejected for detection. Alternatively, the product ions can
be kept inside the trap and another CID reaction can be initiated and this repeat CID reaction can
continue for several times (denoted as MSn in which n is the number of CID reactions). This can
help differentiate molecules with similar structures. However, ion trap analysers cannot provide
precursor scan and neutral loss modes of data acquisition. For quantification, ion trap analysers
are 10 times less sensitive when compared with the tandem quadrupole system operated in multiple
reactions monitoring mode.

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3. The Mass Spectrum

The mass spectrum is a graphical display of the relative a bundance of ion signals against the m/z
ratios. It is a common practice that the highest signal is taken as 100% abundance and all the other
signals are expressed as a percentage of this. Fragmentation of the protonated or de-protonated
molecular ions generated from ESI is generally limited, and the mass spectra are relatively simple.
However, multiple-protonation of proteins and peptides occurs in the ESI process, and the ESI
mass spectra might become more complicated. Figure 5 is the mass spectrum for a highly purified
horse myoglobin preparation (5 μmol/L in 50% acetonitrile containing 0.2% formic acid). A single
protein will show its characteristic cluster ions containing multiple-charged ions. The number of
charges on the protein molecules will depend on the molecular weight of the protein and the
number of accessible basic sites (e.g. arginine, histidine, and lysine). Three basic assumptions are
made in the determination of the molecular weight of a single protein. Namely, that (1) adjacent
peaks in a series only differ by one charge; (2) charging is only due to proton attachment to the
molecular ions; and (3) ionisation occurs to only the intact molecule. To calculate the molecular
weight of the protein, one can use 2 simultaneous equations derived from 2 adjacent ions. As an
example, for the ion with a m/z ratio = 1212, it has an adjacent peak with m/z ratio = 1131. The
adjacent peak must have an additional charge on the molecular ions. Therefore, 2 simultaneous
equations can be set up: m/z = 1212, and m/(z+1) = 1131, solving to z = 14 and m = 16968 for the
molecule.

The situation becomes more complicated if there are mixtures of different proteins present in a
sample. Different MS manufacturers provide their own software for transforming complicated raw
mass spectrum into the individual relative molecular masses. Figure 6 (top) illustrates a mass
spectrum of a mixture of purified alpha-foetal protein glycoforms. The Micromass MaxEnt
programme (Micrormass, Manchester, UK) deconvolutes the mass spectrum into the individual
glycoforms (Figure 6 - bottom). In this way, an ESI-MS system designed for measuring the m/z
ratios of 4000 Da/e is capable of determining the molecular weight of large protein molecules over
100,000 Da. ESI-MS can also be applied to the study of other macromolecules, for example,
oligonucleotides. The accuracy of ESI-MS for protein molecules has been documented to be ±
0.01% when compared with the theoretical amino acids sequence.3

4. Quantitative Analysis

In ESI-MS, the ion signal is proportional to analyte concentration and largely independent of flow
rate and injection volume used for sample introduction.1 The signal is linear from the limit of
detection (usually pmol/L) to around 10 μmol/L of analyte concentration. For quantitative
measurement, it is important to incorporate an internal standard in the procedure to compensate
for losses during sample preparation and variable detection sensitivity of the MS system. The
internal standard should have a structure similar to that of the analyte and the ideal practice is to
synthesise an internal standard by incorporating stable isotopes on the molecules of interest. For
example, for the quantification of free carnitine (m = 162), an internal standard containing 3

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deuterium atoms to replace 3 hydrogen atoms was used (m = 165).4 When an ideal internal
standard is not available, molecules with similar structure can also be used. For example,
ascomycin has been used as an internal standard for ESI-MS/MS analysis of the
immunosuppressant tacrolimus.5

Another critical issue in quantitative ESI-MS is suppression of ionisation due to matrix

interference. A biological sample would give significantly lower ionisation signals compared to
pure standard solutions with similar analyte concentrations. This phenomenon is the result of high
concentrations of non-volatile materials from the biological sample being present in the spray with
the analyte.6 Possible non-volatile interfering solutes are salts and lipids in the biological samples.
To overcome the matrix interference, extensive sample purification processes are required; for
example, liquid-liquid extraction and solid phase extraction by disposable columns. However,
these elaborate procedures are time-consuming and can cause poor recovery. A recent
development is to use short LC columns (or guard columns) and apply a fast HPLC purification
(e.g. for 2–5 minutes) prior to MS analysis.7 The HPLC serves to separate the non-volatile
compounds from the analyte. For HPLC systems with column-switching capability, the analyte in
the biological sample can be purified and concentrated on separate columns before MS analysis.
Such an automated sample purification system utilising affinity chromatography is best illustrated
by a recently published rapid quantification method for transferrin isoforms in serum.8

Clinical Applications

1. Screening for Inborn Errors of Metabolism

One of the most common clinical applications of ESI-MS is in screening for inborn errors of
metabolism (IEM). Patients with IEM inherit defective metabolic pathways leading to either
accumulation of toxic metabolites, or deficiency in metabolites vital for homeostasis. Many of
these defects have serious clinical consequences, including mild to severe mental retardation,
physical handicap, and even fatality. Early diagnosis and management of some of these disorders
has proven very effective in preventing clinical consequences. Among them only a small number
have been successfully screened on nation-wide neonatal screening programmes; for example,
phenylketonuria (PKU) and neonatal hypothyroidism. The problems with the screening methods
include laborious sample preparation, long analytical time, high false-positive rates, and the
narrow disease spectrum covered by each method. The principle of the ESI-MS makes this method
most suitable for measuring the same class of bio-molecules sharing similar molecular structures,
and the MS/MS capability obviates complicated and tedious sample preparation procedures. Since
the early 1990s, the use of ESI-MS/MS has revolutionised neonatal screening for IEM in many
developed countries.9

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I. Neonatal screening for disorders of amino acid and fatty acid metabolism

Currently, nation-wide neonatal screening programmes in North America, Europe and Australia
are using automated ESI-MS/MS for metabolic profiling of amino acids and acylcarnitines from
blood spots. State-of-the-art laboratories can screen several hundred samples per day. Sampling of
blood spots on the Guthrie card, extraction of analytes, formation of derivatives, ESI-MS/MS
analysis, and electronic data processing with computer-alert of abnormal results can all be fully
automated now using micro-plate batch processing.9

Amino acid profiling can detect major aminoacidopathies, such as phenylketonuria (PKU), maple
syrup urine disease, homocystinuria, and galactosaemia. In the past, PKU screening required the
use of blood samples collected on or after Day 5 of birth, so that sufficient phenylalanine had
accumulated for detection by the insensitive Guthrie qualitative bacterial inhibition test. Utilising
ESI-MS/MS measurement of the phenylalanine to tyrosine concentration ratio, screening can now
be performed with high specificity and sensitivity using Day 1 blood spots.10 Acylcarnitines
screening is mainly for fatty acid oxidation defects. Examples of IEM being screened are: medium-
chain and very-long-chain acyl-CoA dehydrogenase deficiencies; glutaric acidemia types I and II;
carnitine palmitoyl transferase type II / translocase deficiencies; primary carnitine deficiency;
isovaleric acidemia / 2-methylbutyryl-CoA dehydrogenase deficiency; and long-chain
hydroxyacyl-CoA dehydrogenase/trifunctional protein deficiencies. Recently, these cost-effective
screening methods have been used to study potential IEM in sudden infant death.11

II. Neonatal screening for galactosaemia

Galactosaemia (incidence, 1: 35,000 in Denmark)12 is a life-threatening IEM with severe

symptoms in the neonatal period. It is most commonly caused by deficiency of the enzyme
galactose-1-phosphate uridyl transferase. In this disorder, galactose derived from ingested lactose
in milk and milk products accumulates in the blood and urine, leading to high intracellular
concentrations of galactose-1-phosphate that is toxic to several tissues, especially the liver, brain,
and renal tubules. Clinical manifestations, including vomiting, diarrhoea, jaundice and lethargy
appear in neonates shortly after milk ingestion, progressing to coma and death from septicaemia,
hepatic or renal failure. Early treatment with a galactose-free diet causes regression of signs and
symptoms within 1–2 weeks. A novel ESI-MS/MS method has been developed for the
measurement of total hexose mono-phosphates on blood spots as a marker for galactose-1-
phosphate.12 The procedure is simple and does not require sample processing for derivative
formation. A retrospective study has demonstrated a diagnostic sensitivity and specificity of 100%
for 12 patients amongst 2055 controls.12

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III. Neonatal screening for cholestatic hepatobiliary diseases

In prolonged neonatal jaundice, it is important to diagnose the presence of cholestatic hepatobiliary

disease such as extrahepatic biliary atresia; a condition that can result in serious morbidity and
mortality. A simple and efficient ESI-MS method has been developed for measuring conjugated
bile acids on blood spots,13 and used in the attempt to set up a neonatal screening programme for
cholestatic hepatobiliary diseases.14 However, the segregation of conjugated bile acid
concentrations between patients and the general population was insufficient to make mass
screening for cholestatic hepatobiliary diseases a feasible option with this method alone. Low birth
weight infants without the pathological conditions could also have significantly high bile acid
concentrations, thereby affecting the specificity of the method. Furthermore, this screening method
is also affected by the significant increases seen in bile acid concentration after feeding. Therefore,
accurate sampling time might be required to improve its usefulness.

IV. Screening for peroxisomal disorders

The peroxisomes are intracellular organelles responsible for the β-oxidation of very long chain and
branched chain fatty acids. Peroxisomal disorders are a heterogeneous group of IEM characterised
by impaired, reduced or totally absent peroxisomes. Examples of such include the Zellweger
syndrome, X-linked adrenoleukodystrophy, acyl-CoA oxidase deficiency, bifunctional protein
deficiency and peroxisomal thiolase deficiency.15 Patients suffering from these disorders may
have abnormal increases in very long chain fatty acids (VLCFA), pristanic or phytanic acids in
serum. Tedious GC/MS methods have been used for the diagnosis of these conditions. A rapid
ESI-MS/MS method was reported for screening peroxisomal disorders.16 VLCFA (C20:0
eicosanoic, C22:0 docosanoic, C24:0 tetracosanoic and C26:0 hexacosanoic acids) in blood spots
or serum need to be converted to dimethylaminoethyl esters. The sample purification, and
derivative formation procedures are relatively simple compared with traditional GC/MS methods.
Since the final step in bile acid biosynthesis takes place in peroxisomes, another HPLC/ESI-
MS/MS method for plasma C27 and C29 conjugated bile acids has also been proposed, to screen
for peroxisomal disorders.17 This method does not require sample processing for derivative
formation, and is similar to the method described above.14

V. Screening for disorders in purine and pyrimidine metabolism

Inborn errors of purine and pyrimidine metabolism have a wide variety of clinical presentations;
for example, gout, anaemia, immunodeficiency, kidney stones, convulsions, mental retardation,
autism and growth retardation.18 Diagnosis is based on the presence of abnormal metabolites or
the absence of normal metabolites in serum, urine or red blood cells. An HPLC/ESI-MS/MS

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screening method has been developed for the quantification of 17 purines and pyrimidines in urine
or urine-soaked filter paper strips with a turn-around-time of 15 minutes per sample.19

2. Identification and Quantification of Haemoglobin Variants

Since the first report on the successful measurement of large bio-molecules by ESI-MS, there has
been a revolution in the identification of protein molecules in biochemical research. In the clinical
laboratory, quantification of glycohaemoglobin (GHb) provides a useful tool for the monitoring of
glycaemic control in diabetic patients. ESI-MS has been the recognised technology for
development of the reference method. In addition, haemoglobin (Hb) variants need to be identified
for the investigation of haemolytic anaemia, methaemoglobinaemia, sickle cell disease and
thalassaemia. Occasionally, these variants are detected incidentally because they interfere with the
measurement of GHb. ESI-MS has also become the preferred technique for a rapid systematic
approach to definitive characterisation of Hb variants.20

I. Quantification of glycohaemoglobin

GHb is the product of irreversible non-enzymatic glycation of Hb. The percentage GHb represents
an integration of the patient’s average plasma glucose concentration over the life span of his/her
erythrocytes (2–3 months). It has been used for monitoring diabetic control for many years. A
recent review reiterated that improved glycaemic control in diabetic patients is strongly associated
with decreased development and/or progression of complications.21 HbA1c is the predominant
species of GHb and is measured in clinical laboratories using either HPLC with ion-exchange or
affinity column, or automated immunoassays. Most of these methods have acceptable analytical
precision and efficiency. However, they suffer from interference by Hb variants, giving falsely
high or low results. Considerable confusion on the accuracy of the methods has been reported due
to the lack of international standardisation. The fundamental issue has been the lack of
understanding of the specific nature of HbA1c. The use of ESI-MS eliminates this problem.

The IFCC working group on international standardisation defines HbA1c as Hb that is irreversibly
glycated at one or both N-terminal valines of the β globin chains.22 Based on this definition, a
candidate reference method was developed using HPLC/ESI-MS.23 It requires an overnight
endoproteinase Glu-C digestion, followed by HPLC separation of the peptides. The ESI signal
intensities of the specific β N-terminal hexa-peptides of Hb0 and HbA1c are used for calculating
the percentage HbA1c in the sample. This method was later adopted as the IFCC reference
method.24 Preliminary reference values reported by IFCC, 3.36 ± 0.48% (mean ± 2 SD), were
significantly lower than those developed by other methods.

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Subsequently, this reference method was modified for adaptation in clinical laboratories. The
separation of peptides by reverse-phase HPLC method took 24 minutes. It was improved by using
a different gradient mobile phase and analysis time was reduced to 8 minutes.25 To improve on
the specificity of the measurement, multiple reactions monitoring signal acquisition was used.
Precision of the method was also improved using signal intensities from both the singly- and
doubly-charged peptide ions.26 To reduce the running cost of the method, an immobilised
endoproteinase Glu-C preparation was also introduced. It has been shown that this reference
method can overcome interferences by Hb variants.27 Despite the various improvements to the
IFCC reference method, it is still too demanding for routine application.

An alternative ESI-MS method for GHb had been developed.28 In this method a whole blood
sample is diluted 500-fold before it is analysed by ESI-MS for intact Hb globin chains. Figure 7
(top) shows a typical ESI mass spectrum of normal Hb. The cluster ions have been deconvoluted
into true molecular weights by computer softwares (Figure 7 - bottom). Normal α and β globin
chains have molecular weights of 15126.4 and 15867.2 respectively. A mass increase of 162 Da
is assigned due to the addition of glucose (mass, 180 Da) and elimination of water (−18 Da). The
advantages of this ESI-MS method are that it is simple and fast, with an analysis time of 3 minutes
per sample, and can be easily automated. Moreover, it is not affected by Hb variants as variant
globin chains and their glycated species can also be measured easily. This proposed method has
also been tested for long-term performance.29 Over a 4-month period of routine operation, the
between-assay imprecision was 1.6–5.0%, with results that were correlated well with the HPLC
ion-exchange method, and the β-glycated Hb percentage agreed closely with established reference
values.29

II. Identification of haemoglobin variants

Electrophoresis and automated HPLC are currently the major methods used in clinical laboratories
for the identification of Hb variants. However, these methods do not provide definitive
characterisation of the variants, especially those with amino acid substitutions not resulting in any
changes to the overall charges on the Hb molecule. In the past, definitive characterisation of Hb
variants involved tedious and time-consuming analytical procedures requiring days and even
months for completion. Recently, a strategy for rapid definitive characterisation of Hb variants
was reported using ESI-MS technique.20 It identified 95% of the Hb variants in over 250 samples
with a turn-around-time of not more than 2 days for each sample. The procedure comprises the
following steps:

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- Molecular weight profiling of intact α and β globin chains by direct ESI-MS on a 500-fold
dilution of the whole blood sample. The cluster ion spectrum is then deconvoluted to a true
molecular weight scale using computer software that is usually supplied with the MS analyser
system. This step can detect Hb variants with molecular weight difference of more than 6 Da when
compared with the wild type globin chains.

- Overnight trypsin digestion for investigation of the amino acid substitution on the Hb variants.
ESI-MS on the tryptic digest can identify the specific peptide harbouring the substituted amino
acid.

- ESI-MS/MS of the target peptide can provide the amino acid sequence of the peptide and thus
the position of the substituted amino acid.

Summary

The large number of ESI-MS methods that have been presented in recent scientific literature and
conferences speak for their increasing applications in the clinical laboratory. They represent the
state-of-the-art technology for precise, accurate and efficient qualitative and quantitative analyses
of normal and pathological metabolites. Although the initial capital investment for an ESI-MS
equipment is substantial compared to our other routine clinical laboratory analysers, its operational
costs are low. This technology is expected to exert an important influence in the future
development and organisation of the clinical laboratory service.

Matrix Assisted Layer Desorption-ionization (MALDI-TOF MS):

MALDI – Principle and Methodology

The sample for analysis by MALDI MS is prepared by mixing or coating with solution of an
energy-absorbent, organic compound called matrix. When the matrix crystallizes on drying, the
sample entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized
in an automated mode with a laser beam. Desorption and ionization with the laser beam generates
singly protonated ions from analytes in the sample. The protonated ions are then accelerated at a
fixed potential, where these separate from each other on the basis of their mass-to-charge ratio
(m/z). The charged analytes are then detected and measured using different types of mass analyzers
like quadrupole mass analyzers, ion trap analyzers, time of flight (TOF) analyzers etc. For

157
microbiological applications mainly TOF mass analyzers are used. During MALDI-TOF analysis,
the m/z ratio of an ion is measured by determining the time required for it to travel the length of
the flight tube. A few TOF analyzers incorporate an ion mirror at the rear end of the flight tube,
which serves to reflect back ions through the flight tube to a detector. Thus, the ion mirror not only
increases the length of the flight tube, it also corrects small differences in energy among ions
(Yates, 1998). Based on the TOF information, a characteristic spectrum called peptide mass
fingerprint (PMF) is generated for analytes in the sample (Figure Figure11).

Identification of microbes by MALDI-TOF MS is done by either comparing the PMF of unknown

organism with the PMFs contained in the database, or by matching the masses of biomarkers of
unknown organism with the proteome database. In PMF matching, the MS spectrum of unknown
microbial isolates is compared with the MS spectra of known microbial isolates contained in the
database. For species level identification of microbes, a typical mass range m/z of 2–20 kDa is
used, which represents mainly ribosomal proteins along with a few housekeeping proteins. The
characteristic pattern of highly abundant ribosomal proteins, which represent about 60–70% of the
dry weight of a microbial cell, in the mass range of 2–20 kDa (Murray, 2012) is used to identify a
particular microorganism by matching its PMF pattern with the PMFs of the ribosomal proteins
contained in an extensive open database. Thus, the identity of a microorganism can be established
down to the genus, and in many cases to the species and strain level (Fagerquist et al., 2010). This
approach is widely used in microbial identification because it is simple and can be conveniently
adopted in a microbial diagnostic laboratory, aided by the availability of many commercial
libraries of organism PMFs. Microbial identification by matching the biomarker masses with the
molecular masses of proteins predicted from the genome sequence is not very popular in
microbiological diagnostic laboratories because it requires knowledge of complete genome
sequence of an organism before a database of its predicted protein molecular masses could be
created.

Although, the culture conditions might profoundly affect the microbial physiology and protein
expression profile (Welker, 2011) they do not influence microbial identification by MALDI-TOF

158
MS. Valentine et al. (2005) cultured three bacterial species on four different culture media and
found that microbial MALDI-TOF MS identification was independent of culture conditions.
Another research group (Carbonnelle et al., 2007) also reported that both the culture conditions
and the culture time did not affect microbial identification by MALDI-TOF MS.

A number of organic compounds have been used as matrices for MALDI-TOF MS but for
microbiological applications, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxy benzoic
acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) have been found to be
the most useful. The matrix solution consists of water and a mixture of organic solvents containing
ethanol/methanol or acetonitrile and a strong acid like trifluoro acetic acid (TFA), which dissolves
the matrix. The solvents penetrate the cell wall of microorganisms and extract out the intracellular
proteins. When the solvent evaporates, ‘co-crystallization’ of protein molecules and other cellular
compounds entrapped within the matrix solution takes place (Horneffer et al., 2001).

The process of sample preparation for identification of microbes by MALDI-TOF MS depends

upon the source from which it was isolated, or on the chemical nature of the constituents of its cell
wall. Investigators have evaluated different sample preparation methods for different groups of
microorganisms. Some microbes might be identified directly by MS, called direct cell profiling,
while for some others whole cell lysates or crude cell extracts are prepared. In direct cell profiling,
a single colony of microorganism is picked and spotted directly on to the sample plate and
immediately overlaid with the matrix solution. Gram negative bacteria like Neisseria spp. (Ilina et
al., 2009), Yersinia spp. (Stephan et al., 2011), and Vibrio spp. (Eddabra et al., 2012) were
identified by MALDI-TOF MS using direct cell profiling. A ‘preparatory extraction’ of microbes
with formic acid (FA) reportedly increased the ability of MALDI-TOF MS in identifying Gram-
positive species. Studies have reported that preparatory extraction was necessary for identification
of Gram-positive bacteria by MALDI-TOF MS, but not for Gram-negative bacteria (Alatoom et
al., 2011; Saffert et al., 2011). A ‘preparatory extraction’ of microbes with formic acid was also
used for sample preparation of sugar-non fermenting bacterial species (Mellmann et al., 2008) and
Staphylococcus spp. (Dubois et al., 2010).

Due to the complex nature of their cell walls aerobic actinomycetes, Nocardia and mycobacteria
require specialized processing procedures prior to MALDI-TOF analysis. Verroken et al. (2010)
described a modified procedure for identification of Nocardia spp. by MALDI-TOF MS. Bacteria
were lysed in boiling water, followed by ethanol precipitation of proteins. The precipitated proteins
were dried, resuspended in 70% formic acid and acetonitrile and analyzed by MALDI-TOF MS.
Researchers have reported different methods for sample preparation for identification of
mycobacteria by MALDI-TOF MS, ranging from direct bacterial profiling to treatment with

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formic acid, safety being a major concern for routine investigations. EI Khéchine et al. (2011)
described a procedure which combined inactivation and processing methods. Mycobacterial
colonies collected in screw-cap tubes containing water and 0.5% Tween 20 were inactivated by
heating at 95°C for 1 h. Inactivated samples were centrifuged and vortexed with glass beads to
disrupt the mycobacterial cell wall, the resultant pellet was re suspended in formic acid,
acetonitrile, and centrifuged again. Finally, the supernatant was deposited onto the MALDI target
plate and overlaid with matrix.

As in bacteria, various sample processing methods have been investigated for identification of
yeasts by MALDI-TOF MS, out of which ‘preparatory extraction’ with formic acid was reported
to be most suitable (Stevenson et al., 2010b; Theel et al., 2012). Cassagne et al. (2014) evaluated
five methods for sample preparation for fungal hyphae and spores. They finally devised a protocol
wherein fungi were cultivated on Sabouraud gentamicin-chloramphenicol agar for 72 h at 27°C,
extracted with formic acid following incubation in ethanol. Acetonitrile was added, the mixture
was centrifuged and supernatant was used for MALDI-TOF MS analysis. Lau et al. (2013)
reported a method based on mechanical lysis for sample preparation of fungal hyphae and spores.
A small specimen from the mold isolate was suspended in ethanol and zirconia-silica beads,
vortexed and centrifuged. The pellet was re suspended in 70% FA, vortexed again, and centrifuged.
The supernatant was used for subsequent analysis by MALDI-TOF MS. Both methods reportedly
gave good results in their respective studies. Analysis of intact cells of members of the genus
Penicillium generated poor MALDI spectra, but re suspension of the conidia and spores in
trifluoroacetic acid-acetonitrile and disruption with glass beads discriminated the species with
100% accuracy (Hettick et al., 2008a).

The discovery of suitable matrices, usage of whole/intact cells for recording PMF of microbes in
the typical mass range (m/z) of 2–20 kDa, followed by availability of dedicated databases for
microbial identification has made MALDI-TOF MS a lucrative alternative for microbial
identification. The first MALDI-TOF MS system capable of microbial identification, termed
“MALDI Biotyper” was developed by Bruker Daltonics. The MALDI Biotyper consisted of a
basic MALDI-TOF platform, operating and analysis softwares, an onsite database and a simple
method for extraction/preparation of sample (Seng et al., 2009). The software and database
integrated easily with the Bruker MALDI-TOF instrument and its associated operating and
analysis softwares. This system also allowed for up gradation by providing option of adding
internal libraries of organisms. Another MALDI platform for microbial identification was
introduced by Shimadzu in collaboration with bioMérieux. Shimadzu supplied the instrumentation
and the analysis software “Launchpad,” while bioMérieux supplied and maintained the centralized
database, “SARAMIS” which could be constantly updated. Later, Andromas introduced a different
kind of database and software for routine bacteriological identification which was compatible with

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both Bruker and Shimadzu instruments (Emonet et al., 2010). The greatest break-through in the
development of MALDI-TOF MS came with its regulatory approval for routine identification of
bacteria and fungi in clinical microbiological laboratories. In the last 2 years, MALDI Biotyper
CA System (Bruker Daltonics Inc.) has been approved by the US Food and Drug Administration
(FDA) for identification of cultured bacteria from human specimens (in vitro diagnosis). Similarly,
VITEK® MS (bioMerieux Inc.) was approved by the US FDA for identification of cultured
bacteria and yeasts (Patel, 2015). The list of microorganisms approved for identification is unique
to the individual system (Patel, 2015). MALDI-TOF MS was first approved for clinical use in
China in 2012 when bioMérieux VITEK MS system was approved by China State FDA for in vitro
diagnostic (IVD) purposes (Luo et al., 2015). Later, China State FDA also approved the Bruker
IVD MALDI Biotyper System for routine identification of microorganisms isolated from human
specimens.

The organism databases are the key components of commercial MALDI platforms. They are
continually increasing in size and are regularly updated by the manufacturers with discovery of
new microbial species and annotations. Both, the Bruker and the Shimadzu systems contained a
large collection of representative organisms in their databases and yielded comparable results with
very low false positive rates (Carbonnelle et al., 2012). With a few exceptions, isolates were rarely
misidentified by these devices. In a few MALDI-based identification studies, some organisms
could not be identified; this failure was attributed to the organism not being included in the earlier
databases rather than to the methodological error (Carbonnelle et al., 2012). A critical drawback
of the proprietary softwares and databases is their limited accessibility to the private researchers
due to their high costs. Nevertheless, a few research groups have developed some open-source
softwares and databases which are freely available for the scientific community. To name a few,
these are, mMASS, Mass-Up, pkDACLASS, MALDIquant, SpectraBank, BIOSPEAN, etc.
(Strohalm et al., 2008; Ndukum et al., 2011; Böhme et al., 2012; Gibb and Strimmer, 2012; Raus
and Šebela, 2013).

Go to:

Applications in Microbial Diagnosis

MALDI-TOF MS in Bacteriology

Clinical Bacteriology

Conventionally diagnosis of bacterial infections in the body fluids is carried out on the basis of
biochemical and metabolic-profiling that requires 24–48 h for identification of the inflicting
bacterial species. In the meantime, patients are administered empirical antibiotics, which are

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sometimes inappropriate. Clinical microbiology laboratories require rapid, reliable, and cost
effective methods for identification of potential pathogens in clinical samples so that appropriate
antimicrobial therapy may be initiated early. A number of researchers have shown that MALDI-
TOF MS can be used for early identification of bacteria in blood cultures, urinary tract infections
(UTIs), cerebrospinal fluids, respiratory tract infections, stool samples etc.

Many studies have shown that MALDI-TOF MS equalled or even surpassed the conventional
diagnostic methods in speed and accuracy in detecting blood stream infections (La Scola and
Raoult, 2009; Stevenson et al., 2010a; Foster, 2013; Haigh et al., 2013; Tadros and Petrich, 2013).
A few studies suggested that additional pre-treatment of body fluids by ammonium chloride
(Prod’hom et al., 2010), formic acid (Christner et al., 2010), or short-term incubation on solid
medium (Idelevich et al., 2014) further improved the diagnostic potential of MALDI-TOF MS.

When conventional methods for identification of urinary tract pathogens for diagnosis of UTIs
were compared with MALDI-TOF MS based identification systems, it was found that MALDI-
TOF MS required minimal processing time and identified bacteria from urine samples in the
presence of even more than two uropathogens (Ferreira et al., 2010; Köhling et al., 2012; Burillo
et al., 2014). A few researchers have suggested procedures involving differential centrifugation of
urine samples (Rosselló et al., 2014) or diafiltration (Demarco and Burnham, 2014) to further
improve the clinical sensitivity and turnaround time of MALDI-TOF MS based diagnosis of UTI.

Diagnosis of infectious diarrhea in laboratory is usually done by culture and identification of

bacteria in the stool samples. This is a costly and time consuming process requiring 3–5 days for
detection and identification of enteric bacterial pathogens. He et al. (2010) performed a
comparative study of identification by MALDI-TOF MS versus routine phenotypic methods for
identification of suspicious colonies from stool samples. They found that the entire procedure for
identification by MALDI-TOF MS, from smear preparation to reporting of the final result was
completed within 30 min, thus shortening the turnaround time of the test by 2–3 days.

Bacterial meningitis is a neurological emergency. Early diagnosis is vital for rapid initiation of
appropriate antimicrobial therapy. MALDI-TOF MS has been used for direct detection of bacteria
causing meningitis in cerebrospinal fluids (Segawa et al., 2014). It has also been used for rapid
identification of atypical, Gram-negative environmental organisms and respiratory tract pathogens
which chronically infect patients with cystic fibrosis (Alby et al., 2013; Baillie et al., 2013).
Recently, a very novel application of MALDI-TOF MS was shown by Guembe et al. (2014) who

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reported that MALDI-TOF MS can perform better than conventional culture methods in diagnosis
of catheter-related bloodstream infections.

Food- and Water-Borne Bacteria

Rapid identification of pathogenic microorganisms is important to ensure safety and quality of

water and food products. MALDI-TOF MS has been shown to be useful for early detection of
bacterial hazards which might contaminate drinking water. The genus Aeromonas which is
indigenous to surface waters is currently composed of 17 species, of which seven can cause severe
water-borne outbreaks. Donohue et al. (2007) used the m/z signature of known strains of
Aeromonas to assign species to unknown environmental isolates. Their results showed that
MALDI-TOF MS rapidly and accurately classified unknown species of the genus Aeromonas,
which was suitable for environmental monitoring.

MALDI-TOF MS has also been applied successfully in food microbiology for various purposes
like, identification and classification of lactic acid bacteria in fermented food (Nguyen et al., 2013),
detection of bacteria involved in spoilage of milk and pork (Nicolaou et al., 2012), identification
of bacteria isolated from milk of dairy cows (Barreiro et al., 2010), identification of bacteria
present in probiotics and yogurt (Angelakis et al., 2011), identification of pathogenic bacteria
contaminating powdered infant formula-food (Stephan et al., 2010), characterization of biogenic
amine-producing bacteria responsible for food poisoning (Fernández-No et al., 2010) and for
identification of causative agents of seafood-borne bacterial gastroenteritis (Hazen et al., 2009;
Böhme et al., 2010, 2011).

Environmental Bacteriology

Tests based on biochemical traits usually fail to identify microbes isolated from environmental
samples, as the diversity of microbes in these habitats is enormous (Torsvik et al., 2002). Various
studies have shown that whole cell MALDI-TOF MS can be used as an efficient tool to identify
and characterize isolates which originate from specific ecosystems. Researchers have reported the
use of MALDI-TOF MS in identification of microbes isolated from sewage sludge (Ruelle et al.,
2004), for grouping of bacteria isolated from marine sponges into different proteotaxanomic
groups (Dieckmann et al., 2005) and for identifying bacteria inhabiting soil contaminated with
polychlorinated biphenyl (Uhlik et al., 2011).

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Another research group performed MALDI-TOF MS analysis of the cultivable fraction of
environmental samples. They acquired strain-specific spectra which helped in grouping aerobic
and moderately halophilic prokaryotes into phenotypic clusters belonging to distinct taxa. They
suggested that, if the culture conditions were not radically different, irrespective of the age or
quality of the culture, MS spectrum of a microbe reflects its taxon-specific phenotypic properties
which results in guaranteed identification (Munoz et al., 2011).

In another study MALDI-TOF MS was used for differentiating bacterial species of the family
Rhizobiaceae. Subdivided within three genera, these bacterial species establish either symbiotic or
saprophytic interactions with plants.The research group even constructed a database that included
the type strains of currently accepted species in the family and validated it by identifying all
rhizobial strains isolated from nodules and tumors (Ferreira et al., 2011).

Detection and Identification of Agents of Biological Warfare

Fast and reliable identification of microbes which pose threats as agents of bioterrorism is required,
not only to combat biological-warfare attacks, but also to prevent natural outbreaks caused by these
organisms. Conventionally, organisms which pose severe threats as agents of bioterrorism have
been identified by phenotypic, genotypic, and immunological identification systems which are
slow, cumbersome and pose significant risk to the laboratory personnel. Recently various
researchers reported MALDI-TOF MS as a simple, rapid and reliable approach to identify highly
pathogenic organisms like Brucella spp., Coxiella burnetti, Bacillus anthracis, Francisella
tularensis, and Y. pestis (Shaw et al., 2004; Pierce et al., 2007; Lasch et al., 2009; Ayyadurai et
al., 2010; Seibold et al., 2010; Lista et al., 2011; Vranakis et al., 2013).

Further work is being carried out to develop safe and MS-compatible protocols for inactivation of
vegetative cells and spores of highly pathogenic organisms, which can be integrated into a routine
microbiological laboratory. Lasch et al. (2008) proposed a tri-fluoro acetic acid (TFA) based
inactivation protocol for vegetative cells and spores of pathogenic organisms, but Couderc et al.
(2012) found that for Yersinia isolates, ethanol inactivation yielded MALDI-TOF MS spectra of
significantly higher quality than TFA inactivation. Jeong et al. (2014) reported a direct in situ
MALDI-TOF MS which allowed a high-throughput detection and identification of aerosolized
Bacillus spores, without any pretreatment process. The spores of the Bacillus spp. were directly
spotted on the MALDI target plate and left for air-drying. These were subsequently coated with
the matrix solution. After the matrix solution air-dried, the spotted samples were analyzed by MS
(Jeong et al., 2013, 2014).

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MALDI-TOF MS has also been shown to be useful for detection of protein toxins, such as
staphylococcal enterotoxin B, botulinum neurotoxins, Clostridium perfringens epsilon toxin, shiga
toxin etc. which can be used as potential agents for bioterrorism when delivered via an aerosol
route (Kull et al., 2010; Alam et al., 2012). In a biological war, early and unambiguous detection
of toxins from aerosols is required to initiate medical countermeasures. Alam et al. (2012)
developed a simple method of sample processing for identification of protein toxins by MALDI-
TOF/TOF MS method. Nebulizer was used to generate aerosols which were collected using a
cyclone collector. Tandem MS data with information from peptide sequences was used for
detecting toxins that originated from organisms of any geographical location.

Detection of Antibiotic Resistance in Bacteria

MALDI-TOF MS has been shown to generate PMFs capable of discriminating lineages of

methicillin-resistant S. aureus strains (Wolters et al., 2011). Under careful experimental
conditions, it has also been shown useful for subtyping methicillin-resistant S. aureus strains
(Croxatto et al., 2012). Similarly, MALDI-TOF MS has been shown to be of great use in
identifying vancomycin-resistant enterococci (Griffin et al., 2012; Nakano et al., 2014). Wang et
al. (2014) suggested that MALDI-TOF MS could be used as a screening tool for discriminating
vancomycin-resistant Enterococcus faecium strains from vancomycin-susceptible E. faecium
strains.

The most common mode of microbial resistance to β-lactams, the largest class of antibiotics, is
their enzymatic hydrolysis by β-lactamases. The production of β-lactamases is detected by
MALDI-TOF MS employing a ‘mass spectrometric β-lactamase (MSBL) assay.’ In this assay,
buffered solution of the antibiotic is mixed with bacterial culture and incubated. The reaction
mixture is centrifuged and supernatant subjected to MALDI-TOF MS analysis. The β-lactamase
producers inactivate the β-lactam ring of the antibiotic by addition of a residue of water. The mass
shift in the non-hydrolyzed and the hydrolyzed forms of the antibiotic confirms the presence or
absence of β-lactamase producing bacteria. The MSBL assay has been applied for detection of
resistance to β-lactam antibiotics like penicillin, ampicillin, piperacillin, cetazidime, cefotaxime,
ertapenem, meropenem, and imipenem. Using MSBL assay researchers have successfully detected
β-lactamase producing organisms like Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Acinetobacter baumanni, Citrobacter freundii, Enterobacter cloaceae, Salmonalla spp.
etc. (Hrabák et al., 2011; Hooff et al., 2012; Sparbier et al., 2012a; Kostrzewa et al., 2013). Given
the accuracy of resistance detection with MSBLs, further innovations and improvements in terms
of enhancing speed of resistance detection and expanding detection of resistance for newer classes
of β-lactam antibiotics in MSBL assays are being envisaged. Recently, Johansson et al. (2014)

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developed a MALDI-TOF MS method for detection and verification of carbapenemase production
in anaerobic bacterium, Bacteroides fragilis as early as 2.5 h. Hoyos-Mallecot et al. (2014)
evaluated a MALDI-TOF MS method for identification and differentiation of carbapenemase
producing clinical strains of Enterobacteriaceae and P. aeruginosa from metallo-β-lactamase
producing strains. Hart et al. (2015) reported a modified strategy for detection of biomarkers of
antibiotic resistance in clinical strains of E. coli using MALDI-TOF MS. They suggested that
instead of using intact bacterial cells for MS, the periplasmic compartment should be extracted
(since β-lactamases are located in the periplasm); in-solution digested with trypsin, separated by
nano-LC before MALDI-TOF MS analysis. Using this approach they reported the peptide
sequence of biomarkers for several classes of β-lactamases like CTX-M-1 group extended
spectrum β-lactamase, TEM β-lactamase, VIM a metallo-β-lactamase and CMY-2 an ampC β-
lactamase. Moreover using this approach they also detected the peptides specific to an
aminoglycoside modifying enzyme Kan-R.

Similar to β-lactams, resistance in microorganisms to aminoglycosides is mainly due to the

enzymatic modification of antibiotics by bacterial acyltransferases, adenyltransferases, and
phosphotransferases. Since these enzymes show different preferentiality for the CoA substrate
present in different aminoglycosides, it has not been possible to establish a universal MS-based
assay for detection of aminoglycoside resistance. Further efforts to develop and standardize
MALDI-TOF MS for detection of aminoglycoside resistance in routine laboratory are still
underway.

Bacterial Strain Typing and Taxonomy

Conventional classification of bacteria has been carried out on the basis of biochemical, metabolic
and antigenic properties. However, currently microbial species are being identified primarily on
the basis of genomic information. The sequencing of 16S rDNA is considered to be the ‘gold
standard,’ because it is present in every prokaryote and allows reconstruction of a global phylogeny
(Woese et al., 1990; Stackebrandt and Goebel, 1994; Pace, 1997). Most DNA fingerprinting
methods, however, do not correlate phylogenetically and fail to give any information regarding the
evolutionary relationship between various species. Genomic techniques like amplified fragment
length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE), and multilocus sequence
typing (MLST) are used for subtyping of bacteria for epidemiological purposes. Though 16S
rDNA sequencing is adequate to assign genus or species to an isolate, in most cases it is not
sufficient for refined typing, e.g., epidemiological studies. PFGE, can be applied for high
resolution typing, but is generally inappropriate for assigning genus or species to microorganisms
(O’Leary et al., 2011).

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Proteomics represents the functional aspect of genomics and can be used as a taxonomic tool. Gel-
based whole cell protein profiling may be as cumbersome and time consuming as any other
genomic technique. On the other hand MALDI-TOF MS intact cell or whole cell PMF based typing
is a rapid and sensitive method for bacterial identification. In many cases it has shown resolution
and reproducibility which is better than gel-based protein or DNA fingerprinting techniques (van
Baar, 2000; Fenselau and Demirev, 2001; Lay, 2001).

Numerous studies have shown that MALDI-TOF MS is a rapid, reliable and cost-effective
technique for identification of bacteria. The method, however, has some lacunae:

(i) proper identification of organisms is possible only when the spectral database contains
information about specific genes like prokaryotic 16S rRNA, gyrB, rpoB, or hsp60 of
strains/species of a particular genus,
(ii) (ii) Databases should be prepared locally for certain taxa (e.g., Streptococcus or
Staphylococcus) in which geographical variations lead to variations in the genotype
and phenotype (Benagli et al., 2011).

During the past few years various researchers have shown applicability of MS for bacterial
identification, taxonomy and strain typing. Haag et al. (1998) used MALDI-TOF MS for rapid
characterization of pathogenic Haemophilus strains. They also determined strain-specific
differences among H. influenzae isolates from several patients. Nilsson (1999), detected strain-
specific biomarkers based on the analysis of six different strains of Helicobacter pylori. Lundquist
et al. (2005) showed differentiation of four subspecies of F. tularensis using this approach.
Carbonnelle et al. (2007) showed that MALDI-TOF MS was a powerful tool for the identification
of clinical isolates of coagulase negative staphylococci. MALDI-TOF MS was used for rapid
identification of ten different species of S. viridans (Friedrichs et al., 2007). When the results of
species identification obtained by MALDI-TOF MS were compared with the
phenotypic/genotypic identification systems, a 100% consonance was achieved. Similarly, a
variety of Staphylococcus spp., pathogenic Neisseria spp., clinically important Yeast species and
Mycobacterium spp. were identified using MALDI-TOF MS (Ilina et al., 2009; Dubois et al., 2010;
Stevenson et al., 2010b; Saleeb et al., 2011). Microorganisms in which intact cell (direct bacterial
profiling) was used for bacterial identification and strain typing.

MALDI-TOF MS is not only used for species identification but has application in strain typing
also. Kumar et al. (2004) showed that MALDI-TOF MS may be a useful tool for discriminating
strains of beta hemolytic streptococci, and also for characterization of untypable strains of
streptococci group A. Eddabra et al. (2012) used MALDI-TOF MS to discriminate 30
environmental strains of Vibrio spp. Stephan et al. (2011) used MALDI-TOF MS for rapid
identification of Y. enterocolitica strains to the species, and subtyping to the biotype level.

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MALDI-TOF MS based methods for the discrimination and typing of mycobacteria, typing
multidrug-resistant K. pneumonia, and typing strains responsible for nosocomial outbreak of A.
baumannii have been described (Shitikov et al., 2012; Berrazeg et al., 2013; Mencacci et al., 2013).

MALDI-TOF MS spectrum of an individual microbe is the taxon-specific property of that

organism, which is independent of its geographical location, culture conditions (which should not
be drastically different) or sample preparation methodology. With this approach, not only
identification of new isolates as members of existing species is possible if their type strains have
been previously studied (Ruelle et al., 2004), recognition of coherent phenotypic patterns reflecting
taxonomic identities is also possible. The ease and rapidity in identification of large numbers of
isolates can be used to study taxonomic and inter- and intra-specific diversity (Feli and Dellaglio,
2007).

MALDI-TOF MS in Virology

Clinical Virology

Viruses were traditionally detected by cell culture, which in spite of being the gold standard, often
took days or even weeks before any results were available. It was later replaced or complemented
by lesser sensitive immunological methods, based on antibody analysis (by immunoassays or
immunofluorescence) and by more sensitive molecular methods based on PCR and dot blot
hybridization. The use of MALDI-TOF MS in virology has advanced less as it has in bacteriology
or mycology. This might be a consequence of the relatively low protein content of viruses (Kliem
and Sauer, 2012), higher molecular weight of viral proteins (>20,000 Da) and a probable carryover
of debris of the cell substrate in which viruses are cultured in vitro. Nevertheless, many researchers
have proved the utility of MALDI-TOF MS for diagnosis of various infectious viruses in clinical
samples like influenza viruses, enteroviruses, human papilloma viruses (HPVs), herpes virus,
hepatitis virus etc. (Sjöholm et al., 2008; Yi et al., 2011; Piao et al., 2012). Interestingly in most
of the studies, the viral genetic material was amplified by PCR and the amplicons were analyzed /
Identified by MALDI. Sjöholm et al. (2008) developed an efficient MALDI-TOF MS based
screening method for multiplex detection of all human herpes viruses which were present in
different archival biological samples. The sensitivity and the detection limit of viruses by MALDI-
TOF MS method were high and comparable to reference methods like oligonucleotide microarrays
and multiplex PCR (Sjöholm et al., 2008).

Yi et al. (2011) reported the use of a PCR-based MS method for detection of high-risk HPVs, a
prime cause of human cervical cancer. They claimed that the high-throughput and cost-
effectiveness associated with the method made it suitable for diagnosis in routine clinical settings
and for epidemiological studies. Piao et al. (2012) combined the multiplex PCR with MALDI-

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TOF MS and developed a PCR-Mass assay which simultaneously detected eight distinct viruses
associated with enteric infections in humans. Later, Peng et al. (2013) proved the utility of
multiplexed MALDI-TOF for type-specific detection of human enteroviruses associated with
hand, foot and mouth diseases. Recently, Calderaro et al. (2014) reported that MALDI-TOF MS
was an effective, rapid and inexpensive tool which identified various poliovirus serotypes from
different clinical samples. They further reported that through MALDI-TOF MS, specific viral
biomarkers could be detected which were helpful in differentiating virus-infected cells from
healthy cells.

Viral Genotyping, Subtyping, and Epidemiological Studies

Apart from viral identification, MALDI-TOF MS has also been used in virology for genotyping
of JC polyomaviruses (Bayliss et al., 2010), hepatitis B and hepatitis C viruses (Ilina et al., 2005;
Ganova-Raeva et al., 2010) and for detection of mutations in hepatitis B viruses (Luan et al., 2009).
Also, many researchers have demonstrated the application of MALDI-TOF MS for screening of
influenza virus subtypes and for tracking epidemiology of influenza viruses (Schwahn et al., 2009,
2010; Fernandes and Downard, 2014). Newer strains of influenza viruses frequently originate by
mixing of genetic material of several strains in a common host. Thus, detection of ever evolving
influenza viruses is a challenge for PCR based molecular methods, because the primers fail to
anneal to their respective target sequences and need to be redesigned repeatedly. Similarly, the
antibody-based immunological assays fail to identify the antigenically distinct, reassorted virus
strains. Rapid identification and characterization of the inflicting influenza viral strains is
necessary to initiate an early, effective therapy and to prevent a probable pandemic. MALDI-TOF
MS has emerged as a rapid and reliable approach to screen highly evolving influenza virus types
and subtypes. Chou et al. (2011) reported the use of MALDI-TOF MS in combination with
antibody-magnetic nanoparticles for detection and rapid screening of influenza virus subtypes.
Later, Downard (2013) described a method for detection of strains of influenza viruses using whole
virus protein digests. This ‘proteotyping approach’ was successful in typing, subtyping, and tracing
the lineage of human influenza viruses. He even described the success of his ‘proteotyping
approach’ in characterization of strains of parainfluenza virus, another respiratory infectious agent.

Detection of Antiviral Resistance

The studies in this area are scanty. However, MALDI-TOF MS has proved efficacy in detecting
drug resistance to ganciclovir in cytomegaloviruses which frequently infect transplant recipients
(Zürcher et al., 2012).

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MALDI-TOF MS in Mycology

Clinical Mycology

Conventional methods for identification of fungi are based on morphological, biochemical, and/or
immunological properties which might span 2–5 days, or more, and often require combining
several phenotypic methods for conclusive interpretations. The molecular methods based on
analysis of genes encoding 18S rRNA and the internal transcribed spacer regions 1 and/or 2 (ITS
1/2) are tedious and time consuming (Sendid et al., 2013). Rapid and accurate identification of the
inflicting fungal species is required for early initiation of antifungal therapy.

Fungal identification by MALDI-TOF MS in the medical mycology laboratory has moved at a

slower pace than bacterial identification, owing to their inherent biological complexity which
makes their study as a whole difficult, and also due to co-existence of different fungal phenotypes
(hyphae and/or conidia) in the same organism (Santos et al., 2010). In order to obtain reproducible
PMF results, parameters like culture media, quantity/type of colony material and incubation time,
need to be carefully standardized. Also, the fungal cells might require additional treatment with
trifluoroacetic acid, formic acid, or acetonitrile along with beating with beads to disrupt strong cell
walls.

The first study describing the use of MALDI-TOF MS for identification and characterization of
single-celled fungus, Saccharomyces cerevisiae was reported in Amiri-Eliasi and Fenselau (2001).
Among fungi, highly reproducible PMF spectra have been reported for the ascomycetous and
basidiomycetous yeasts including organisms like Candida, Cryptococcus, and Pichia. These
genera of yeast form uniform colonies on agar plates which can be lysed efficiently with the
standard procedure (as used in bacteria) for MALDI-TOF sample preparation (Bader, 2013).
However, Cassagne et al. (2014) evaluated various pretreatment procedures for MALDI-TOF MS
identification of yeasts and reported that although labor-intensive, complete extraction with formic
acid/acetonitrile yields better identification results. Various researchers have reported that
MALDI-TOF MS was a reliable and time-saving approach for identification of various yeast
species in bloodstream infections (Spanu et al., 2012; Yaman et al., 2012; Lavergne et al., 2013).
Several researchers have described the use of MALDI-TOF MS for detection of various human
fungal pathogens.

Detection of Antibiotic Resistance in Fungi

Identification/prediction of resistance for antifungals by MALDI-TOF MS has not advanced as

much asSit has, in predicting resistance in bacteria. This might be attributed to the fact that
antimycotic resistance in fungi is not as frequent as in bacteria due to absence of drug degrading

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enzymes. Only a few fungal species like C. glabrata or C. krusei and C. parapsilosisis have been
reported to be intrinsically resistant to azoles and echinocandins respectively (Bader, 2013).
Similarly, a species-specific resistance to antimycotic agents is observed in many molds and
zygomycetes (Pfaller et al., 2002; Alastruey-Izquierdo et al., 2010). Thus, drug resistance in fungal
isolates may be predicted simply by identification of the inflicting fungal species by MALDI-TOF
MS (Bader, 2013).

Fungal Strain Typing and Taxonomy

The use of MALDI-TOF MS for strain typing of fungal isolates is still in infancy and not as
successful as in bacteria. In contrast to yeasts, it has been difficult to type molds since they have
complicated phylogenetic relationships (Samson and Varga, 2009) and more complicated
morphology. Strain typing by MALDI-TOF MS was, however, reported to be feasible with C.
albicans and C. parapsilosis (Qian et al., 2008; Pulcrano et al., 2012).

Future Perspectives

Although attempts for MS based bacterial identification and diagnosis date back to mid 1970s it is
only in the recent years that microbiologists have realized the potential and applicability of
MALDI-TOF MS in routine microbiological laboratories. The current gold standard for microbial
identification – 16S rRNA and 18S rRNA gene sequencing is not favorable in terms of both cost
and time. Bizzini et al. (2011) claimed that including the cost of consumables, salaries, and
apparatus-depreciation, identification of a single bacterial isolate by 16S rRNA sequencing costed
approximately 100 US dollars in their laboratory and the results were available after 48 h. On the
other hand identification of a single bacterial isolate by MALDI-TOF MS could be done in minutes
and costed only a few US dollars (Seng et al., 2009; Cherkaoui et al., 2010).

Comparison of PMF of unknown isolate to reference mass fingerprints present in the database is
the most crucial step for species identification which requires a database containing not only
reference mass fingerprints of all species of interest, but also mass fingerprints of multiple strains
of each species (Lartigue et al., 2009). The MALDI-TOF MS technology failed to distinguish E.
coli and Shigella spp. (Bizzini et al., 2010) or discriminate among species of the mitis complex of
Streptococci (Seng et al., 2009; van Veen et al., 2010). The former failure has been explained by
the fact that E. coli and Shigella were one species phylogenetically but due to historical and clinical
constraints microbiologists have classified them into two species. The reason for improper
delineation of S. pneumoniae and S. mitis is the close relationship between the two species (a
microbiological constraint) which cannot be distinguished by any database update, except tests
like optochin sensitivity or bile solubility (Kilian et al., 2008). Such constraints of MALDI-TOF
MS certainly need to be addressed in future.

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The major constraint against using MALDI-TOF MS in routine microbiological diagnosis is the
reproducibility of the PMFs of the same microbial species during different experiments in the same
laboratory or during different experiments in different laboratories employing the same/different
MALDI-TOF equipment. The intra-laboratory reproducibility studies have shown a higher level
of concordance in PMFs during repeated experiments with the same MS equipment using similar
sample preparation technique (Walker et al., 2002). Another study reported that intra-laboratory
reproducibility of PMFs depended only on the quality of the microbial samples and was
independent of the sample preparation technique (Croxatto et al., 2012). However, studies on
reproducibility of PMFs during inter-laboratory studies have yielded conflicting results. Two
independent research studies showed a higher level of reproducibility in PMFs of identical
microorganisms identified in separate laboratories employing similar sample preparation
technique, the same MS equipment and the same analysis software (Wang et al., 1998; Walker et
al., 2002). On the other hand, another study reported poor inter-laboratory concordance during
MALDI-TOF analysis of identical aliquots of E. coli by three separate laboratories using similar
sample preparation techniques, but different commercial MALDI-TOF equipment (Wunschel et
al., 2005). This research group further combined the PMF spectra of individual laboratories and
formed a ‘multiple laboratory master PMF.’ When this multiple laboratories’ master PMF spectra
were used to identify E. coli, 100 percent identification was observed in each laboratory (Wunschel
et al., 2005). Thus, this study provided a solution to a very important question that is, the potential
of MALDI-TOF MS for microbial identification in different geographical locations with different
MALDI-TOF equipment. By employing a standard protocol for MALDI-TOF MS analysis and
creating a library/database containing PMFs from multiple laboratories (with different MALDI-
TOF equipment), this constraint could be overcome easily.

In recent years there has been an overabundance of information about MALDI-TOF MS and its
application to a broad spectrum of microbes ranging from Gram positive to Gram negative
bacteria, from clinical samples to extreme halophiles and from BSL-3 organisms to environmental
isolates. Beyond the realms of microbial world, recent studies indicate that MALDI-TOF MS can
even be applied to identify algae (von Bergen et al., 2009), mosquitoes (Yssouf et al., 2014),
nematodes (Perera et al., 2005), and insects (Feltens et al., 2010; Hoppenheit et al., 2013).
Independence of the culture conditions, culture formulations, cultivation time, quantity of
inoculum required for identification, have made MALDI-TOF MS as the technique of choice in
routine microbiological laboratories. The provision for integrating in-built databases in the public
databases of MALDI-TOF MS, as well as development of inexpensive and user-friendly softwares
for comparisons and analyses would further increase the credibility of MALDI-TOF MS in future.
What seemed an exaggeration sometimes back has now become a reality? MALDI-TOF MS has
become a valuable tool for a microbiological laboratory, which might potentially replace molecular
identification techniques in near future.

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Surface Enhanced Laser Desorption-Ionization (SELDI):

Surface-enhanced laser desorption/ionization (SELDI) is a variation of MALDI and has been

widely used in discovery studies to identify new protein molecular markers.28 The basic principle
is that the sample is exposed to chips with different active surfaces that enrich certain groups of
proteins. These are then eluted onto a MALDI plate and analyzed using a time-of-flight mass
spectrometer. SELDI is a combination of affinity chromatography and time of flight mass
spectrometry on a single platform. This system enables protein capture, purification, ionization
and analysis of complex biological mixture directly on protein chip array. SELDI system
comprises of (a) Protein chip array (b) Protein chip reader (c) Specific software. Although the
system is highly integrated and easy to use, it is prone to producing artifacts.75–77 The most likely
reason is that the time-of-flight mass spectrometer that was included in the SELDI system lacked
appropriate resolution.28 Also, due to the selectiveness of the activated surfaces, only a fraction
of the proteome is analyzed and potentially critical information may be missed. A significant
source of variability and artifacts is that binding of proteins to the active surfaces is not very robust
and is easily influenced by even small variations in pH, salt concentrations and interfering
compounds. Due to these shortcomings, SELDI has lost its importance.

A limitation of common fragmentation technologies is that they result in cleavage of post-

translational modifications before the protein or peptide backbone is cleaved; however, cleavage
of the backbone with post-translational modifications attached will yield valuable structural
information. Electron capture and electron transfer dissociation are technologies that are
complementary to classical fragmentation, and they tend to result in fragmentation more evenly
distributed over the entire peptide backbone. This makes them particularly useful in localizing
post-translational modifications.

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 CHAPTER: 13
Molecular Techniques
Comet assay analysis
Introduction:

The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid
technique for quantifying and analyzing DNA damage in individual cells. As such, this is one of
the techniques used in the area of cancer research for the evaluation of genotoxicity and
effectiveness of chemoprevention. Swedish researchers Ostling & Johansson developed this
technique in 1984. Singh, et al, later modified this technique, in 1988, as the Alkaline Comet
Assay. The resulting image that is obtained resembles a "comet" with a distinct head and tail. The
head is composed of intact DNA, while the tail consists of damaged (single-strand or double-strand
breaks) or broken pieces of DNA. While most of the applications of the Comet Assay have been
to study animal eukaryotes, there have been reports of successful application in the study of plant
cells.

Individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are
then removed from the cells by lysing. The DNA is allowed to unwind under alkaline/neutral
conditions. Following the unwinding, the DNA undergoes electrophoresis, allowing the broken
DNA fragments or damaged DNA to migrate away from the nucleus. After staining with a DNA-
specific fluorescent dye such as ethidium bromide or propodeum iodide, the gel is read for amount
of fluorescence in head and tail and length of tail. The extent of DNA liberated from the head of
the comet is directly proportional to the amount of DNA damage.

The Comet Assay can be used to detect DNA damage caused by double strand breaks, single strand
breaks, alkali labile sites, oxidative base damage, and DNA cross-linking with DNA or protein.
The Comet Assay is also used to monitor DNA repair by living cells.3

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Procedure:

The Comet Assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive and relatively
simple method for detecting DNA damage at the level of individual. It combines the simplicity of
biochemical techniques for detecting DNA single strand breaks (strand breaks and incomplete
excision repair sites), alkali labile sites and cross-linking, with the single cell approach typical of
cytogenetic assays. The assay has also been successfully implemented in plant cells under
laboratory conditions (Gichner et al. 2004, 2006). The main advantages of the Comet Assay
include:

(a) The collection of data at the level of the individual cell, allowing more robust statistical
analyses,

(b) The need for a small number of cells per sample (<10,000),

(c) Sensitivity for detecting DNA damage and

(d) use of any eukaryote single cell population both in vitro and in vivo, including cells obtained
from exposed human populations and aquatic organisms for eco-genotoxicological studies and
environmental.

The importance of this assay has also been realized in regulatory genotoxicological and there is a
move to replace some traditional assays (e.g. liver unscheduled DNA synthesis assay) in regulatory
genotoxicological studies with in vivo Comet assay. In combination with certain bacterial enzymes
(e.g. formamidopyrimidine glycosylase, endonuclease III, uracil-DNA glycosylases, etc.), which
recognize oxidised purines and pyrimidine bases, this assay has been used to determine oxidative
DNA damage that has been implicated in several health conditions. This assay has also been used

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to show protective effects of different dietary factors in chemo-preventive. In combination with
the fluorescence in situ hybridisation (FISH) technique (Comet-FISH), the application of this assay
has also been extended to determine sequence or gene-specific damage and repair as well as of
possible diagnostic use. In addition, the assay is being used in translational research to assess
whether tumor radio-sensitivity (Fisher et al. 2007) and chemo-sensitivity (Smith et al. 2007) can
be determined. This would allow clinicians to individualise patient management, allocating cancer
therapy to those for whom it will be of most benefit and reducing the likelihood of patients
receiving toxic (and as such ineffective) therapy.

The Comet Assay is based on the ability of negatively charged loops/fragments of DNA to be
drawn through an agarose gel in response to an electric field. The extent of DNA migration
depends directly on the DNA damage present in the cells. It should be noted that DNA lesions
consisting of strand breaks after treatment with alkali either alone or in combination with certain
enzymes (e.g. endonucleases) increase DNA migration, whereas DNA–DNA and DNA–protein
cross-links result in retarded DNA migration compared to those in concurrent controls (Tice et al.
2000).

In this assay, a suspension of cells is mixed with low melting point agarose and spreads onto a
microscope glass slide. After lysis of cells with detergent at high salt concentration, DNA
unwinding and electrophoresis is carried out at a specific ph. Unwinding of the DNA and
electrophoresis at neutral pH (7–8) predominantly facilitates the detection of double strand breaks
and cross links; unwinding and electrophoresis at pH 12.1–12.4 facilitates the detection of single
and double strand breaks, incomplete excision repair sites and cross-links

The determination of the shape, size and amount of DNA within comets is therefore a very
important attribute of the assay if the induced damage is to be evaluated accurately. In parallel
with several technical and procedural evolutions to make the assay more robust, several
approaches have also evolved to quantify the extent of damage more reliably, reproducibly and
meaningfully. Such quantification includes both visual examinations (i.e., photographic,
occulometer or non-specific image analysis systems) or by use of commercially available or public
domain specific image analysis software packages. Although visual examinations give a fairly
good indication of DNA damage and can be used in situations that are considered appropriate or
where specific software packages are not available, the use of specific image analysis software is
considered to be reliable, reproducible, which provide simultaneously a range of parameters and
additional information (e.g. the distribution of DNA within the comet tail, total cellular DNA
content), and may also indicate different phases of cell cycle distribution, which can be useful in
the interpretation of the data. Such specific software packages also facilitate easy statistical
analyses, plotting and documentation of the data.

Despite being a very popular choice to determine DNA damage, there are still some concerns over
the methodology used, and the type and quality of data produced using this assay. Given the

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importance of different measurements in determining the extent of DNA damage (and repair) in
this very widely used assay,

Developmental perspectives of measurement procedures:

The Comet Assay was first introduced by Ostling and Johanson in 1984. This was a neutral version
of the Comet Assay, and interestingly, they used quite sophisticated techniques of image analysis
for quantification of the comets, using acridine orange (AO) as the DNA binding dye. The
fluorescence was measured with a Leitz MPV2 microscope photometer with a ×40 objective using
a Phloemopak filter block H2 giving excitation at 390–490 nm. The emitted light from individual
cells passed an emission filter (long pass 525 nm). Green fluorescence was then measured using a
circular diaphragm first over the head and then over different positions on the comet tail. The
background fluorescence adjacent to the cells was subtracted. The time of illumination and
between measurements were standardized to minimize fading bias. They presented their results in
terms of the ratio of fluorescence (FX) at distance × micrometer on the tail versus fluorescence at
the centre of the head (FO). Based on their observation, they concluded that 50 μm gives the best
resolution of the method used. Based on our current understanding however, as mentioned later,
this measurement is not considered to be robust.

Singh et al. (1988) developed the alkaline version of the Comet Assay in which they used the
length of DNA migration (tail length) to quantify the extent of damage. Subsequently, several
research groups published papers in which various Comet Assay parameters were used (Table).
However, with time, most of them were not of frequent or wide use. Of notable importance is the
publication by Olive et al. (1990), who used the concept of the tail moment to describe DNA
migration. The tail moment calculated by Olive et al. (1990) came to be known as the Olive tail
moment (OTM). This parameter is considered to be particularly useful in describing heterogeneity
within a cell population, as OTM can pick up variations in DNA distribution within the tail.

Although image analysis on comets has been preferable for continuity in assessing DNA damage
by this method, some groups have been working on simple, less time consuming visual scoring
methods that do not require special image analysis software. Collins et al. (1995) published a visual
scoring method that classifies comets from grades 0–4. In this approach, for example, if 100 comets
are scored and each comet assigned a value of 0 to 4 according to its class, the total score for the
sample gel will be between 0 and 400 “arbitrary units.” Visual scoring is rapid as well as simple
and should appeal to those exploring the usefulness of the technique without the need to invest in
expensive analytical equipment or software packages (Collins 2004).

Significance of staining procedures in comet measurements:

Whether the comets are scored by image analysis or visual scoring, good staining of comets is of
paramount importance. Various fluorochromes, which were traditionally used to stain DNA,
chromosomes or nuclei, are being used to stain the comets (i.e. ‘head’ and ‘tail’). Ethidium
bromide (EB) is most commonly used to stain the DNA on Comet Assay slides (Singh et al. 1988),

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followed by 4, 6-diamidino-2-phenylindole (DAPI, Gedik et al. 1992). EB is an intercalating dye
that binds more efficiently to double-stranded DNA than to single-stranded DNA. DAPI binds
predominantly to the major groove of the DNA. The amount of dye binding to the DNA is
proportional to the amount of DNA present and, hence, the amount of light emitted after excitation
with ultraviolet light of appropriate wavelength. It is important that we use very low concentrations
of the dye, as higher concentrations will saturate the system. If the light emitted by the comets is
very intense, the image analysis software cannot accurately define certain Comet Assay
measurements like OTM, because the centre of gravity (CG) of DNA distribution is not defined
correctly (Fig. 4). Other dyes used are SYBR® green/gold (Tice et al. 1998), AO, YOYO dye
(Singh et al. 1994) and propidium iodide. In addition, non-fluorescent staining, such as silver stain,
has also been used by some workers (Kizilian et al. 1999; Garcia et al. 2007).

For general genotoxicity testing purpose, EB is an excellent choice of Comet Assay stain. EB
produces a bright fluorescence, which does not fade easily. Unlike the SYBR® green stain, EB
does not fade during the process of image capturing. This gives the option to work in dim light
rather than in completely dark rooms. Furthermore, EB at concentrations in the range of 2–20 μg
ml−1 gives good quality staining of DNA with a low background signal. YOYO dye gives a strong
fluorescence signal and is particularly useful when cells with low DNA content are used in the
Comet Assay. However, YOYO dye is expensive and cannot be stored for longer periods of time.
SYBR® green/gold also gives a bright fluorescence, but fading during the process of scoring is
definitely a problem. SYBR® gold stains both double-stranded and single-stranded DNA and is
considered better than SYBR® green.

Principles of image analysis in Comet Assay:

It is difficult to ascertain who used commercially available software for the first time. Presently,
different software packages are used for measurement of comet parameters on the basis of image
analysis. Although these software packages may differ slightly the way they calculate DNA
damage, the underlying principles of image analysis are the same.

The first step is to visualize the comets under the fluorescence microscope fitted with appropriate
filters (depending on the fluorochromes used) and capture the image using a camera. The camera
records the intensity of light emitted from each point on the comet and converts it into electrical
signals. These electrical signals are then sent to the computer along with their coordinates, and the
computer decodes these signals and displays the image on the screen (Fig.). An image is composed
of small dots called pixels. Each pixel represents one light sensitive element of the camera, and
the numbers corresponding to light intensity detected for each pixel (grey values) are stored in a
computer file (Fig.). Once the image is converted into numbers as shown in Fig., the actual image
analysis is initiated. The next step is to define the head and the tail of the comet. Different software
packages use different approaches to detect the head and the tail (Fig.). Once the ‘head’ and ‘tail’
region are defined, the tail length is measured in terms of pixels, which are then converted into
microns. The light intensities originating from the head and tail parts of the comet are used to

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calculate different comet parameters (Fig.). The background fluorescence is subtracted from head
and tail intensities to get the true fluorescence as described in Table.

Standardization of Comet Assay measurements:

The Comet Assay has mainly remained an assay of academic and scientific interest until quite
recently. Currently, the Comet Assay has however the potential to be used as a tool in genotoxicity
testing and regulatory submissions for new chemicals and mixtures. In an attempt to make the
assay more sensitive and reliable, several research groups have come out with unique procedures
and specialized measures of DNA migration. Attempts have been made to make this assay widely
acceptable, by correlating the results with other well-established assays (e.g. micronucleus assay)
whilst determining the genotoxicity (Raisuddin and Jha 2004). Before it can be accepted as a

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regulatory tool, this assay has to be harmonized in terms of its methodology and interpretations
and should be demonstrated to be reliable, accurate and transferable between laboratories.

Kumaravel and Jha (2006) defined the most reliable comet measurements that would truly reflect
the extent of DNA damage induced by low linear energy transfer ionising radiation. The authors
approached this question by performing alkaline Comet Assay on human peripheral blood
lymphocytes irradiated with graded doses of 137Cs gamma radiation and correlating the various
comet measurements with the radiation dose. As DNA damage produced is directly proportional
to the radiation dose, any change in dose should be reflected in proportional change in the comet
measurements. They concluded that only a few comet measurements provided by the image
analysis software correlated well with gamma radiation dose. Further retrospective analysis from
in vitro and in vivo experiments using chemicals also suggested that OTM and % Tail DNA gave
good correlations with the dose of genotoxic agents used and were the most reliable comet
measurements. Statistically, the authors did not find much difference between OTM and % Tail
DNA in analysing extent of DNA damage.

Although OTM appeared to be the most statistically significant measurement (Kumaravel and Jha
2006), the inter-laboratory comparison of results seems to be difficult for this parameter. OTM is
calculated as a product of two factors: the percentage of DNA in the tail (%Tail DNA) and the
distance between the intensity centroids (centre of gravity) of the head and the tail along the x-axis
of the comet. Hence, OTM is an absolute parameter with a measurement unit μm (Vilhar 2004;
http://www.botanika.biologija.org/exp/comet/Comet-principles). This requires that the image
analysis system is geometrically calibrated before comet measurement (i.e. the number of pixels
per micrometer is known for different microscope objectives). If the system is not calibrated, inter-
laboratory comparisons are difficult

Identification and measuring the ‘Clouds’

‘Clouds’ or ‘hedgehogs’ are important observations in most Comet Assay experiments. These are
cells with extensive DNA migration that are outside the measurement capabilities of the image
analysis system or may give inappropriate measurements when image analysis is used. Clouds are
therefore scored only by visual analysis. They are characterised by a small or absent head with a
highly diffused tail that is physically separate from the head. Accurate identification of clouds
comes with practice and experience; hence, it is important that clouds are identified correctly
whilst scoring slides using image analysis systems. Ideally, they should be scored visually and
recorded alongside the results of image analysis.

The exact origin of clouds is not clear, but it is assumed that apoptotic cells lead to clouds. This
has been observed in Rat-1 cells exposed to irradiation with 10 Gy gamma rays, where the cells
underwent apoptosis 24 h after irradiation and produced clouds in Comet Assay (Kumaravel TS;
unpublished observations). Interestingly, cells treated with hydrogen peroxide for approximately
5 min and processed immediately (assuming that apoptotic process cannot be initiated and

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completed in 5 min) also gave rise to clouds. Moreover, cells treated with high doses of gamma
radiations and processed immediately gave a similar response. Clouds are routinely seen in in vitro
and in vivo Comet Assay experiments (particularly where cells are collected by scraping, e.g.
stomach). These observations suggest that, in addition to apoptosis, clouds are also induced by
high levels of DNA damage as well as in necrotic cells. It is also observed that, sometimes,
identical experimental conditions can result either in measurable comets or in clouds. This
phenomenon is particularly seen in treatment with hydrogen peroxide and methyl
methanesulphonate (Collins 2004; Speit et al. 2004) when used in conjunction with enzymes such
as endonucleases. To confirm whether clouds really represent apoptotic cells, more experiments
under different exposure conditions are required bearing in mind that apoptosis is an irreversible
process. There is insufficient information in literature on this issue, and more studies are required
to elucidate this phenomenon.

Comet assay combined with FISH:

The Comet Assay has also been combined with FISH technique (Comet-FISH) to investigate the
localisation of specific gene domains within an individual cell (e.g. p53, her-2). The position of
the fluorescent hybridisation spots in the comet head or tail indicates whether the sequence of
interest lies within or in the vicinity of a damaged region of DNA. Although not many studies have
been performed using this technique, it has a number of potential uses in DNA repair and genomic
instability studies. The measurements that can be collected from Comet-FISH experiments are the
position (either in head or tail) and number (number of fluorescence spots) of FISH signals after
DNA damage. In the assay, depending on the probe region and probe length, the signals can be
split or just migrate to the tail. The location of FISH signals (either split or intact) in either head
or tail appears to be the best indicator for DNA damage using Comet-FISH. The splitting of signals
appears to be random events depending on whether the DNA damaging agent targets the vicinity
of the gene/locus specific indicator of interest. The p53 gene is a well-studied example where the
signals split and migrate to the tail immediately after irradiation (McKenna et al. 2003; Kumaravel
and Bristow 2005). When allowed to repair for a period of time, the signals return back to the head.
More work is necessary to standardise the measurements for the Comet -FISH technique before it
is adopted for routine use.

Use of control cells in Comet Assay measurements:

In a typical Comet Assay, electrophoresis methods and differences in cell preparations create a
significant source of variation for the measurements. Such variation sometimes makes it difficult
to compare results between laboratories, and even within the same laboratory. To overcome this
problem, use of slides prepared from cells containing a known amount of DNA damage (also called
control cells) are included in every electrophoresis run. Control cells consistently produce comets
with predetermined DNA migration in the tails. Some researchers define acceptance criteria for
experiments based on DNA migration observed in control cells. If the DNA migration in control
cells does not fall within the laboratory’s historical control values, the data generated from that

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electrophoresis run are rejected. Some researchers use the data for DNA migration in control cells
to normalise with those from other samples. Control cells are produced from stable cells that have
been irradiated with known amount of gamma radiation, aliquoting them in small cryovials
followed by immediate flash freezing. There are some commercial sources of control cells such as
Trevigen®, who prepare their control cells by treating them with known concentrations of
etoposide. There was no recommendation for use of control cells in any of the IWGT workshops.
We will recommend the use of control cells in each and every electrophoresis run as a best practice
to generate high quality Comet Assay data.

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 CHAPTER: 14
Real Time-Polymerase chain Reaction
[RT-PCR]
Real-time PCR has become one of the most widely used methods of gene quantitation because it
has a large dynamic range, boosts tremendous sensitivity, can be highly sequence-specific, has
little to no post-amplification processing. The advent of real-time PCR and real-time reverse
transcription PCR (real-time RT-PCR) has changed the field of measuring gene expression. Real-
time PCR is the technique of collecting data throughout the PCR process as it occurs, thus
combining amplification and detection into a single step. This is achieved using a variety of
different fluorescent chemistries that correlate PCR product concentration to fluorescence
intensity. Reactions are characterized by the point in time (or PCR cycle) where the target
amplification is first detected. This value is usually referred to as cycle threshold (Ct), the time at
which fluorescence intensity is greater than background fluorescence. Consequently, the greater
the quantity of target DNA in the starting material, the faster a significant increase in fluorescent
signal will appear, yielding a lower Ct.

There are many benefits of using real-time PCR over other methods to quantify gene expression.
It can produce quantitative data with an accurate dynamic range of 7 to 8 log orders of magnitude
and does not require post-amplification manipulation. Real-time PCR assays are 10,000- to
100,000-fold more sensitive than RNase protection assays (1000-fold more sensitive than dot blot
hybridization and can even detect a single copy of a specific transcript. In addition, real-time PCR
assays can reliably detect gene expression differences as small as 23% between samples and have
lower coefficients of variation (cv; SYBR® Green at 14.2%; TaqMan® at 24%) than end point
assays such as band densitometry (44.9%) and probe hybridization (45.1%). Real-time PCR can
also discriminate between messenger RNAs (mRNAs) with almost identical sequences, requires
much less RNA template than other methods of gene expression analysis, and can be relatively
high-throughput given the proper equipment. The major disadvantage to real-time PCR is that it
requires expensive equipment and reagents. In addition, due to its extremely high sensitivity, sound
experimental design and an in-depth understanding of normalization techniques are imperative for
accurate conclusions. The general steps performed during a real-time PCR experiment, from RNA
isolation to data analysis, are outlined in Figure.

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 Figure: Steps for measuring gene expression using real-time PCR.

RNA is first isolated and characterized for quantity and integrity. If performing a one-step reaction,
RNA is used as a template for the real-time PCR assay, and reverse transcription occurs during the
assay. During a two-step reaction, cDNA is first synthesized and then used as a PCR template. The
steps performed on the real-time PCR machine are shown in blue, the time during which raw
fluorescence data are collected, adjusted, and manipulated to generate the output data used for
analysis. For normalizing results with multiple housekeeping genes, a normalization factor must
be calculated for each individual sample. Dividing the fluorescent data by its normalization factor
produces the normalized data, which is followed by statistical analysis.

Theory of Real-Time PCR

PCR can be broken into four major phases (Figure 2): the linear ground phase, early exponential
phase, log-linear (also known as exponential) phase, and plateau phase (9). During the linear
ground phase (usually the first 10–15 cycles), PCR is just beginning, and fluorescence emission at
each cycle has not yet risen above background. Baseline fluorescence is calculated at this time. At
the early exponential phase, the amount of fluorescence has reached a threshold where it is
significantly higher (usually 10 times the standard deviation of the baseline) than background
levels. The cycle at which this occurs is known as Ct in ABI PRISM® literature (Applied
Biosystems, Foster City, CA, USA) or crossing point (CP) in LightCycler® literature (Roche
Applied Science, Indianapolis, IN, USA) (2,10). This value is representative of the starting copy
number in the original template and is used to calculate experimental results (2). During the log-
linear phase, PCR reaches its optimal amplification period with the PCR product doubling after
every cycle in ideal reaction conditions. Finally, the plateau stage is reached when reaction

184
components become limited and the fluorescence intensity is no longer useful for data calculation
(11).

Figure 2. Phases of the PCR amplification curve.

The PCR amplification curve charts the accumulation of fluorescent emission at each reaction
cycle. The curve can be broken into four different phases: the linear ground, early exponential,
log-linear, and plateau phases. Data gathered from these phases are important for calculating
background signal, cycle threshold (Ct), and amplification efficiency. Rn is the intensity of
fluorescent emission of the reporter dye divided by the intensity of fluorescent emission of the
passive dye (a reference dye incorporated into the PCR master mix to control for differences in
master mix volume). ΔRn is calculated as the difference in Rn values of a sample and either no
template control or background, and thus represents the magnitude of signal generated during PCR.

One-Step versus Two-Step Real-Time PCR:

When quantifying mRNA, real-time PCR can be performed as either a one-step reaction, where
the entire reaction from cDNA synthesis to PCR amplification is performed in a single tube, or as
a two-step reaction, where reverse transcription and PCR amplification occur in separate tubes.
There are several pros and cons associated with each method. One-step real-time PCR is thought
to minimize experimental variation because both enzymatic reactions occur in a single tube.
However, this method uses an RNA starting template, which is prone to rapid degradation if not
handled properly. Therefore, a one-step reaction may not be suitable in situations where the same

185
sample is assayed on several occasions over a period of time. One-step protocols are also
reportedly less sensitive than two-step protocols (12).

Two-step real-time PCR separates the reverse transcription reaction from the real-time PCR assay,
allowing several different real-time PCR assays on dilutions of a single cDNA. Because the
process of reverse transcription is notorious for its highly variable reaction efficiency (13), using
dilutions from the same cDNA template ensures that reactions from subsequent assays have the
same amount of template as those assayed earlier. Data from two-step real-time PCR is quite
reproducible with Pearson correlation coefficients ranging from 0.974 to 0.988 (14). A two-step
protocol may be preferred when using a DNA binding dye (such as SYBR Green I) because it is
easier to eliminate primer-dimers through the manipulation of melting temperatures (Tms) (14).
However, two-step protocols allow for increased opportunities of DNA contamination in real-time
PCR.

Types of Real-Time Quantification

Absolute Quantitation

Absolute quantitation uses serially diluted standards of known concentrations to generate a

standard curve. The standard curve produces a linear relationship between Ct and initial amounts
of total RNA or cDNA, allowing the determination of the concentration of unknowns based on
their Ct values. This method assumes all standards and samples have approximately equal
amplification efficiencies. In addition, the concentration of serial dilutions should encompass the
levels in the experimental samples and stay within the range of accurately quantifiable and
detectable levels specific for both the real-time PCR machine and assay.

The PCR standard is a fragment of double-stranded DNA (dsDNA), single-stranded DNA

(ssDNA), or cRNA bearing the target sequence. A simple protocol for constructing a cRNA
standard for one-step PCR can be found in Fronhoffs et al., while a DNA standard for two-step
real-time PCR can be synthesized by cloning the target sequence into a plasmid, purifying a
conventional PCR product, or directly synthesizing the target nucleic acid. The standard used must
be a pure species. DNA standards have been shown to have a larger quantification range and
greater sensitivity, reproducibility, and stability than RNA standards. However, a DNA standard
cannot be used for a one-step real-time RT-PCR due to the absence of a control for the reverse
transcription efficiency.

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Relative Quantitation

During relative quantitation, changes in sample gene expression are measured based on either an
external standard or a reference sample, also known as a calibrator. When using a calibrator, the
results are expressed as a target/reference ratio. There are numerous mathematical models
available to calculate the mean normalized gene expression from relative quantitation assays.
Depending on the method employed, these can yield different results and thus discrepant measures
of standard error. Table shows a comparison of the different methods, with an explanation of each
method to follow.

Amplification efficiency

Amplification efficiency of the reaction is an important consideration when performing relative

quantitation. Past methods of calculating gene expression have assumed the amplification
efficiency of the reaction is ideal, or 1, meaning the PCR product concentration doubles during
every cycle within the exponential phase of the reaction (24). However, many PCRs do not have
ideal amplification efficiencies, and calculations without an appropriate correction factor may
overestimate starting concentration (22). Current mathematical models make assumptions of
reaction kinetics and usually require its accurate measurement (7,21,22,25,26).Traditionally,

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amplification efficiency of a reaction is calculated using data collected from a standard curve with
the following formula (27):

The amplification efficiency of the reaction varies from being relatively stable in the early
exponential phase and gradually declining to zero. This decay is due to the depletion of PCR
components, the decline of polymerase activity, and competition with PCR products. Calculation
of amplification efficiency using a standard curve is not reflective of this changing efficiency and
may overestimate efficiencies. Because PCR results are based on Ct, which are determined very
early in the exponential phase of the reaction, these differences in amplification efficiency usually
generate only minor differences in Ct value. Nonetheless, after 26 cycles, a 5% difference in
amplification efficiency can result in a 2-fold difference of PCR product concentration .

There are several alternate methods of calculating amplification efficiency based on raw data
collected during PCR (7,9,22,25,30). During the exponential phase, the absolute fluorescence
increase at each PCR cycle for each individual sample reflects the true reaction kinetics of that
sample. Consequently, data collected during the exponential phase can be log-transformed and
plotted with the slope of the regression line representing the sample's amplification efficiency. In
the Liu and Saint (22) method, the individual researcher designates which cycles have exponential
characteristics, while the method proposed by Tichopad et al. (9) uses a statistical calculation to
define the period of exponential growth. Amplification efficiency calculated from raw data
analysis is reportedly more accurate than when derived from a standard curve (9,25).

Standard curve method for relative quantification

The quantity of each experimental sample is first determined using a standard curve and then
expressed relative to a single calibrator sample. The calibrator is designated as 1-fold, with all
experimentally derived quantities reported as an n-fold difference relative to the calibrator.
Because sample quantity is divided by calibrator quantity, standard curve units are eliminated,
requiring only the relative dilution factors of the standards for quantification. This method is often
applied when the amplification efficiencies of the reference and target genes are unequal. It is also
the simplest method of quantification because it requires no preparation of exogenous standards,
no quantification of calibrator samples, and is not based on complex mathematics. However,
because this method does not incorporate an endogenous control (usually a housekeeping gene),
results must still be normalized.

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Comparative Ct (2-ΔΔCt) method

The comparative Ct method is a mathematical model that calculates changes in gene expression as
a relative fold difference between an experimental and calibrator sample. While this method
includes a correction for noni deal amplification efficiencies, the amplification kinetics of the
target gene and reference gene assays must be approximately equal because different efficiencies
will generate errors when using this method. Consequently, a validation assay must be performed
where serial dilutions are assayed for the target and reference gene and the results plotted with the
log input concentration for each dilution on the x-axis, and the difference in Ct (target-reference)
for each dilution on the y-axis. If the absolute value of the slope of the line is less than 0.1, the
comparative Ct method may be used. The PCR product size should be kept small (less than 150
bp) and the reaction rigorously optimized. Because the comparative Ct method does not require a
standard curve, it is useful when assaying a large number of samples since all reaction wells are
filled with sample reactions rather than standards.

Pfaffl model

The Pfaffl model combines gene quantification and normalization into a single calculation. This
model incorporates the amplification efficiencies of the target and reference (normalization) genes
to correct for differences between the two assays. The relative expression software tool (REST©),
which runs in Microsoft® Excel, automates data analysis using this model. REST uses the Pairwise
Fixed Reallocation Randomization Test© to calculate result significance and will indicate if the
reference gene used is suitable for normalization.

Q-Gene

Q-Gene is a fully comprehensive Microsoft Excel-based software application that aids in the entire
process of a real-time PCR experiment, from experimental planning and setup through data
analysis and graphical presentation. Q-Gene calculates the mean normalized gene expression with
standard errors using two different mathematical models, both correcting for amplification
efficiencies. The calculated expression values are then compared between two matched groups to
determine the expression of a sample relative to a calibrator. The program also includes several
statistical tests such as the paired or unpaired Student's t-test, a Mann-Whitney U-test, Wilcoxon
signed-rank test, together with Pearson's correlation analysis to fully assess the significance of
experimental results. When running large or complex real-time PCR experiments, having an
organized and automated method such as Q-Gene can significantly expedite data processing and
management.

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Gentle et al

Gentle et al., designed one of the first models in which both fold changes between samples and
amplification efficiencies of experimental versus control samples are calculated without the use of
standard curves. Linear regression analyses of the mean of the raw log fluorescence data collected
during the exponential phase of the PCR are used to calculate the amplification efficiency of each
sample. By graphing the control and experimental samples together, they show that the vertical
distance between the control and experimental lines is the log of the fold difference between the
two, with the slopes of the lines representing the log of their amplification efficiencies.

Liu and Saint

Liu and Saint developed a sigmoidal mathematical model to quantitate and normalize gene
expression. Similar to Gentle et al., this method calculates amplification efficiencies from the
actual slope of the amplification plot rather than a standard curve. The authors found this method
was more accurate than the comparative Ct method with regard to the varying amplification
efficiency throughout the PCR because the user defines which PCR cycles experience exponential
growth and are used for the calculation.

Amplification plot method

The amplification plot method uses a simple algorithm to calculate the amplification efficiency of
every sample individually within the real-time PCR assay. These data are then used in the
calculation for expression quantitation. To ease data handling, Peirson et al., have developed a
Microsoft Excel workbook entitled Data Analysis for Real-Time PCR (DART-PCR) that quickly
calculates all results from raw data.

Absolute or Relative Quantitation: Pros and Cons

Absolute quantitation is considered to be more labor-intensive than relative quantitation because

of the necessity to create reliable standards for quantitation and include these standards in every
PCR. However, when performing relative quantitation, the data (Ct) used for comparison are
arbitrary values and only applicable to the samples run within the same PCR. To compare samples
between two different PCRs, it is necessary to include a reference control in every plate or run. In
cases where data compared are assayed on different days or in different laboratories, absolute
quantitation may be preferred because results are based on a constant. In terms of fold-change data,
absolute and relative quantitation methods produce comparable results.

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Controls

There are several types of controls that ensure the integrity of every step of the real-time PCR
process. DNA contamination in the sample may be accounted for with a minus reverse
transcription control. However, when one has numerous samples, an alternate method to prevent
the detection of genomic DNA is to design the target PCR product to span an exon/exon boundary.
Variation in the efficiency of the reverse transcriptase as well as the amount of RNA added into
the reaction can be accounted for using an endogenous control, which is a nucleic acid already
present in an individual sample. The use of endogenous controls is discussed in detail in the section
entitled Normalization. PCR master mix volume has been shown to be a factor in PCR
amplification efficiency such that differences in master mix volume in reactions using the same
amount of starting template have different amplification efficiencies. A passive reference dye
(such as ROX) is often included in the master mix to account for subtle differences in PCR master
mix volumes as well as non-PCR-related fluctuations in fluorescence signal. Problems with the
PCR master mix itself can be accounted for using an exogenous control, which is a synthesized
construct of characterized RNA or DNA spiked into each reaction.

Normalization

Normalization of gene expression data is used to correct sample-to-sample variation. Starting

material obtained from different individuals usually varies in tissue mass or cell number, RNA
integrity or quantity, or experimental treatment. Under ideal conditions, mRNA levels can be
standardized to cell number, but when using whole tissue samples, this type of normalization is
impossible. Therefore, real-time PCR results are usually normalized against a control gene that
may also serve as a positive control for the reaction. The ideal control gene should be expressed
in an unchanging fashion regardless of experimental conditions, including different tissue or cell
types, developmental stage, or sample treatment. Because there is no one gene that meets this
criterion for every experimental condition, it is necessary to validate the expression stability of a
control gene for the specific requirements of an experiment prior to its use for normalization.

Housekeeping genes (mRNA)

Traditionally, genes thought to have stable expression have been employed as controls in gene
expression assays. Due to the increased sensitivity and dynamic range of real-time PCR over
traditional quantitation techniques, many of the well-known housekeeping genes such as GAPDH
and β-actin have been shown to be affected by different treatments, biological processes, and even
different tissues or cell types. Consequently, normalization with a single housekeeping gene can
falsely bias results. When using a housekeeping gene for normalization, it is absolutely imperative

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to validate its stability with one's own samples rather than relying on previously published
materials.

Ribosomal RNA (rRNA)

rRNA is another possible reference gene for normalization. Of the two main rRNAs, 28S and 18S,
28S is considered more representative of mRNA integrity because 18S may remain intact in
samples with degraded mRNA. There are several problems with using 28 S rRNA to normalize
mRNA gene measurements. rRNAs are transcribed with a different polymerase than mRNAs, so
changes in polymerase activity may not affect both types of RNA expression equally. This is likely
reflected in the fact that rRNA expression tends to be less affected by treatments that significantly
alter mRNA expression . Varying ratios of rRNA to mRNA have been reported and, given the
extreme abundance of 28S rRNA in a total RNA sample [in a 10-µg total RNA sample, on average
2 µg are 18S rRNA and 5.5 µg are 28S rRNA (Technical Bulletin #151,
www.ambion.com/techlib/tb/tb_151.html;], it may be impossible to accurately measure both 28S
and a rare transcript in the same RNA or cDNA dilution. Lastly, rRNA, which lacks a poly A) tail,
cannot be measured if an oligo(dT) or gene-specific primer has been used for reverse transcription.

Total RNA

Gene expression measurements may be normalized against total RNA concentration. RNA
quantitation can be performed via RiboGreen® RNA (Molecular Probes, Eugene, OR, USA)
quantification or the Agilent 2100 Bio Analyzer (Agilent Technologies, Palo Alto, CA, USA);
spectrophotometry may not have the sensitivity and accuracy required for this measurement. There
are several inherent problems with this approach: total RNA levels are affected by cellular
processes, RNA quality and reverse transcription efficiency are not considered, normalization is
only as accurate as the RNA quantification and, in situations where RNA is extracted from a micro
dissected tissue, all recovered RNA may be needed for the real-time PCR assay itself.

Multiple mRNAs

Given the many disadvantages of using cell number, mRNA, rRNA, or total RNA for
normalization purposes, a new method of employing multiple housekeeping genes has emerged to
minimize these problems. Multiple housekeeping genes are assayed and a normalization factor is
calculated from the geometric mean of their expression levels. In this method, the expression
stability of several different housekeeping genes in the samples of interest are measured to identify
the genes most suitable for an individual experiment. Microarray results may be exploited to
identify potential normalization candidates. A list of housekeeping genes can be found in

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Vandesompele et al., The expression stability of candidate control genes is determined with either
geNorm or BestKeeper, which are both Microsoft Excel applets that estimate gene stability
through numerous pair-wise comparisons. GeNorm can be downloaded at
medgen.ugent.be/∼jvdesomp/genorm, and BestKeeper can be downloaded at www.gene-
quantification.info. While the use of multiple housekeeping genes may be the most labor-intensive
method, it is also the most conservative method of data normalization.

Detection Chemistries

A diagram of all real-time PCR detection chemistries discussed in this review can be seen in Figure
3, with a comparison of their characteristics in Table 2.

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Figure : Real-time PCR detection chemistries.

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Probe sequences are shown in blue while target DNA sequences are shown in black. Primers are
indicated by horizontal arrowheads. Not all unlabeled PCR primers are shown. Oligo,
oligonucleotide.

DNA Binding Dyes

DNA binding dyes emit fluorescence when bound to dsDNA (Figure 3A). As the double-stranded
PCR product accumulates during cycling, more dye can bind and emit fluorescence. Thus, the
fluorescence intensity increases proportionally to dsDNA concentration. This technique is very
flexible because one dye can be used for different gene assays. Consequently, multiplexing
reactions is not possible. Because DNA binding dyes do not bind in a sequence-specific manner,
these assays are prone to false positives. Accurate results demand a specific PCR, which can be
confirmed via dissociation curve analysis, where the presence of different PCR products is
reflected in the number of first-derivative melting peaks or gel analysis. A protocol for SYBR
Green I PCR master mix can be found in Ramos-Payen et al.

Hybridization Probes

Hybridization probes can be utilized in either a four or three oligonucleotide manner (Figure). The
four oligonucleotide method consists of two PCR primers and two sequence-specific probes that
bind adjacent to each other in a head-to-tail arrangement. The upstream probe is labeled with an
acceptor dye on the 3′ end, and the downstream probe with a donor dye on the 5′ end, allowing the

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donor and acceptor fluorophores to experience an increase in fluorescence resonance energy
transfer (FRET) when bound. The three oligonucleotide method is similar to the four
oligonucleotide method, except that the upstream PCR primer is labeled with an acceptor dye on
the 3′ end, and thus replaces the function of one of the probes from the four oligonucleotide
method.

In both cases, the downstream probe can be designed to cover a mutation site and discriminate
between known alleles and detect new alleles simultaneously. Alleles are identified and
differentiated via dissociation curve. A single melting curve can distinguish up to four different
Tms, and six differently labeled probes may be multiplexed, theoretically allowing a run of 24
assays in a single tube. While multiplex reactions are theoretically a simple way to increase the
efficiency of data collection, in reality it is a very technically challenging process that requires
extensive optimization to ensure that reactions do not compete with each other.

Hydrolysis Probes

Hydrolysis probes, exemplified by the TaqMan chemistry, also known as 5′ nuclease assay,
fluoresce upon probe hydrolysis to detect PCR product accumulation (Figure 3C). The sequence-
specific probe is labeled with a reporter dye on the 5′ end and a quencher dye on the 3′ end, which
allows the quencher to reduce the reporter fluorescence intensity by FRET when the probe is intact.
While both hydrolysis and hybridization probes rely on FRET to alter the intensity of fluorescence
emission, the energy transfer works in opposite manners in these two chemistries. FRET reduces
fluorescence intensity in hydrolysis probes and increases intensity in hybridization probes. When
annealed to the target sequence, the bound and quenched probe will be degraded by the DNA
polymerase's 5′ nuclease ability during the extension step of the PCR. Probe degradation allows
for separation of the reporter from the quencher dye, resulting in increased fluorescence emission.

Minor groove binders (MGBs), such as dihydrocyclopyrroloindole tripeptide (DPI3), may be

added to these probes to increase their Tm and allow the use of a shorter probe. These probes are
not only less expensive to produce but have reduced background fluorescence and a larger dynamic
range due to increased efficiency of reporter quenching.

Hairpin Probes

Molecular beacons Consisting of a sequence-specific region (loop region) flanked by two inverted
repeats, molecular beacons are the simplest hairpin probe (Figure 3D). Reporter and quencher dyes
are attached to each end of the molecule, causing a reduction in fluorescence emission via contact
quenching (FRET) when the beacon is in hairpin formation (free in solution). When bound to the

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target, the quencher and reporter are separated, allowing reporter emission. Hairpin probes tend to
have greater specificity than linear probes because the probe-target complex must be
thermodynamically more stable than the hairpin structure itself, a property often exploited for
allele discrimination. To increase fluorescence emission, “wavelength-shifting molecular
beacons” have been developed, which fluoresce in a number of colors from a single
monochromatic light source.

Scorpions

Scorpions combine the detection probe with the upstream PCR primer and consist of a fluorophore
on the 5′ end, followed by a complementary stem-loop structure (also containing the specific probe
sequence), quencher dye, DNA polymerase blocker (a nonamplifiable monomer that prevents
DNA polymerase extension), and finally a PCR primer on the 3′ end. The probe sequence
contained within the hairpin allows the scorpion to anneal to the template strand, which separates
the quencher for the fluorophore and results in increased fluorescence. Because sequence-specific
priming and probing is a unimolecular event, scorpions perform better than bimolecular methods
under conditions of rapid cycling such as the Light Cycler. Cycling is performed at a temperature
optimal for DNA polymerase activity instead of the reduced temperature necessary for the 5′
nuclease assay. Scorpions are specific enough for allele discrimination and may be multiplexed
easily.

The scorpion chemistry has been improved with the creation of duplex scorpions in which the
reporter dye/probe and quencher fragment are on separate, complementary molecules. The duplex
scorpions still bind in a unimolecular event, but because the reporter and quenchers are on separate
molecules, they yield greater signal intensity because the reporter and quencher can separate
completely.

Sunrise™ primers

Created by Oncor (Gaithersburg, MD, USA), Sunrise primers are similar to scorpions in that they
combine both the PCR primer and detection mechanism in the same molecule (Figure). These
probes consist of a dual-labeled (reporter and quencher fluorophores) hairpin loop on the 5′ end,
with the 3′ end acting as the PCR primer. When unbound, the hairpin is intact, causing reporter
quenching via FRET. Upon integration into the newly formed PCR product, the reporter and
quencher are held far enough apart to allow reporter emission.

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LUX™ fluorogenic primers

Light upon extension (LUX) primers (Invitrogen, Carlsbad, CA, USA) are self-quenched single-
fluorophore labeled primers almost identical to Sunrise primers (Figure). However, rather than
using a quencher fluorophore, the secondary structure of the 3′ end reduces initial fluorescence to
a minimal amount. Because this chemistry does not require a quencher dye, it is much less
expensive than dual-labeled probes. While this system relies on only two oligonucleotides for
specificity, unlike the SYBR Green I platform in which a dissociation curve is used to detect
erroneous amplification, no such convenient detection exists for the LUX platform. Agarose gels
must be run to ensure the presence of a single PCR product, a step that is extremely important not
only for the LUX primers but also for the Sunrise primers and scorpions because PCR is priming
and probe binding are not independent in these chemistries.

Causes of Variation

In theory, PCR is quite robust and predictable, but in actuality, minor variations in reaction
components, thermal cycling conditions, and mispriming events during the early stages of the
reaction can lead to large changes in the overall amount of amplified product. Due to the high
sensitivity of the real-time PCR assay and the numerous steps that may introduce experimental
error, awareness of the causes of variation helps produce the most accurate data possible.

Whether using a one- or two-step process, cDNA synthesis can greatly affect the overall real-time
PCR results. Both reverse transcriptase enzyme and dithiothreitol (DTT) are PCR inhibitors that
may affect reaction kinetics in a one-step process or when carried over during a two-step reaction.
In addition, many samples from complex biological sources often have other PCR inhibitors that
may be carried over during sample preparation. Inhibitor carryover can be avoided using a cDNA
precipitation protocol, while DTT may be omitted from the reaction.

The oligonucleotides used for reverse transcription priming affect overall cDNA levels. Gene-
specific primers yield the most efficient reaction, oligo (dT) primers have an intermediate
efficiency, and random hexamers are the least efficient. Gene-specific priming is often not ideal
because one cannot assay both a target and a normalization gene from the same cDNA template,
while with oligo (dT) priming, one may not effectively transcribe the 5′ end of long transcripts.
The use of random and specific hexamers has been reported to overestimate mRNA copy number
up to 19-fold and 4-fold, respectively, in comparison to 22-mer gene-specific primers (64).
Consequently, one solution is to use a mixture of both oligo (dT) and random hexamer primers
during the reverse transcription reaction.

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The structure and concentration of the RNA template and the reverse transcriptase enzyme itself
are other sources of variation during cDNA synthesis. RNA secondary structure and protein
complexes present on the target RNA can interfere with the reaction by causing enzyme pausing,
dissociation, or skipping over looped regions. Raising reaction temperature above 47°C may
minimize this problem (65). Different reverse transcriptase enzymes have differing abilities to read
through secondary structure. For example, SuperScript™ RT II (Invitrogen) has greater efficiency
and accuracy than Sensiscript® (Qiagen, Valencia, CA, USA).

As mentioned in the Normalization section, the biological sample itself is often a source of much
variation. In cases where whole tissue is assayed, measuring several different cell types within a
single sample yields an average expression value of the different cell types. Techniques such as
laser-capture microdissection (LCM) may be utilized to extract a pure subpopulation of cells from
a heterogeneous source.

Variation during PCR can be incurred from several sources including assay design, PCR reagents,
PCR equipment, and human error. Assay design, particularly primer stability and specificity as
well as PCR product size, is crucial for an accurate result because amplification efficiency can
greatly affect overall results. When using a block thermal cycler versus capillary tubes, it is
important to measure any positional effects because slight variations in temperature when
measuring fluorescence can lead to a variation in the amount detected, especially when using a
DNA binding dye. If a service contract is used to maintain the realtime PCR machine, these effects
are usually monitored as part of the routine maintenance. Variation in annealing temperature can
also affect the enzymatic ability of the polymerase, primer binding, and formation or melting of
secondary structure, etc., all which have compounding effects on the overall PCR.

Variation can occur from the PCR reagents even when using premade master mixes from the same
manufacturer. Bustin reported a significant Ct value difference from a single template assayed with
two different batches of the TaqMan EZ RT-PCR system (a one enzyme/tube system; Applied
Biosystems) master mix that translated into a 2.5-fold difference in median mRNA copy number.
Different probe lots synthesized within 6 months of each other also generated significant
differences in Ct value, resulting in a 7-fold difference in mRNA copy number. Probes
manufactured from different sources vary in stability. Bustin reported that Applied Biosystems
produces the most stable probes.

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Nevertheless, the most likely source of variation is the person performing the experiment. Three
different people used the same micropipettes, master mix, laboratory, template, and machine (ABI
PRISM 7700; Applied Biosystems) to quantitate the same target and found initial copy numbers
ranging from 8.7×105 to 2.7×103. Even the most careful pipetting technique may have a 1%
relative error. With a 10-fold dilution, this original error will result in a 1% error in amplification
efficiency. Consequently, precision pipetting and pipet calibration are also essential for preventing
cumulative error. Running a standard curve during every reaction can help alleviate this issue
because the standard will be affected to the same extent as the unknowns. Using the same batch of
enzymes, buffers, master mixes, pipets, and especially the same person will all help reduce
experimental variability.

Calculating Variation

Because experimental variation is unavoidable, it is important to validate assay results by

measuring intra- and inter-assay variation. Variation should not be calculated using Ct values
because these are logarithmic units and will misrepresent true variability. Therefore, data used for
calculation must be a linear value (such as copy number) to obtain accurate measurements of
coefficients of variation.

Intra-assay variation quantifies the amount of error seen within a single assay when the same
template is run multiple times on the same plate with the same reagents. Intra-assay variation can
be calculated for every single sample of every reaction if the real time PCR experiments are
performed in triplicate, with a pooled variance for all sets of PCR triplicates representing statistical
power. This variation is thought to be both primer and template dependent, with lower
concentrations of starting template tending to have higher intra-assay variability. PCR
reproducibility is influenced by distribution statistics and stochastic effects (Poisson's Law).
However, several reports have found no correlation between initial template copy number and
overall variability.

Inter-assay variation should be quantified in cases where comparisons are made of results from
two separate assays run on either the same or different days. Variation can be measured by running
the same sample on every plate used during a single experiment. This calculation may often be
performed using data from either a calibrator or standard sample because these are often already
included on all plates.

Given the number of choices available for every aspect of real-time PCR, it may be difficult to
determine what detection chemistry, quantitation method, normalization gene, etc., to use.

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Although every experimental situation is unique and requires specialized consideration, some
general guidelines can be suggested. In terms of quantitation method (absolute versus relative), the
majority of users will not require absolute data such as copy number of transcripts or nanograms
of DNA, and therefore, relative quantitation will suffice. As discussed, there are many
mathematical models available for relative quantitation. Larger projects would benefit greatly by
using a method with an associated Excel worksheet such as Pfaffl, Q-Gene, or DART-PCR. While
amplification efficiency may be more efficiently calculated from raw fluorescence data instead of
a standard curve, using a set of serial dilutions is recommended not only to check the dynamic
range of the assay but also to ensure the accuracy of the quantitation. In addition, inclusion of a
standard curve would allow results to be calculated using any of the relative quantitation methods
available. The choice of detection chemistry is highly dependent on the characteristics of an
individual experiment. During the validation of microarray results, which tends to have only a few
samples and several target genes, it is reasonable to use a DNA binding dye. However, in situations
where it may be difficult to design a specific PCR (perhaps due to the presence of processed
pseudo-genes), a sequence-specific probe-based method would have increased reaction specificity.
Of the many probe-based techniques available, a well-established system such as the hybridization
TaqMan probes may be the best choice. This system has very well-written guidelines and protocols
and is fairly error-proof when designed and run according to protocol.

In terms of normalization, the use of multiple housekeeping genes is the most accurate method.
Nevertheless, when one has only a few genes to assay or a sample set with low diversity (such as
cell culture), it may not be feasible to run multiple housekeeping genes. If a single gene is used,
its stability should be validated in an assay similar to the one used to rank gene stability in geNorm.
Because real-time PCR is now a common method for measuring gene expression, it is increasingly
important for users to be aware of the numerous choices available in all aspects of this technology.
Unlike traditional PCR, there are many complexities with real-time PCR that can affect overall
results. However, with a well-designed experiment performed with the proper controls, real-time
PCR can be one of the most sensitive, efficient, fast, and reproducible methods of measuring gene
expression.

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 CHAPTER: 15
Random Amplified Polymorphic DNA (RAPD) Marker

RAPD
Randomly amplified polymorphic DNA, or RAPD, marker analysis utilizes short PCR primers
consisting of random sequences usually in the size range of 8 to 15 nucleotides in length. Complex
patterns of PCR products are generated as these random sequence primers anneal to various
regions in an organism’s genome. RAPD suffers from poor reproducibility between laboratories
largely because of the requirement of consistent PCR amplification conditions including thermal
cycler ramp speeds. The complex patterns of RAPD also prevent mixture interpretation and
provide challenges in consistent scoring of electrophoretic images even in single-source samples.

RAPD refers to polymorphism found within a species in the randomly amplified DNA generated
by restriction endonuclease enzyme. RAPDs are PCR based DNA markers. RAPD marker assays
are performed using single DNA primer of arbitrary sequence.

RAPD primers are readily available being universal. They provide moderately high genotyping
throughput. This technique is simple PCR assay (no blotting and no radioactivity). It does not
require special equipment. Only PCR is needed. The start-up cost is low.

How It Works:

Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not require any specific
knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will
not amplify a segment of DNA, depending on positions that are complementary to the primers'
sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the
primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at
the site that was previously complementary to the primer, a PCR product will not be produced,
resulting in a different pattern of amplified DNA segments on the gel.

Example

RAPD is an inexpensive yet powerful typing method for many bacterial species.

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Silver-stained polyacrylamide gel showing three distinct RAPD profiles generated by primer
OPE15 for Haemophilus ducreyi isolates from Tanzania, Senegal, Thailand, Europe, and North
America.

Selecting the right sequence for the primer is very important because different sequences will
produce different band patterns and possibly allow for a more specific recognition of individual
strains.

Limitations of RAPD

Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA
segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-
dominant RAPD markers, observed as different-sized DNA segments amplified from the same
locus, are detected only rarely.

PCR is an enzymatic reaction, therefore the quality and concentration of template DNA,
concentrations of PCR components, and the PCR cycling conditions may greatly influence the
outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully
developed laboratory protocols to be reproducible.

Mismatches between the primer and the template may result in the total absence of PCR product
as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to
interpret.

Advantages:

RAPD marker assays can be performed using very small DNA samples (5 to 25 ng per sample).
RAPD primers are universal and can be commercially purchased. RAPD markers can be easily
shared between laboratories. Locus-specific, co-dominant PCR-based markers can be developed
from RAPD markers. It provides more polymorphism than RFLPs.

Disadvantages:

The detection of polymorphism is limited. The maximum polymorphic information content for
any bi-allelic marker is 0.5. This technique only detects dominant markers. The reproducibility of
RAPD assays across laboratories is often low. The homology of fragments across genotypes
cannot be ascertained without mapping. It is not applicable in marker assisted breeding
programme.

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Uses:

This technique can be used in various ways such as for varietal identification, DNA fingerprinting,
gene tagging and construction of linkage maps. It can also be used to study phylogenetic
relationship among species and sub-species and assessment of variability in breeding populations.

Applications of RAPD marker in plants. Also learn about its demerits.

1. RAPD is used to distinguish between variety is based on difference in DNA sequence. RAPD
have been used to identify nearly 15 commercial sunflower varieties. The new bean varieties
(Phaseolus vulgaris) which are difficult to distinguish based on mor-phological trait have been
used as ideal candidate for the application of RAPD marker methods when DNA was extracted
from each varieties. The 12 samples were analysed using 60 primers. This produced 296 markers
and that could be scored. Al-most 85% similarity was predicted. For example, DNA from plant
allows the ampli-fication of sequences a, c, d but not b. This indicates that in plant 1, primer sites
for the primers used not found at sequence b. Similarly, a DNA sequence alternation at one of the
primer binding site (priming) for a sequence ‘a’ has prevent it from being amplified when DNA
from plant 2 is used.

RAPD have been extensively used for number of horticultural crops in variety iden-tification,
genetic purity and sex determination. A specific RAPD marker has been used to select for high
and low β-glucan content between barley varieties.

2. RAPD markers are employed in the construction of genetic maps. Genetic maps of several plants
including model plant Arabidopsis and tobacco have been constructed. RAPD markers have been
used to construct 15 linkage groups in coffee. Both genomic and chloroplast DNA provided the
source of probes.

RAPD markers are used for the selection of segregating populations more or less indirectly, during
plant breeding. These markers also accelerate back crossing process and allow the selection of
individually with more of recurrent genome at each generation facilitate breeding programme to
be completed within few generations.

3. RAPD molecular marker used in the direct selection of desirable trait. Molecular marker linked
to the trait of interest can be screened for at any stage in the breeding programme.

4. RAPD and other molecular markers have great value in the selection for desirable trait in long-
lived species which takes long time for maturity and show phenotypic character. For example,
avacado (Persea americana) fruit quality can be assessed in seedling itself using RAPD molecular
marker.

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5. RAPD markers have been used to identify several disease resistant genes in plants. The rp94
gene is responsible for resistance to stem rust (Puccinia gramnis) in barley. RAPD markers
identified to link to this gene. Similarly, RAPD markers linked to heat smut resistance gene have
been characterized. Controlling of height in barley plant by specific gene has been used to locate
dwarfism gene by RAPD marker.

6. In tissue culture work, somatic hybrids involving protoplast fusion requires thorough screening.
However, screening of somatic hybrid is cumbersome. Therefore, RAPD markers can be exploited
in identifying somatic hybrids. RAPD analysis provides an important tool for the characterization
of biodiversity.

Identification of areas rich in endemic genotypes helps in habitat conservative and prevents species
extinction. Molecular analysis of genetic diversity using RAPD or RFLP in plant genetic
germplasm collection facilitate better management especially space and resources are serious
constraints.

RAPD analysis has been used for the identification of duplicates in germplasm. These duplicates
are then discarded once no morphological differences were detected. RAPD analysis has been
implicated in the analysis of rice genome collections held at the International Rice Research
Institute, Phillipines.

Genetic diversity was carried out in a set a 63 tetraploid wheat genotype. Which comprises 24
duran land races, 18 duran cultivars and nine diococcum cultivars, two wild tetraploid species?
The duran and dicoccum wheat genotypes are part of the germplasm used in Indian tetraploid
wheat breeding programme.

RAPD scoring analysis reveals 78% were polymorphic in different categories of Indian tetraploid
wheat. These indicate that RAPD diversity data can be used in breeding improved cultivars and
maintaining genetic diversity in germplasm. Similarly RAPD markers were generated from 6
groups of 23 varieties of Tibetian barley. Nearly 23 RAPD and 29 genes loci were identified on
72 chromosomes.

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RAPD have also been used in variety identification and purity in grain processing in food industry.
For example, particular duran variety of wheat is used in the prepara-tion of food products.
Contamination of other variety can be identified by using these molecular markers.

Demerits of RAPD:

1. Constraints about reproducibility of results.

2. Since RAPD markers are dominant, only half the genetic informations are co-domi-nant
markers.

3. Null alleles not directly detected.

References:

» Mbwana J, et al. Molecular characterization of Haemophilus ducreyi isolates from different

geographical locations. J Clin Microbiol. 2006 Jan;44(1):132-7. PMID: 16390960

» Williams JG, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic
markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531-5. PMID: 1979162

https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/

John M. Butler, in Advanced Topics in Forensic DNA Typing: Methodology, 2012

Patricia J. Simner, ... Nancy L. Wengenack, in Molecular Medical Microbiology (Second Edition),
2015

https://www.biologydiscussion.com/dna/dna-markers/top-10-types-of-dna-markers-
genetics/37969#Random_Amplified_Polymorphic_DNA_RAPDs

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/rapd

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 CHAPTER: 16
Restriction Fragment Length Polymorphism
(RFLPs)
The eukaryotic genome is very large and there is no simple way to observe genetic polymorphisms
of individual genes or sequences. The property of complementary base pairing allowed for method
to be developed where by small pieces of DNA is used as molecular probes to reveal
polymorphisms in just the sequences homologous to the molecular probe. The genetic systems
based on DNA-DNA hybridization were developed during beginning of 1980s and by using this
approach RFLPs genetic marker system developed.

Introduction:

Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English
scientist Alec Jeffrey’s during research into hereditary diseases. It is used for the analysis of unique
patterns in DNA fragments in order to genetically differentiate between organisms. these patterns
are called Variable Number of Tandem Repeats (VNTRs). Genetic polymorphism is defined as
the inherited genetic differences among individuals in over 1% of normal population. The RFLP
technique exploits these differences in DNA sequences to recognize and study both intraspecies
and interspecies variation.

RFLPs basic Principle:

Restriction endonucleases enzymes are type of genetic scissors that cut large DNA molecules into
small pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand
and therefore cuts at different sites. The distance between the cleavage sites of a certain restriction
endonuclease differs between individuals. Hence, the length of the DNA fragments produced by a
restriction endonuclease will differ across both individual organisms and species. The similarity
of the patterns generated can be used to differentiate species (even strains) from one another so

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this technique is mainly based on the special class of enzymes i.e. Restriction endonucleases. In
RFLPs, DNA polymorphism is detected by hybridizing a chemically labeled DNA probe to a
Southern blot of DNA digested by restriction endonucleases, resulting in differential DNA
fragment profile.

Process of RFLPs:

RFLP is performed using a series of steps briefly outlined below:

DNA Extraction:

The process begins with DNA extraction from different samples and purified.

DNA Fragmentation:

The purified DNA is digested using restriction endonucleases. The recognition sites of these
enzymes are generally 4 to 6 base pairs in length. The shorter the sequence recognized, the greater
the number of fragments generated from digestion. For example, if there is a short sequence of
GAGC that occurs repeatedly in a sample of DNA. The restriction endonuclease that recognizes
the GAGC sequence cuts the DNA at every repetition of the GAGC pattern. If one sample repeats
the GAGC sequence 4 times whilst another sample repeats it 2 times, the length of the fragments
generated by the enzyme for the two samples will be different.

Gel Electrophoresis:

The restriction fragments produced during DNA fragmentation are analyzed using gel
electrophoresis. The fragments are negatively charged and can be easily separated by
electrophoresis, which separates molecules based on their size and charge. The fragmented DNA
samples are placed in the chamber containing the electrophoretic gel and two electrodes. When an
electric field is applied, the fragments migrate towards the positive electrode. Smaller fragments
move faster through the gel leaving the larger ones behind and thus the DNA samples are separated
into distinct bands on the gel.

Visualization of Bands:

The gel is treated with luminescent dyes in order to make the DNA bands visible.

Formation of RFLPs:

Each organism inherits its DNA from its parents. Since DNA is replicated with each generation,
any given sequence can be passed on to next generation. An RFLP is a sequence of DNA that has
a restriction site on each end with a target sequence in between. A target sequence is any segment
of DNA that binds to a molecular probe by forming complementary base pairs. A molecular probe
is a sequence of single-stranded DNA that is tagged with radioactivity or an enzyme so that the
probe can be detected. When a probe base pairs to its target, the investigator can detect this binding

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and know where the target sequence is since the probe is detectable. RFLP produces a series of
bands when southern blot is performed with a particular combination of restriction enzyme and
probe sequence. For example, let's follow a particular RFLP that is defined by the restriction
enzyme EcoR I and the target sequence of 20 bases GCATGCATGCATGCATGCAT. EcoR I
bind to its recognition sequence GAATTC and cut the double-stranded DNA as shown:

In the segment of DNA shown below, you can see the elements of an RFLP; a target sequence
flanked by a pair of restriction sites. When this segment of DNA is cut by EcoR I, three restriction
fragments are produced, but only one contains the target sequence which can be bound by the
complementary probe sequence (purple).

Let's look at two people and the segments of DNA they carry that contain this RFLP (for clarity,
we will only see one of the two stands of DNA). Since Jack and Jill are both diploid organisms,
they have two copies of this RFLP. When we examine one copy from Jack and one copy from Jill,
we see that they are identical:

Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks
an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics).

Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-

GAATTC-

Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-

GAATTC-

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Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band
in common and one band that does not match the other's in molecular weight:

Paternity Case:

Let's use RFLP technology to determine if Jack is the father of Jill's child named Payle. In this
scenario, DNA was extracted from white blood cells from all three individuals and subjected to
RFLP analysis. The results are shown below:

In this case, it appears that Jack could be the father, since Payle inherited the 12.4 kb fragment
from Jill and the 4.3 fragment from Jack. However, it is possible that another man with similar
RFLP pattern could be as well. To be certain, several more RFLP loci would be tested. It would
be highly unlikely that two men (other than identical twins) would share multiple RFLP patterns
and so paternity could be confirmed.

In a different scenario, DNA was extracted from white blood cells from all three individuals and
subjected to RFLP analysis. The results are shown below:

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This time, it can be determined that Jack is NOT the father of Payle since Payle has a band of about
6 kb and Jack does not. Therefore, it is very probable that Payle's father is not Jack, though it is
possible that Payle carries a new mutation at this locus and a different sized band was produced.
What could you do as an investigator to be more certain that Jack was not the father of Payle?

Disease Status:

In this example, we want to know if a person carries any cystic fibrosis (CF) alleles and if so, how
many. Because CF is a recessive disease, anyone with CF must be homozygous for disease alleles.
From pedigree information, we can often determine who in this family is a carrier. However, if a
couple comes to a genetic counselor, often an RFLP analysis is performed on the couple's DNA.

RFLPs are known for CF and so it would be easy to determine if a person were homozygous wild-
type (wt), heterozygous "carrier", or homozygous disease alleles and thus have CF.

For couples expecting a child, it would be simple to test both parents and make a prediction about
the eventual disease status of their fetus. For example, if both parents were homozygous wt, then
all of their children would also be homozygous wt:

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However, if both parents were heterozygous, they could have children with any of the three
genotypes, though heterozygous children would be twice as likely as either of the homozygous
genotypes.

With increasing genomic sequence information, increasing numbers of genetic disease can be
predicted from RFLP analyses

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Genetic Mapping

To calculate the genetic distance between to loci, you need to be able to observe recombination.
Traditionally, this was performed by observing phenotypes but with RFLP analysis, it is possible
to measure the genetic distance between two RFLP loci whether they are a part of genes or not.

Let's look at a simple example in fruit flies. Two RFLP loci with two RFLP bands possible at each
locus:

These loci are located on the same chromosome for the female (left) and the male (right). The
upper locus can produce two different bands called 1 and 3. The lower locus can produce bands
called 2 or 4. The male is homozygous for band 1 at the upper locus and 2 for the lower locus. The
female is heterozygous at both loci. Thier RFLP banding patterns can be seen on the Southern blot
below:

The male can only produce one type of gamete (1 and 2) but the female can produce four different
gametes. Two of the possible four are called parental because they carry both RFLP bands from
the same chromosome; 1 and 2 from the left chromosome or 3 and 4 from the right chromosome.
The other two chromosomes are recombinant because recombination has occurred between the
two loci and thus the RFLP bands are mixed so that 1 is now linked to 4 and 3 is linked to 2.

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When these two flies mate, the frequency of the four possible progeny can be measured and from
this information, the genetic distance between the two RFLP loci (upper and lower) can be
determined.

In this example, 70% of the progeny were produce from parental genotype eggs and 30% were
produced by recombinant genotype eggs. Therefore, these two RFLP loci are 30 centiMorgans
apart from each other.

Applications of RFLPs:

RFLP has been used for several genetic analyses since its invention. Some of these applications of
RFLP are listed below:

• To determine the status of genetic diseases such as Cystic Fibrosis in an individual.

• To determine or confirm the source of a DNA sample such as in paternity tests or criminal
investigations.
• In genetic mapping to determine recombination rates that show the genetic distance
between the loci.
• To identify a carrier of a disease-causing mutation in a family.
Disadvantages of RFLPs:

RFLP has been a widely used genome analysis techniques employed in forensic science, medicine,
and genetic studies. However, it has become almost obsolete with the advent of relatively simple
and less expensive DNA profiling technologies such as the polymerase chain reaction (PCR). The

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RFLP procedure requires numerous steps and takes weeks to yield results, while techniques such
as PCR can amplify target DNA sequences in a mere few hours. Additionally, RFLP requires a
large DNA sample, the isolation of which can be a laborious and time-consuming process. In
contrast, PCR can amplify minute amounts of DNA in a matter of hours. Due to numerous reasons
such as these, the PCR technique has largely replaced RFLP in most applications requiring DNA
sequencing such as paternity testing or forensic sample analysis. Furthermore, the identification of
single-nucleotide polymorphisms in the Human Genome Project has almost replaced the need for
RFLP in disease status analysis.

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 CHAPTER: 17
ARDRA

ARDRA is the extension of RFLP to the gene encoding the 16S rRNA of bacteria (Smit et al.,
1997). The technique involves an enzymatic amplification using primers directed at the conserved
regions at the ends of the 16S gene, followed by digestion using tetracutter restriction enzymes
(e.g., AluI and HaeIII). Amplified ribosomal DNA restriction analysis (ARDRA) or PCR-RFLP
analysis of the rRNA gene(s) could be designated more briefly as ‘restriction analysis of the rRNA
gene’, but this name has been used previously for ribotyping (e.g., Grimont & Grimont, 1986;
Martinetti-Lucchini & Altwegg, 1992; see Chapter 5), which actually consists of selective
restriction fragment hybridisation with the rRNA cistron as the probe, and which has technically
and theoretically nothing in common with ARDRA (Vaneechoutte, 1996). To avoid confusion it
has been suggested that ARDRA is used to designate PCR-RFLP analysis of the rRNA genes
(Vaneechoutte et al., 1992). Adding to the confusion is the fact that ‘PCR-ribotyping’ has been
used as a name for amplification of the spacer region between the 16S and 23S rRNA genes
without subsequent restriction digestion (Kostman et al., 1992), and also as a name for ARDRA
of the complete rRNA cistron (Smith-Vaughan et al., 1995). Further confusion is caused by the
fact that ribotyping has been commercialised under the name of ‘riboprinting’ (Riboprinter;
QualiCon Europe, Birmingham, UK), a term which was already in use for ARDRA of eukaryotes
(Clark, 1993; 1997; Stothard et al., 1998) and also bacteria (Mas-Castella et al., 1996).

As a result of this nomenclatural confusion, ARDRA appears to be at present the only

unambiguous name in use to denote PCR-RFLP analysis of rRNA genes, while it also provides
the best description of the different technical aspects involved. Names like PCR-ARDRA (Giraffa
et al., 1998) are tautological. ARDREA is a variant name that has also been used for ARDRA
(Selenska-Pobell et al., 1998).

Amplified ribosomal DNA restriction analysis (ARDRA)

Amplification of 16S rRNA gene of cyanobacteria 16S rRNA gene fragment was amplified with
universal primers FD1: 5′-AGAGTTTGATCCTGGCTCAG-3′ and RP2: 5′-
ACGGCTACCTTGTTACGACTT-3′ (Weisburg et al. 1991; Lyra et al. 1997). The polymerase
chain reaction was carried out in a final volume of 25 µl, having 1X TAE buffer containing 2 mM
MgCl2, 10 mM deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP), 16S primers (FD1
and RP2) 2.5 pmol each and 1 U of Taq DNA polymerase (Fermentas, USA) and 50 ng of genomic
DNA. Amplification was achieved in a Master Cycler Gradient (Eppendorf) programmed for
initial denaturation (94 °C for 5 min) followed by 35 cycles, composed of denaturation (94 °C for
30 s), primer annealing (64 °C for 45 s), extension (72 °C for 2 min) followed by a final extension
of 5 min at 72 °C and subsequent cooling at 4 °C temperature. Amplified PCR product was
separated along with a molecular weight marker (GeneRuler, 1 kb, Fermentas, USA) by

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electrophoresis on 1.5% agarose (Vivantis, USA) gel run in 1X TAE (Tris–acetate–EDTA) buffer,
stained with ethidium bromide for a period of 1 h at 75 V. These were visualized under UV light
and gel photographs were scanned through Gel Doc System (MiniBis Bioimaging System, USA)
and the amplification product sizes were evaluated using software AlphaEaseFC (FluorChem
5500) (Alfa Innotech Corporation, USA).

Selection of restriction enzymes Initially seven restriction enzymes, viz. EcoRI, EcoRV, HindIII,
HaeI, HaeIII, HinfI and PstI were selected, but to make sure which of these may be more suitable
for analyzing the diversity, 10 16S rDNA sequences of different Nostoc strains were downloaded
from the EMBL database (Table 3) and in silico restriction digestion was carried out using the
Cleaver software (Jarman 2006). Analyzing the restriction fragments, it was found that there was
no recognition site for HaeI and HindIII (Table 3) and among the rest of the five enzymes, HinfI
and HaeIII were comparatively performing better than the others to produce differential restriction
fragmentation pattern for the selected sequences (Table 3). So, finally HinfI and HaeIII were
selected for restriction digestion of the 16S rRNA gene.

Restriction digestion of amplified 16S rRNA gene 8 µl of amplified PCR products were digested
overnight at 37 °C with 5 U of each of the restriction enzymes namely HaeIII and HinfI, procured
from New England Biolabs (Rasmussen and Svenning 2001). Digested products were separated
along with a molecular weight marker (100 bp ladder, Vivantis, USA) by electrophoresis on 3%
agarose (Vivantis, USA) gel run in 1X TAE buffer, stained with ethidium bromide for a period of
3 h at 75 V and gel photographs were visualized under UV light and gel photographs were scanned
through Gel Doc System (MiniBis Bioimaging System, USA) and the amplification product sizes
were evaluated using software AlphaEaseFC (FluorChem 5500) (Alfa Innotech Corporation,
USA).

Statistical analysis and dendrogram construction

Fingerprints generated by RAPD and ARDRA from different cyanobacterial strains were
compared and all bands were scored. The presence or absence of fragments was converted into
binary data. Pairwise genetic similarities among the genotypes under study were determined using
Jaccard’s coefficient (Jaccard 1908), J = N11/(N11 + N10 + N01), where N11 is the number of
bands present in both individuals I and j, N10 is the number of bands present in the individual i
and N01 is the number of bands present in the individual j. Cluster analyses were carried out on
similarity estimates using the unweighted pair-group method with arithmetic average (UPGMA)
using NTSYS-pc, version 1.80 (Rohlf 1995).

ARDRA

217
As expected after selecting the restriction enzymes through in silico analysis, HaeIII and HinfI
produced differential pattern of restriction fragments for all the 20 Nostoc strains studied. HaeIII
produced a total of 58 fragments ranging from 143 to 925 bp in size while HinfI produced 57
fragments ranging from 152 to 1030 bp in size (Table). Dendrogram (Fig.) based on ARDRA data
for all the 20 Nostoc strains revealed two major clusters, viz. one (MC I) with only N.
spongiaeforme (CCC110) from Cochin, Kerala, and other one (MC II) with rest of the 19 strains
of Nostoc. Degree of similarity among the Nostoc strains as revealed by ARDRA ranged from
22.5 to 100%. In MC II, there were two subclusters comprising two strains (SC I: CCC63 and
CCC139) and rest of the 17 strains (SC II). In SC II, it was found that CCC42 (Nostoc sp. from
Jammu and Kashmir) was placed in a completely separate clade and rest of the 16 strains placed
in another cluster showed more than 50% similarity among themselves. CCC92 and CCC48 were
placed in the same clade in SCII with 80% similarity. In SC II, CCC184 (Nostoc sp.) and CCC94
(N. carneum) were placed in the same clade with 100% similarity. It will be worthy to mention
here that both of these strains were geographically related as their origin belongs to Kerala.
Similarly, two Nostoc strains, viz. CCC150 and CCC282 isolated from IARI fields were also found
to be placed in the same clade with 100% similarity. N. verrucosum (CCC90) and N. piscinale
(CCC88) isolated from VIB, Nimpith, West Bengal, also showed 100% similarity and were placed
in the same clade despite of their specific designation based on morphological features. In the same
clade, N. linckia (CCC62) was also placed with 100% similarity although it was obtained from
Baharaich, UP, N. commune (CCC89) isolated from VIB, Nimpith, also showed almost 90%
similarity with the clade comprising CCC88, CCC90 and CCC62. CCC131 (N. paludosum) and
CCC151 (Nostoc sp.) isolated from IARI were also placed in same clade with 67.5% similarity.
Not for all the strains, but for some of the strains (8 strains) ARDRA analysis revealed clustering
based on geographical origin.

Table :

Restriction fragments produced by digestion with HaeIII and HinfI

Sl. Cyanob
no. acterial ARDRA
strains
As expected after selecting the restriction enzymes through in silico
analysis, HaeIII and HinfI produced differential pattern of restriction
fragments for all the 20 Nostoc strains studied. HaeIII produced a total of
58 fragments ranging from 143 to 925 bp in size while HinfI produced 57
fragments ranging from 152 to 1030 bp in size (Table 5). Dendrogram
(Fig. 3) based on ARDRA data for all the 20 Nostoc strains revealed two
major clusters, viz. one (MC I) with only N. spongiaeforme (CCC110) from
Cochin, Kerala, and other one (MC II) with rest of the 19 strains of Nostoc.

218
Degree of similarity among the Nostoc strains as revealed by ARDRA
ranged from 22.5 to 100%. In MC II, there were two subclusters
comprising two strains (SC I: CCC63 and CCC139) and rest of the 17
strains (SC II). In SC II, it was found that CCC42 (Nostoc sp. from Jammu
and Kashmir) was placed in a completely separate clade and rest of the 16
strains placed in another cluster showed more than 50% similarity among
themselves. CCC92 and CCC48 were placed in the same clade in SCII with
80% similarity. In SC II, CCC184 (Nostoc sp.) and CCC94 (N. carneum)
were placed in the same clade with 100% similarity. It will be worthy to
mention here that both of these strains were geographically related as their
origin belongs to Kerala. Similarly, two Nostoc strains, viz. CCC150 and
CCC282 isolated from IARI fields were also found to be placed in the same
clade with 100% similarity. N. verrucosum (CCC90) and N.
piscinale (CCC88) isolated from VIB, Nimpith, West Bengal, also showed
100% similarity and were placed in the same clade despite of their specific
designation based on morphological features. In the same clade, N.
linckia (CCC62) was also placed with 100% similarity although it was
obtained from Baharaich, UP, N. commune (CCC89) isolated from VIB,
Nimpith, also showed almost 90% similarity with the clade comprising
CCC88, CCC90 and CCC62. CCC131 (N. paludosum) and CCC151
(Nostoc sp.) isolated from IARI were also placed in same clade with 67.5%
similarity. Not for all the strains, but for some of the strains (8 strains)
ARDRA analysis revealed clustering based on geographical origin.

Table 5
Restriction fragments produced by digestion with HaeIII and HinfI

Sl. Cyanobacterial Fragments (bp) generated after

no. strains restriction digestion

HaeIII HinfI

1 CCC42 196, 355, 978 169, 1140

2 CCC92 193, 551, 848 137, 163, 925

219
3 CCC48 190, 567, 873 158, 284, 894

4 CCC94 186, 301,926 158, 284, 991

5 CCC184 185, 301, 925 158, 284, 994

6 CCC150 185, 301, 584 163, 284, 1026

7 CCC282 185, 301, 584 163, 288, 1030

8 CCC100 175, 285, 899 285, 326, 752

9 CCC62 175, 275, 592 169, 294, 610

10 CCC89 180, 286, 560, 175, 285, 610

11 CCC133 160, 239,825 163, 265, 1063

12 CCC90 173, 277, 590 169, 294, 610

13 CCC131 151, 233, 849 460, 1080

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14 CCC63 143, 226, 536 188, 294, 778

15 CCC125 148, 240, 779 169, 284, 470

16 CCC139 147, 247, 552 163, 569, 805

17 CCC88 173, 276, 587 163, 292, 610

18 CCC110 825, 395 530, 1010

19 CCC151 152, 269, 637 158, 265, 520

20 CCC130 636, 415 152, 304, 980

Total fragments 58 57

Open in a separate window

221
Fig. 3
Dendrogram generated using ARDRA profile of the studied Nostoc strains.
Dendrogram was constructed following the UPGMA method
Go to:

Discussion
With the advent of sequencing technologies available with the advantages
of minimal costs and less time, 16S rDNA sequencing has become the most
powerful tool to identify novel taxa for bacterial domain. Other powerful
tools like DNA–DNA hybridization (DDH), G+C contents are also equally
important for this purpose as 16S rDNA-based identification does not
guarantee species identity (Fox et al. 1992). Hence, 16S rDNA gene
sequencing to reveal intrageneric or intraspecific diversity of bacteria, may
not always be a tool of choice. PCR-based methods like RAPD, PCR–
RFLP, ERIC-PCR, REP-PCR have been widely used for analysis of
diversity in wide range of bacteria. RAPD has an advantage over most of

222
the other PCR-based methods in that it covers the whole genome as a
random primer can bind anywhere in the genome; however, reproducibility
of the RAPD data sometimes makes it inconvenient for use. Careful
optimization of the RAPD conditions and use of a sufficient number of
primers depending on the number of isolates being studied can make this
tool very useful for diversity analysis. On the other hand, the PCR–RFLP of
16S rDNA also has its own advantages and limitations. Choice of
restriction enzymes for digestion of the DNA becomes the key factor for
the analytical power of this tool.
RAPD has been a widely used tool for analyzing the genetic diversity of
various cyanobacterial groups. Casamatta et al. (2003) used RAPD to study
the genetic variability in Phormidium retzii. In 2008, Shalini and Gupta
carried out phylogenetic analysis of 30 Calothrix strains using single and
multiplex RAPD which showed its superior discriminatory power for
analyzing variability. Singh (2008) also used RAPD to analyze the genetic
diversity of Spirulina and related genera. Genetic relatedness
of Phormidium-like strains originating from distinct geographical sites was
determined using RAPD (Palinska et al. 2011). Arima et al. (2012) showed
that RAPD profiles generated through HIP primers can be useful to
distinguish among the genotypes of N. commune. Similarly, Singh et al.
(2014) also showed that HIP-based DNA fingerprinting technique could
produce strain-specific and unique banding pattern for heterocystous
cyanobacteria belonging to Subsection IV and V. Neilan (1995) used
multiplex RAPD for determining genetic heterogeneity and generating
unique identifying genetic profile of bloom-forming members of the
genera Anabaena and Microcystis. The RAPD profile generated in the
study was able to distinguish up to strain level among all the cyanobacteria
studied.
In the present study, degree of heterogeneity revealed by the RAPD was
enormous which is clear from the fact that in RAPD study, similarity
coefficient ranged from 0.125 to 0.25 (except 0.70 for CCC92 and CCC48),
while ARDRA showed similarity coefficient in the range of 0.225–1.00
where 13 strains showed more than 50% similarity among themselves.
Intra-generic or intra-specific diversity of the studied Nostoc strains has
been clearly revealed by the RAPD which indicates highly heterogeneous
nature of this genus while diversity revealed by ARDRA is very limited.
The strains (CCC94 and CCC184; CCC150 and CCC282; CCC62, CCC88
and CCC90), which showed 100% similarity in ARDRA, were distantly
related in the dendrogram generated by RAPD. The strains, viz. CCC62 (N.
linckia from Uttar Pradesh), CCC90 (N. piscinale from West Bengal) and
CCC88 (N. verrucosum from West Bengal) showing 100% similarity point
out an important and interesting fact that despite having identical 16S
rDNA sequence, the strains may not be clonal which is evident from the
different RAPD profiles of these strains. This fact is also supported by the
differential specific designation based on the morphology. However, the

223
cultured strains of cyanobacteria often show little similarity to natural
populations as some phenotypic traits are apparently not expressed under
the controlled conditions. This problem may lead to the misidentification of
cultures (Palinska et al. 1996; Wilmotte and Herdman 2001) and can reduce
the value of phylogenetic reconstructions and other analyses of
evolutionary interrelationships. Although some of the Nostoc strains
showed clustering based on geographical origin, it was not conclusive
enough to infer that the conservativeness of 16S rDNA is influenced by the
geographical region or geographically related strains are also
phylogenetically related.
An interesting fact was revealed from this study that CCC92 (N. muscorum)
and CCC48 (N. punctiforme) were genetically very closely related as
evident from the RAPD-banding profiles. However, ARDRA was able to
sufficiently discriminate between these two strains. These two strains were
reported morphologically distinct in terms of the shape of their vegetative
cells; heterocyst shape, frequency; akinete shape, size and frequency
(Chakdar and Pabbi 2012) and they were placed in two different species
due to such significant variations in morphological attributes. It appears
from the results of the present study that the taxonomic placement of these
two strains based on their morphology does not correlate with their genetic
relatedness and hence, they appear to be two different strains of the same
species; however, it needs to be clarified following a polyphasic approach.
It is clear from the present study that for analyzing the intra-generic or
intra-specific diversity of cyanobacteria, RAPD is a far better tool
compared to ARDRA. The strains which may show high similarity or may
be identical in ARDRA pattern, are not necessarily the same as they may
have significant variations distributed over the genome. Particularly, for a
genus like Nostoc which is highly diverse in nature, it will not be wise to
choose tool like ARDRA which may underestimate the diversity among the
different strains. However, other genera of cyanobacterial group may be
explored for testing the intrageneric discriminatory power of ARDRA.
In silico tools have been used for ARDRA of bacterial groups like
Phytoplasma followed by identification, but only very limited reports
(Iteman et al. 2002) are available for its use in cyanobacteria. Wei et al.
(2007) were able to classify 800 Phytoplsma isolates into 28 groups by
following the in silico approach. A careful choice of REs enabled the use of
the ARDRA technique to discriminate
among Lactobacillus, Streptococcus and Bifidobacterium at the genus level,
but not at species level (Collado and Hernandez 2007). From the present
study, we will also suggest to go for in silico ARDRA (can be done by a
number of programs) which can really reduce the efforts of the researchers
for selecting the appropriate REs for ARDRA. The availability of huge
number of 16S rDNA sequences in different databases all over the world
along with a number of freely available in silico analytical tools have
empowered the researchers to dig out more and more information with
224
 minimum effort. Not only time will be saved through this, but also the
consumables can be saved.
Fragments (bp) generated after restriction digestion
HaeIII HinfI
1 CCC42 196, 355, 978 169, 1140
2 CCC92 193, 551, 848 137, 163, 925
3 CCC48 190, 567, 873 158, 284, 894
4 CCC94 186, 301,926 158, 284, 991
5 CCC184 185, 301, 925 158, 284, 994
6 CCC150 185, 301, 584 163, 284, 1026
7 CCC282 185, 301, 584 163, 288, 1030
8 CCC100 175, 285, 899 285, 326, 752
9 CCC62 175, 275, 592 169, 294, 610
10 CCC89 180, 286, 560, 175, 285, 610
11 CCC133 160, 239,825 163, 265, 1063
12 CCC90 173, 277, 590 169, 294, 610
13 CCC131 151, 233, 849 460, 1080
14 CCC63 143, 226, 536 188, 294, 778
15 CCC125 148, 240, 779 169, 284, 470
16 CCC139 147, 247, 552 163, 569, 805
17 CCC88 173, 276, 587 163, 292, 610
18 CCC110 825, 395 530, 1010
19 CCC151 152, 269, 637 158, 265, 520
20 CCC130 636, 415 152, 304, 980
Total 58 57
frag
ment
s
Dendrogram generated using ARDRA profile of the studied Nostoc strains. Dendrogram was
constructed following the UPGMA method

Discussion

With the advent of sequencing technologies available with the advantages of minimal costs and
less time, 16S rDNA sequencing has become the most powerful tool to identify novel taxa for
bacterial domain. Other powerful tools like DNA–DNA hybridization (DDH), G+C contents are
also equally important for this purpose as 16S rDNA-based identification does not guarantee
species identity (Fox et al. 1992). Hence, 16S rDNA gene sequencing to reveal intrageneric or
intraspecific diversity of bacteria, may not always be a tool of choice. PCR-based methods like
RAPD, PCR–RFLP, ERIC-PCR, REP-PCR have been widely used for analysis of diversity in
wide range of bacteria. RAPD has an advantage over most of the other PCR-based methods in that
it covers the whole genome as a random primer can bind anywhere in the genome; however,
reproducibility of the RAPD data sometimes makes it inconvenient for use. Careful optimization
of the RAPD conditions and use of a sufficient number of primers depending on the number of
225
isolates being studied can make this tool very useful for diversity analysis. On the other hand, the
PCR–RFLP of 16S rDNA also has its own advantages and limitations. Choice of restriction
enzymes for digestion of the DNA becomes the key factor for the analytical power of this tool.

RAPD has been a widely used tool for analyzing the genetic diversity of various cyanobacterial
groups. Casamatta et al. (2003) used RAPD to study the genetic variability in Phormidium retzii.
In 2008, Shalini and Gupta carried out phylogenetic analysis of 30 Calothrix strains using single
and multiplex RAPD which showed its superior discriminatory power for analyzing variability.
Singh (2008) also used RAPD to analyze the genetic diversity of Spirulina and related genera.
Genetic relatedness of Phormidium-like strains originating from distinct geographical sites was
determined using RAPD (Palinska et al. 2011). Arima et al. (2012) showed that RAPD profiles
generated through HIP primers can be useful to distinguish among the genotypes of N. commune.
Similarly, Singh et al. (2014) also showed that HIP-based DNA fingerprinting technique could
produce strain-specific and unique banding pattern for heterocystous cyanobacteria belonging to
Subsection IV and V. Neilan (1995) used multiplex RAPD for determining genetic heterogeneity
and generating unique identifying genetic profile of bloom-forming members of the genera
Anabaena and Microcystis. The RAPD profile generated in the study was able to distinguish up to
strain level among all the cyanobacteria studied.

In the present study, degree of heterogeneity revealed by the RAPD was enormous which is clear
from the fact that in RAPD study, similarity coefficient ranged from 0.125 to 0.25 (except 0.70 for
CCC92 and CCC48), while ARDRA showed similarity coefficient in the range of 0.225–1.00
where 13 strains showed more than 50% similarity among themselves. Intra-generic or intra-
specific diversity of the studied Nostoc strains has been clearly revealed by the RAPD which
indicates highly heterogeneous nature of this genus while diversity revealed by ARDRA is very
limited. The strains (CCC94 and CCC184; CCC150 and CCC282; CCC62, CCC88 and CCC90),
which showed 100% similarity in ARDRA, were distantly related in the dendrogram generated by
RAPD. The strains, viz. CCC62 (N. linckia from Uttar Pradesh), CCC90 (N. piscinale from West
Bengal) and CCC88 (N. verrucosum from West Bengal) showing 100% similarity point out an
important and interesting fact that despite having identical 16S rDNA sequence, the strains may
not be clonal which is evident from the different RAPD profiles of these strains. This fact is also
supported by the differential specific designation based on the morphology. However, the cultured
strains of cyanobacteria often show little similarity to natural populations as some phenotypic traits
are apparently not expressed under the controlled conditions. This problem may lead to the
misidentification of cultures (Palinska et al. 1996; Wilmotte and Herdman 2001) and can reduce
the value of phylogenetic reconstructions and other analyses of evolutionary interrelationships.
Although some of the Nostoc strains showed clustering based on geographical origin, it was not

226
conclusive enough to infer that the conservativeness of 16S rDNA is influenced by the
geographical region or geographically related strains are also phylogenetically related.

An interesting fact was revealed from this study that CCC92 (N. muscorum) and CCC48 (N.
punctiforme) were genetically very closely related as evident from the RAPD-banding profiles.
However, ARDRA was able to sufficiently discriminate between these two strains. These two
strains were reported morphologically distinct in terms of the shape of their vegetative cells;
heterocyst shape, frequency; akinete shape, size and frequency (Chakdar and Pabbi 2012) and they
were placed in two different species due to such significant variations in morphological attributes.
It appears from the results of the present study that the taxonomic placement of these two strains
based on their morphology does not correlate with their genetic relatedness and hence, they appear
to be two different strains of the same species; however, it needs to be clarified following a
polyphasic approach.

It is clear from the present study that for analyzing the intra-generic or intra-specific diversity of
cyanobacteria, RAPD is a far better tool compared to ARDRA. The strains which may show high
similarity or may be identical in ARDRA pattern, are not necessarily the same as they may have
significant variations distributed over the genome. Particularly, for a genus like Nostoc which is
highly diverse in nature, it will not be wise to choose tool like ARDRA which may underestimate
the diversity among the different strains. However, other genera of cyanobacterial group may be
explored for testing the intrageneric discriminatory power of ARDRA.

In silico tools have been used for ARDRA of bacterial groups like Phytoplasma followed by
identification, but only very limited reports (Iteman et al. 2002) are available for its use in
cyanobacteria. Wei et al. (2007) were able to classify 800 Phytoplsma isolates into 28 groups by
following the in silico approach. A careful choice of REs enabled the use of the ARDRA technique
to discriminate among Lactobacillus, Streptococcus and Bifidobacterium at the genus level, but
not at species level (Collado and Hernandez 2007). From the present study, we will also suggest
to go for in silico ARDRA (can be done by a number of programs) which can really reduce the
efforts of the researchers for selecting the appropriate REs for ARDRA. The availability of huge
number of 16S rDNA sequences in different databases all over the world along with a number of
freely available in silico analytical tools have empowered the researchers to dig out more and more
information with minimum effort. Not only time will be saved through this, but also the
consumables can be saved.

227
 CHAPTER: 18
Fluorescence in Situ Hybridization
(FISH)

FISH is a common cell biological technique and a golden standard in diagnostics. The earliest
implementation of ISH was achieved in 1969. The introduction of fluorescence in situ
hybridization (FISH) almost 30 years ago marked the beginning of a new era for the study of
chromosome structure and function. Conceptually, FISH is a very straightforward technique that
essentially consists in hybridizing a DNA probe to its complementary sequence on chromosomal
preparations previously fixed on slides. Probes are labeled either directly, by incorporation of
fluorescent nucleotides, or indirectly, by incorporation of reporter molecules that are
subsequently detected by fluorescent antibodies or other affinity molecules. Probes and targets
are finally visualized in situ by microscopy analysis. As a combined molecular and cytological
approach, the major advantage of this visually appealing technique resides in its unique ability to
provide an intermediate degree of resolution between DNA analysis and chromosomal
investigations, while also retaining information at the single-cell level. FISH gained widespread
recognition as a physical mapping technique to support large-scale mapping and sequencing
efforts related to the human genome project; however, its accuracy and adaptability were
simultaneously, or soon after, exploited in other areas of biological and medical research. As a
result, a wealth of diverse applications and FISH-based diagnostic assays have been developed
within different fields of investigation, including clinical genetics, neuroscience, reproductive
medicine, toxicology, microbial ecology, evolutionary biology, comparative genomics, cellular
genomics, and chromosome biology. The diversification of the original FISH protocol into the
impressive number of procedures available these days has been promoted through the years by a
number of interconnected factors, such as the improvement in sensitivity, specificity, and
resolution of the technique, brought about by a better understanding of the chemical and physical
properties of nucleic acids and chromatin, together with the advances in the fields of
fluorescence microscopy and digital imaging, and the growing availability of genomic and
bioinformatic resources. In situ hybridization

In situ hybridization (ISH) is a cytogenetic technique allowing high-resolution detection,

quantification, and localization of nucleic acid targets inside cells or tissues. The method is based
on the hybridization of sequence-specific complementary probes (typically DNA sequences) to
their target inside the cell. Figure shows schematically how a fluorescently-labeled nucleic acid
probe enters the cell nucleus where it hybridizes to its complementary target in the DNA. Probes
can be targeted at DNA (metaphase and interphase chromosomes) as well as RNA, making the
study of genomic sequences and transcriptomic expression profiles of individual cells possible.
The reaction can be visualized directly using radioactively or fluorescently labeled probes or
indirectly via histochemical chromogens, antigen binding, or biotin-streptavidin interactions.

228
Binding of complementary probes to the target of interest thus enables its detection and
visualization. This visualization can be leveraged for medical applications such as prenatal
diagnostics or oncology. Using probes targeted to different chromosomes or chromosomal
regions (chromosome painting), chromosomal aberrations such as differences in enumeration,
translocations, insertions, or deletions can be analyzed. Some examples of diseases diagnosed
using ISH include cystic fibrosis, Duchenne's muscular dystrophy, Prader-Willi syndrome,
Angelman syndrome, Lejeune's syndrome, trisomies 13, 16, 18, 21, monosomy X, Turner
syndrome, and different types of leukemia. Furthermore, gene-specific targets can be used to
detect gene amplifications often found in tumor cells. An example is the detection of human
epidermal growth factor receptor 2 (HER2) gene copy number changes as prognostic and
predictive biomarkers in breast and ovarian cancer. ISH additionally plays a role in the species
identification of (pathogenic) microbes.

Therefore, ISH is a common tool in cell biological research as well as in diagnostic applications,
as it provides valuable quantitative information about the nucleic acid distribution inside
individual cells. A large advantage of the technique is its ability to provide spatial information of
cellular content, thus providing the possibility of investigating spatial heterogeneities.

A historical view on in situ hybridization

ISH was developed and matured much later than immunofluorescence-based protein detection
methods. While immunofluorescence was first used by Coons et al., in 1941 and started to be
applied regularly in the 1950s, in situ hybridization was introduced ~30 years later (Fig.).
Melting and re-hybridization of DNA and complementary RNA-DNA binding were first
described in the 1960s. In 1969, Pardue and Gall then tagged rRNA probes with H3 to
autoradiographically visualize rRNA-encoding genes in cytological preparations of Xenopus
laevis oocytes. In parallel, John et al., and Buongiorno-Nardelli and Amaldi independently
developed in situ hybridization techniques for the detection of rDNA in Xenopus and paraffin-
embedded tissues. A few years later, Gall et al. visualized satellite elements in heterochromatic
regions of mouse chromosomes demonstrating the use of ISH in mammalian cells. Limitations of
these early ISH experiments were the low sensitivity and the limited availability of sequence-
specific probes. Although radioisotope labeling is still considered the most sensitive method of
detection, it requires long exposure times (up to weeks for the detection of H3), the spatial
resolution is low (in the range of Mega bases), and the background levels are high. These
disadvantages led to the development of non-isotopic labeling approaches for ISH probes.

229
 Figure: Timeline of fluorescence in situ hybridization developments

The earliest record of in situ hybridization is found by Gall and Pardue in 1969. First fluorescent
versions of the technique (FISH) appeared in the 1970s, followed by direct probe labeling twenty
years later. ‘Modern’ FISH further includes developments in the probe design and production as
well as background reduction. The combination of microfluidics and FISH first appeared in the
early 21st century. Events directly related to the development of FISH are shown in boxes, while
other findings influencing FISH are displayed as text only.

From radioactive to fluorescent probe labels

In the mid-1970s, a range of non-radioisotopic probes were developed, allowing for new
detection options of the probe-target hybrids. In one approach, a poly (rA)-poly (dT) antibody
was used to detect RNA-DNA hybrids on Drosophila chromosomes. The signal was created
using a rhodamine-conjugated secondary antibody. Others used rRNA-biotin, which was then
indirectly detected via an avidin-based detection system, and visualized using a scanning
electron microscope. Langer-Safer and Ward (1982) on the other hand used nick-translation to
incorporate biotin-labeled bases into DNA, which were then detected by primary and
fluorescently- or enzymatically-conjugated secondary antibodies.

Direct fluorescent detection of chromosomal targets was reported by Bauman et al. in the 1980s,
who labeled the 3′ ends of the probes with a fluorochrome. The combination of direct
fluorophore-labeling of nucleic acids and enzymatic incorporation of fluorophore-modified bases
from these publications lay the ground for the facilitated preparation of fluorescent probes.
Probes with fluorescein-labeled bases incorporated using nick-translation showed detection

230
sensitivities of 50–100 kb and were used for first multicolor FISH experiments. The development
of amino-allyl modified bases for the conjugation with haptens or fluorophores further facilitated
the production of a large variety of FISH probes. Owing to sparse labeling of long probe
sequences for specific hybridization, FISH was still limited by the detection sensitivity. Indirect
detection using secondary reporter systems based on biotin-avidin interactions or antibody
binding could amplify the generated signal.

The chemical synthesis of DNA probes finally allowed the incorporation of enough fluorophores
for direct detection. A range of different probe labeling techniques followed for generating
densely labeled FISH probes, such as digoxigenin labeling of the probe, cis‑platinum complex-
mediated labeling or polymerase chain reaction (PCR) and degenerate oligonucleotide primed
PCR, among many others. This enabled efficient generation of probes carrying various labels for
multiplex FISH, which led to new methods such as comparative genomic hybridization (CGH)
for cytogenetic analysis of tumors, or spectral karyotyping by chromosome painting. Recent
developments include the use of quantum dots and click chemistry.

The commercialization of fluorescence microscopes beginning in the 1970s and the

developments in the fields of confocal or multiphoton microscopy in parallel to the design of
new labeling schemes paved the way for highly sensitive detection of FISH signals.

FISH probe design

From the early developments of ISH to the late 1970s, polytene chromosomes or satellite
sequences were the ISH targets of choice, which, owing to their repetitive sequences, enabled
ISH signal detection in the absence of sensitive probes or detectors. With the development of
gene cloning and new probe labeling strategies, the specific design of FISH probes became
possible improving their specificity significantly. While first probes were generated by isolation
of crude RNA to target large fragments of chromosomes in the late 1980s, gene-specific FISH
probes could be generated from gene libraries from 1986 onwards. However, libraries generated
from microdissected chromosomes contain repetitive elements, which are spread across the
genome. Thus, FISH probes had the tendency to not only hybridize to the specific target but also
to off-target sequences in the cell. Methods for masking such repetitive elements with salmon
sperm or Cot-1 deoxyribonucleic acids (DNA) were developed, and recently, Swennehuis et al.
reported a method for enzymatic removal of repetitive elements to create unique, repeat-free
FISH probes from artificial chromosomes.

Thanks to advances in the Human Genome Project, bioinformatics tools and oligonucleotide
synthesis, synthetic FISH probes are now being increasingly used as they can be easily designed
and synthesized. The use of oligonucleotide libraries as a source for probes offers the possibility
of detecting chromosomal regions in the range of tens of kilobases to megabases. Bioinformatic
pipelines for the selective design and de novo synthesis of oligonucleotides facilitate the

231
development of chromosome-specific painting probes. Synthetic oligonucleotide probes are
particularly powerful, as they can be specifically designed to be sensitive enough to detect
splicing variants and even single nucleotide polymorphisms. Lately, in situ hybridization-based
single-cell RNA sequencing approaches were developed using oligonucleotide probes flanked
with primers for rolling circle amplification. In addition to synthetic oligonucleotide-based FISH
probes, also DNA analogous probes such as peptide nucleic acid and locked nucleic acid probes
are regularly used in FISH experiments. As, in contrast to DNA, they do not contain a charged
backbone, these nucleic acid analogues hybridize more efficiently and specifically than DNA
owing to minimal electrostatic interactions with the sample.

The advances in the field of probe design and labeling strategies allowed FISH to become a
highly versatile technique addressing a broad range of cellular targets with high specificity and
sensitivity. FISH could thus be used to enumerate chromosomes, detect chromosomal aberrations
and investigate specific chromosomal regions or transcriptional levels. These aspects are not
only interesting for cytogenetic research applications, but additionally provide the basis for
molecular diagnostics in a range of medical fields.

FISH hybridization kinetics

A key aspect of the FISH assay is the hybridization of the probe to its target. The hybridization
reaction of the free probes to chromosomal targets in the nucleus can be described using second-
order reaction kinetics

Target in the nucleus, cPT is the concentration of bound probes, and kon and koff are the on- and
the off-rates of the hybridization reaction, respectively.

With the assumption that cP > > cT, the characteristic hybridization time of a FISH reaction can
be defined as Thus, a high concentration of FISH probes in the nucleus enhances the
hybridization rate. Standard FISH reactions are typically performed using a bench-top assay by
pipetting the FISH hybridization mix onto the cytological sample. The diffusion-based transport
of FISH probes in solution and in the cell is slow, and the diffusion coefficient decreases with
increasing probe length. This leads to typical incubation times of up to 16 h for low copy number
targets and 48 to 94 h for entire genome hybridization.

232
This rate-limiting step can be overcome by performing a flow-based FISH probe incubation step,
resulting in a convection-enhanced transport of FISH probes into the cells and thus an increased
FISH probe concentration cP in the cell. Thus, the hybridization time and with this the assay time
can be reduced using microfluidic devices employing an active delivery of FISH probes to the
cells, as demonstrated by the microfluidic community. The microfluidic delivery ensures a
constant replenishment of probes on the surface of the permeabilized cells and tissues. The
reaction is therefore limited by the diffusion inside the tissue and the hybridization reaction.

Conventional implementations of FISH

A conventional FISH reaction is typically performed as a bench-top assay by pipetting the FISH
hybridization mix, containing the probes, onto the cytological sample and incubating it (Figure).
FISH can be performed on suspended cells followed by cell sorting procedures for separating the
fluorescent signal as well as on cultured cells and frozen or formalin-fixed paraffin-embedded
tissue sections. During fixation of the sample, it is important to preserve nucleic acid integrity
and cell morphology. The actual experimental FISH procedure includes several preparatory
steps, the hybridization reaction itself, and the removal of unbound probes. A short outline of the
procedure is illustrated below.

Figure: Schematic representation of the experimental FISH assay procedure

The sample of interest is fixed and pretreated to facilitate the penetration of FISH probes. A
potential denaturation step is followed by hybridization with the FISH probes under stringent
buffer and temperature conditions. Unbound probes are removed by washing to ensure imaging of
specifically stained regions only. Counter staining of specific cellular structures aids in cell
selection. Signal detection is performed on a fluorescence microscope followed by a qualitative or
quantitative analysis.

Experimental procedure

Depending on the sample of interest, pretreatment of the sample can be performed to facilitate
the entry of FISH probes and thus increase their hybridization efficiency. In the case of cultured
cells, these typically include fixation and cell permeabilization using organic solvents or
formaldehyde. For tissue sections, formalin cross-linking might have to be removed before

233
subsequent protease treatment revealing the target sequences and improving FISH probe
penetration into the tissue.

The actual hybridization reaction then takes place by incubating the FISH probes with the sample
of interest at a temperature providing specificity for the hybridization reaction for several hours.
The concentration of formamide in the hybridization buffer changes the stringency of the
hybridization reaction and needs to be chosen probe-specifically. The buffer composition as well
as the hybridization temperature determine the sensitivity and specificity of the reaction and are
typically defined empirically based on the length of the probes and the sample of interest.
Unspecific charge-charge interactions of the probe with the tissue can be reduced further using a
mixture of acetic anhydride and triethanolamine.

Several stringent washing steps after the hybridization ensure removal of unbound FISH probes
and specificity of the detected signal. Additionally, nuclear staining is often performed before
FISH signal detection on an epifluorescent or confocal microscope or even using super-
resolution microscopy. For high-quality images, a large magnification is used, which results in
the collection of a large number of images with small fields of view.

Here, we have assembled in a glossary format many of the FISH applications published so far.
Although we intend this review to celebrate the versatility of this technique, it is of course
impossible to cover every modification of FISH, and therefore we have limited ourselves to
variants that are named by combining a prefix with the acronym FISH. As seen in the many
flavors of FISH described in the following, this flexibility has allowed the underlying technique
to remain at the forefront of biomedical research over the last three decades.

ACM-FISH

ACM-FISH is a multicolor FISH assay for the simultaneous detection of numerical and structural
chromosomal abnormalities in sperm cells. The abbreviation ACM refers to the concurrent
hybridization of DNA probes for the alpha (centromere), classical (1q12), and midi (1p36.3)
satellites of chromosome 1 for the specific detection of duplications and deletions of 1pter and
1cen, and for the identification of chromosomal breaks within the 1cen-1q12 region. The ACM
technique originated from the integration of technical aspects and biological findings that emerged
from previous FISH investigations of chromosome rearrangements in sperm, as well in other cell
types (e.g., the assessment of breaks in 1q12 in human lymphocytes. Its application has led to
significant discoveries in the occurrence of chromosomal abnormalities in the sperm of healthy
men, showing that spontaneous structural defects are more frequent than numerical ones and that
chromosomal breaks preexist in human sperm before fertilization, and also providing evidence for
reproducible donor differences in breakpoint locations within 1q12. The assay has also been
successfully used for the analysis of sperm of infertile men to show that oligozoospermic men
carry a higher burden of transmissible chromosome damage; these results raising the question of

234
possible elevated levels of chromosomal defects following intracytoplasmic sperm injections
(ICSI) treatments.

Arm FISH

Arm FISH is a 42-color M-FISH variant (see below for M-FISH) that allows the detection of
chromosomal abnormalities at the resolution of chromosome arms (p- and q-arms of all 24 human
chromosomes, except the p-arm of the Y and acrocentric chromosomes). The protocol combines a
commercially available M-FISH kit with an arm kit or a set of chromosome arm-specific painting
probes. It is a straightforward, but significant, improvement to the standard M-FISH technique,
the most obvious advantage being the increased resolution to the level of chromosome arms and
the resulting ability to detect pericentric inversions. The assay has been successfully applied to
reveal widespread chromosomal instability in glioma cell lines.

CARD-FISH

CARD-FISH, which stands for catalyzed reporter deposition-FISH, refers to the signal
amplification obtained by peroxidase activity through the deposition of a large number of
fluorescently labeled tyramine molecules in which the horseradish peroxidase (HRP)-labeled
probe has bound (see also T-FISH). Improvements have been made to the technique to aid the
delineation of bacterial sequences, with one aspect of improvement being collecting bacteria on
filters (9–11). There has also been a combination of CARD-FISH with microautoradiography that
has been termed MICRO-CARD-FISH (12–14). Tritium is the radioisotope that is usually used
and is taken up by active cells as 3H-aspartic acid. The filters on which the bacteria are captured
are submitted to CARD-FISH, and they are then placed onto photographic emulsions. Cells can
be assessed using a fluorescence microscope that has transmission light in addition. This allows
both fluorescent, positive cells to be viewed by excitation of UV light and co-localization of silver
grains by white light.

Cat FISH

Cellular compartment analysis of temporal (cat) activity by FISH is an ingenious experimental

approach devised to investigate the dynamic interactions of neuronal populations associated with
different behaviors or cognitive challenges. The method, based on RNA-FISH on cryosections
followed by confocal analysis, was originally applied to study the environment-specific expression
of the neural activity-regulated, immediateearly gene (IEG) Arc, and to monitor its cellular and
subcellular distribution in the whole brain in rat. As a functional brain imaging technique, the
uniqueness of catFISH resides in its ability to confer both temporal and cellular resolution to the
analysis of gene expression patterns in brain, an important combination for the study of the
dynamics of information processing.

CB-FISH

235
CB-FISH involves hybridization on binucleated cells in which cytokinesis has been blocked by
treatment with cytochalasin-B (CB). The term CB-FISH was coined by a research group
investigating the mechanism by which the ratio of mosaic diploid cells in vivo increased in trisomy
21 cases. However, protocols combining FISH with the CB blocking assay for the cytological
analysis of micronucleation and aneuploidy events had already appeared in a number of earlier
molecular cytogenetic studies (18–21), including an investigation of chromosome 21
malsegregation in Alzheimer's patients. Analysis of the chromosomal content of micronuclei can
be facilitated by combining the standard CB-FISH protocol with the 24-color SKY technology,
since in one hybridization step, the DNA from any chromosome within the micronuclei can be
identified.

CO-FISH

Chromosome orientation or CO-FISH is the name given to a FISH technique that uses single-
stranded DNA probes to produce strand-specific hybridization. The technique relies on labeling
by 5′-bromodeoxyuridine (BrdU) incorporation one strand of the sample cells’ DNA during S-
phase. Metaphase chromosomes are prepared a number of hours after the BrdU pulse. Following
Hoechst staining and UV irradiation, the newly synthesized DNA becomes nicked at the sites of
BrdU incorporation. These nicks are enlarged using ExoIII, and the newly replicated DNA is
removed, leaving the parental strand as a single-stranded template for the hybridization procedure.
Initially, CO-FISH were designed to determine the orientation of tandem repeats within
centromeric regions of chromosomes. This technique had also been useful in assessing aspects of
translocated chromosomes, specifically Robertsonian, and also chromosomal inversions, since an
inversion obviously changes the orientation of the involved chromosome segment.

COBRA-FISH

The prefix COBRA stands for combined binary ratio. This particular FISH protocol brings together
combinatorial labeling with ratio labeling. The ratio labeling method allows different ratios of label
to distinguish between probes. This permits the use of fewer fluorochromes to produce more
pseudocolors, allowing the resolution of more than 24 colors within a specimen. The novel aspect
of the COBRA technique is that two sets of probes are ratio-labeled identically with three
fluorochromes (usually two sets of 12 chromosomes for a human karyotype), but then one set is
further labeled with an additional fourth fluorochrome. Indeed, a further fifth fluorochrome can be
used to make up to 48 color combinations for differential painting of human chromosome arms.
The capacity to view more than 24 colors allows the delineation of additional sequences such as
viral genome inserts or single-copy genes.

COD-FISH

Although COD-FISH is an abbreviation that has been used to describe three different hybridization
techniques, the most common use is for chromosome orientation and direction-FISH. This protocol
is similar to CO-FISH (see above), but when combined with the information about the directional

236
organization of telomeric sequences, the technique can be termed COD-FISH. COD-FISH can also
stand for concomitant oncoprotein detection-FISH, a technique to visualize not only the loci
signals for a particular oncogene, but also the protein product derived from this gene. The
development of such a protocol was aimed at performing quantification of gene copy number and
amount of protein product to help elucidate interesting mechanisms involved in the transcription
and translation of a particular message. Others have called this type of regimen Immuno-FISH,
but the COD protocol does add a semiquantitative aspect to it. Tubbs and colleagues went on to
improve their method using nanogold visualized with bright field microscopy. Another technique
that has been termed COD-FISH is the combined CaCO3 optical detection-FISH, in which FISH
is used to detect calcifying microrganisms in Open Ocean.

COMBO-FISH

COMBO-FISH is a method with no requirement for sample denaturation prior to hybridization.

The prefix COMBO stands for combinatorial oligonucleotide. The technique utilizes sequence
information to identify regions of the genome where there are stretches of purines or pyrimidines
and uses homopurine or homopyrimidine probes that are able to form triple helices with duplex
genomic DNA. Homopurine or homopyrimidine regions of DNA 14 bp in length or more compose
1% to 2% of the human genome, with an average of 200 of such stretches in a 250-kb segment of
the genome. Accordingly, specific probe sets can be constructed to target genomic regions of
interest in that size range. The omission of the denaturation step makes the hybridization procedure
less harsh on nuclear architecture, rendering this technique ideal for three-dimensional (3-D)
analysis of genome organization.

Comet-FISH

Comet-FISH is a combination of the comet assay and FISH analysis. The comet assay, also called
single-cell gel electrophoresis or the single-cell gel test, is used to evaluate the amount of DNA
breakage within single cells by running the DNA out of the nuclei into an agarose gel. The
combined Comet-FISH method, consisting in releasing, by electrophoresis, the DNA onto agarose-
coated microscope slides prior to in situ hybridization, allows specific sequences to be delineated
in the comet head or tail, thus permitting the assessment of whether specific genomic regions are
sensitive to DNA damage and breakage. With this technique, researchers have demonstrated that
DNA damage susceptibility is associated with the gene density of a chromosome rather than the
chromosome size. Further, damage to specific genes can be detected (44–47). The sensitivity of
telomeres to damage has also been assessed successfully using this method.

Cryo-FISH

Cryo-FISH is a promising in situ hybridization technique that makes use of ultrathin cryosections
(150 nm thick) of well-fixed, sucrose-embedded cells. The technique was recently devised and
successfully applied to the study of spatial interrelationships of chromosome territories in the cell
nucleus, a muchdebated aspect of chromosome organization in interphase. By combining a robust

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cell fixation procedure and ultracryomicrotome sectioning with two-dimensional (2-D)
microscopy analysis (wide-field), this innovative technique maintains to preserve chromatin
nanostructure while simultaneously presenting with a better efficiency of hybridization and
resolution than canonical 3-D FISH, and provides an alternative, and possibly more userfriendly
approach, to the study of genome organization in the nuclear context. Cryo-FISH was also recently
used to validate results obtained by chromosome conformation capture on chip (4C) technology to
demonstrate long-range chromosomal interactions of functional significance.

D-FISH

D-FISH is an enhanced version of the fusion signal-FISH protocol for the detection of recurring
chromosomal translocations in hematological malignancies. The prefix D stands for double fusion,
since in this particular protocol the use of two (or two sets of) differentially labeled, large probes,
each spanning one of the two translocation breakpoints, allows the simultaneous visualization of
both fusion products, significantly reducing the impact of false-negative results, a reason of
concern in single fusion FISH. D-FISH was initially devised to improve detection of the double
BCR/ABL fusion in chronic myeloid leukemia (CML) patients. Subsequently, D-FISH probes
were developed for the 8; 21 translocation and used for the assessment of minimal residual disease
in acute myeloid leukemia (AML) patients during remission and also for the visualization of
PML/RARA double fusion in acute promyelocytic leukemia (APL), the DEK/CAN double fusion
resulting from t (6; 9) in AML, and the PBX1/E2A double fusion in pediatric patients with acute
lymphoblastic leukemia (ALL). A wide range of probes for D-FISH, as well as Fusion-Signal and
Split-Signal FISH for the chromosomal analysis of hematological cancer, are now commercially
available.

DBD-FISH

DBD-FISH stands for DNA breakage detection FISH, a technique developed by Gosálvez and
colleagues. Basically, this adaptation of the FISH procedure permits any sites of DNA
damage/breakage in the sample genome to be analyzed in situ by means of an alkali DNA
unwinding solution and protein removal. Cells are normally stabilized in agarose beads, but the
technique can be applied to DNA comets. The incubation with the unwinding buffer leads to the
presence of single-stranded DNA in the sample that can be hybridized with the appropriate probes.
The technique has been used successfully to test the DNA fragmentation levels in sperm samples.

e-FISH

e-FISH is a BLAST-based FISH simulation program able to accurately predict the outcome of
hybridization experiments. The program was developed as one of the bioinformatics resources to
be available from the Database of Genomic Variants, aimed at simplifying the choice of
appropriate genomic probes for hybridization experiments and facilitating the interpretation of the
results. This virtual FISH approach includes a repeat masking step mimicking in silico the COT-1
blocking of repetitive sequences. The program is freely accessible at projects.tcag.ca/efish.

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Fiber-FISH

Fiber-FISH is a technique that allows high resolution mapping of genes and chromosomal regions
on fibers of chromatin or DNA, permitting physical ordering of DNA probes down to a resolution
of 1000 bp, and that also allows assessment of gaps and overlaps in contigs and analysis of
segmental duplications and copy number variants. In practice, the method consists of releasing
chromatin/DNA fibers from cell nuclei, usually by means of salt or solvent extraction, and
stretching and fixing them on a microscope slide prior to hybridization. Similar variants of the
technique, in which the chromatin is run down the slide and is stretched by fluid flow, were initially
set up more or less concomitantly by different research groups. However, the specific Fiber-FISH
terminology was introduced slightly later. A significant improvement in terms of DNA stretching
uniformity and reproducibility was provided by the implementation of the molecular combing
protocol, which uses the action of a receding air/water meniscus to extend and align DNA
molecules attached at one end to a glass surface.

Flow-FISH

In the Flow-FISH technique, as described by Lansdorp and colleagues in 1998, PNA-labeled

telomere probes are used to visualize and measure the length of telomere repeats, as in the
Quantitative-FISH technique (see below for Q-FISH and PNA-FISH), but the analysis combines
in situ hybridization with flow cytometry for measurement of the telomeric signals from cells in
suspension. This permits large numbers of cells to be analyzed rapidly. Lansdorp and his
collaborators have developed this technique, and his and other laboratories have used it for a
number of different cell types and clinical applications. Indeed, Flow-FISH has been used in aging
studies, telomere maintenance, and in clinical applications for ex vivo suspension cells
(hematopoietic). Also, combining telomere Flow-FISH with fluorescent immunodetection of cell
surface markers has advanced the understanding of the behavior of stem cell populations.
Researchers have expanded the Flow-FISH technique to permit assessment of different strains of
bacteria.

Fusion-Signal FISH

The Fusion-Signal FISH technique was initially devised for the identification of the 9; 22
Philadelphia translocation in peripheral blood and bone marrow cells of CML patients to detect
minimal residual disease after bone marrow transplantation. BCR and ABL gene fragments, each
flanking one of the two breakpoints, were used as probes for the detection of the BCR/ABL fusion
product, hence the fusion-signal appellation. Since then, sets of probes to detect fused gene signals
originating from a range of critical translocation events in hematological malignancies, for instance
the PML and RARA fusion product resulting from t(15:17) in APL, have been designed by
different research groups. With FISH assays available for CML and APL, as well as AML, non-
Hodgkin's lymphoma, mantle cell lymphoma, childhood B-lineage acute lymphoblastic leukemia,
and infant leukemia, it would be difficult to deny the major impact of the fusion signal technique

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as a diagnostic and prognostic tool for blood cancer. However, soon after its conception, concerns
started to emerge that the interpretation of the results was complicated by the variable occurrence
of false-positive and false-negative signals. To overcome these difficulties, an ingenious dual-
fusion variant of the technique was devised for the detection of the 9; 22 translocation in CML.
The protocol involves the use of large probes, spanning the two breakpoints, for the simultaneous
visualization of both fusion signals BCR/ABL and ABL/BCR. This modified and improved
version of the fusion signal technique was named D-FISH.

Halo-FISH

Halo-FISH is an acronym that describes FISH performed on cells that are first permeabilized and
then extracted with high salt to remove soluble proteins. Indeed, chromatin/DNA that is not fixed
to an internal structure within cell nuclei is released, forming a halo around a residual nucleus.
FISH can then be performed on these preparations using any type of probe to delineate specific
DNA sequences. Researchers have used α-satellite, telomeres, scaffold attachment regions
(SARs), matrix attachment regions (MARs), gene loci, and whole chromosomes. DNA halo
preparations can be used for high-resolution mapping, since such long extended loops of DNA are
created. A number of groups have used DNA halo preparations to analyze sperm chromatin, as it
makes it easier to access sperm DNA, which is normally very compact due to its association with
protoamines. Some have even described this type of analysis as SpermHalo-FISH, whereby sperm
nuclei are spun onto glass microscope slides and treated with dithiothreitol for permeabilization
followed by high salt.

Harlequin-FISH

Harlequin-FISH is a method for cell cycle-controlled chromosome analysis in human lymphocytes

that allows a precise quantification of induced chromosome damage for human biodosimetry
purposes. The approach combines FISH painting with differential replication staining after BrdU
treatment of lymphocyte cultures (or harlequin staining). The principle, on which differential
replication staining is based, is that after two rounds of replication in the presence of the base
analog BrdU, sister chromatids will stain differentially (with either Giemsa and/or fluorescent
dyes). This allows the identification of the two chromatids and the observation of sister chromatid
exchanges (SCEs), that after a few cell divisions confer to the chromosomes an asymmetrically
striped appearance, to which the term harlequin refers. Since for accurate cytogenetic
measurements of genetic damage, cells must be analyzed in their first mitosis following exposure,
the most relevant aspect of the harlequin technique in combination with FISH for biodosimetry
studies is that, according to sister chromatid staining patterns, cells in different division cycles can
be distinguished, allowing chromosomal analysis to be carried out selectively.

Immuno-FISH

Immuno-FISH is a combination of two techniques, one being standard FISH, either on flattened
chromosome preparations (2-D FISH) or on three-dimensionally preserved nuclei (3-D FISH), and

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the other indirect or direct immunofluorescence. The latter technique permits the visualization of
antigens within the sample, so that both DNA and proteins can be analyzed on the same sample. It
is often used to investigate co-localization of genomic regions with proteinaceous entities within
interphase nuclei such as nucleoli or promyelocytic leukemia (PML) bodies. Not all antigens will
be preserved after the various steps in the FISH protocols, but it is possible to apply the primary
or primary and secondary antibodies and proceed with a fixation step prior to the FISH procedure.
The term Immuno-FISH was first coined by Brown et al. in 1997, in which co-localization of
active or inactive gene loci were assessed with nuclear structures containing the Ikaros protein,
although others had combined the two methodologies previously for RNA-FISH with splicing
speckles and for anti-CENP C staining in combination with α-satellite probes. The combination of
these techniques has been used for great effect, even helping to position chromosomes in
interphase nuclei, and is now being used with multiple probes and colors, such as in the paper from
Cremer's group, whereby they look at chromosomal regions, gene expression, and histone
methylation.

LNA-FISH

Locked nucleic acids (LNAs) are a class of RNA analogs with exceptionally high affinity toward
complementary DNA and RNA. Because of the LNA chemical makeup, heteroduplexes between
LNA oligonucleotides and their complementary DNA oligonucleotides show a shift in structure
from a B-like helix toward an A-type helix. This results in a higher thermal stability of the LNA-
DNA heteroduplexes. LNA-FISH refers to the use of LNA-modified oligonucleotides in FISH
experiments for improved resolution and sensitivity.

M-FISH

The invention of M-FISH (or Multiplex-FISH), a protocol for 24-color karyotyping, based on
combinatorial labeling and aimed at facilitating the analysis of complex chromosomal
rearrangements and marker chromosomes, has signified a groundbreaking development in
molecular cytogenetics, particularly for the study of tumors and prenatal diagnosis. Dissimilarly
from ratio-labeling based multicolor approaches, in which chromosome-specific probes are
characteristically labeled with different proportions of fluorochromes and accurate measurements
of relative fluorescence intensity are required, the M-FISH technique (like the related SKY
technique, see below) consists of labeling each probe with a unique combination of five spectrally
separable fluorochromes in a 1:1 ratio. Accordingly, although relying on the use of narrow band-
pass fluorescence filters appropriately set in a motorized filter wheel and on digital imaging
software more sophisticated than the standard setup for FISH analysis, the interpretation of the
results is relatively straightforward (that is, the fluorochrome is either present or absent). The
technique was originally devised for use with and simultaneous detection of the 24 human
chromosome painting probes (22 autosomes and the X and Y chromosomes), but has been
subsequently used to analyze specific chromosomal subregions, like centromeres and
subcentromeres in protocol variants for the characterization of small supernumerary marker

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chromosomes with no euchromatin. Examples include cenM-FISH, CM-FISH, and subcenM-
FISH, telomeres for the identification of subtle subtelomeric rearrangements as in M-TEL and
chromosome arm-specific probes for the detection of pericentric inversions. Similar in principle
and application to the M-FISH technique is the spectral karyotyping technique or SKY, in which
chromosomes are classified on the basis of their unique emission spectra. For image acquisition
and analysis, SKY requires specific hardware and software, comprising a custom-designed single
triple-band-pass filter and an interferometer able to retrieve spectral information for every pixel in
a digital image. For comprehensive reviews on 24-color FISH analysis see Kearney et al. (2006)
and Schrock et al. (2006). M-FISH is also used as a term to mean Multicolor-FISH.

Multilocus or ML-FISH

The word multilocus (subsequently abbreviated with the acronym ML) refers to the simultaneous
use in multicolor FISH of multiple probes. This FISH assay was initially designed to screen for
multiple microdeletion syndromes in patients with unexplained developmental delay and/or mental
retardation. The original multilocus panel designed included Prader-Willi, Angelman, Williams,
Di George/velocardiofacial, and Smith-Magenis syndromes. Based on the same principle, a
multilocus strategy with locus-specific DNA probes for chromosomes 13 and 21 was more recently
applied to the study of nondisjunction in mature human oocytes.

PCC-FISH

PCC-FISH is a FISH application used for biodosimetric analysis that relies on the use of
chromosome-specific painting probes to determine chromosome damage after irradiation. The
acronym PCC stands for premature chromosome condensation and refers to the effect obtained by
virusmediated cell fusion or phosphatase inhibitors (either calyculin A or okadaic acid) to
prematurely condense the chromosomes of cells in G1 and G2 phases, a central aspect of the
procedure that overcomes the need to culture cells in vitro as required for conventional metaphase
chromosome analysis. PCC-FISH was initially devised as an assay to estimate/predict the in situ
radiation sensitivity of individual human tumors. It has subsequently been used to estimate the
effect of whole-body high- or low-dose exposure to human peripheral lymphocytes and carried out
on skin fibroblasts in the case of acute localized irradiation following accidental overexposure.
PCC-FISH has also been used to establish the radiosensitizing effect of drugs in experimental
cancer treatments. The technique is used additionally in in vitro studies for general radiation
research and cancer research purposes.

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PNA-FISH

Peptide nucleic acids (PNAs) are synthetic analogs of DNA in which the deoxyribose phosphate
backbone supporting the nucleic acid bases is replaced by a noncharged peptide backbone. As a
result of this unique structural property, there is no electrostatic repulsion when PNA oligomers
hybridize to complementary DNA or RNA sequences, and PNA-DNA and PNA-RNA duplexes
are more stable than the natural homo- or heteroduplexes. FISH with PNA probes or PNA-FISH
was first used by Landsdorp and collaborators to measure individual telomere lengths on
metaphase chromosomes. Quantitative telomere analysis by PNA-FISH was subsequently carried
out on interphase cells. The discovery that PNA-DNA hybridization is more significantly affected
by base mismatches than DNA-DNA hybridization and that PNA probes could discriminate
between two centromeric repeats that differed only by a single base pair endorsed this as an
important development in the field. Thus, short (15- to 18-mer) PNA probes for α-satellite domains
of specific chromosomes were designed, and their power of discrimination at a single-base level
was used for unique chromosome identification in metaphase and interphase. As well as on
lymphocytes, amniocytes and fibroblasts, PNA-FISH has also been successfully carried out on
human spermatozoa and isolated oocytes, polar bodies, and blastomeres, a strong indication of the
potential of PNA probes for preimplantation cytogenetic diagnosis. Larsen and collaborators tested
the suitability of PNA-FISH for noninvasive prenatal diagnosis by detecting γ-globin messenger
RNA (mRNA) in fetal nucleated red blood cells from maternal blood. Also, as a result of the
relative hydrophobic character of PNA compared with DNA, that allows better diffusion through
the cell wall, PNA-FISH has also had wide application in microbiology, for both research and
clinical purposes. Because of its high binding specificity quality, PNA technology is expected to
provide a platform for the design of allele-specific probes for in situ hybridization, a technical
development longed for by many in the genetic, cytogenetic, and epigenetic ranks.

Q-FISH

Quantitative-FISH methodology permits the measurement of probe signal intensity. Q-FISH was
initially invented by Lansdorp and collaborators and was shown to work in combination with flow
cytometry. This method has been used mainly for measuring the number of telomere repeats on a
particular chromosome, using PNA-conjugated probes. Typically, metaphases are imaged and then
analyzed using software such as TFL-TELO. The lengths of chromosomal regions can be
measured at the resolution of 200 bp. Q-FISH has advanced and is now used on interphase cells
and tissue sections. Indeed, in interphase cells, a similar methodology with a different acronym
has been used (i.e., single telomere length analysis or STELA). Q-FISH has also been used to
quantify telomere length in interphase cells and has even been given the acronym IQ-FISH for
Interphase Q-FISH. Q-FISH has become an important tool in studying the role of telomeres in
aging and cancer.

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QD-FISH

QD-FISH refers to the recently pioneered utilization of quantum dot-conjugates in FISH protocols.
Quantum dots are nanometer-sized inorganic fluorophores, characterized by photostability and
narrow emission spectra that have been successfully used for FISH analysis on human metaphase
chromosomes, human sperm cells, bacterial cells, and also to detect subcellular mRNA distribution
in tissue sections. While initial QD-FISH protocols entailed hybridization of biotinylated probes
and subsequent detection using streptavidin-conjugated QDs, more recent adaptations of the
procedure, aimed at multicolor imaging, involve direct labeling of oligonucleotide probes.

Rainbow-FISH

Rainbow-FISH is an advanced digital imaging procedure that allows the simultaneous detection
and quantification of up to seven different microbial groups in a microscopic field. The technique
was designed to improve quantitative analytical studies of microbial communities from various
aquatic environments. Based on similar principles to the multi-FISH technique previously
developed for the analysis of microbes in human feces, the rainbow protocol brings together the
use of specific 16S ribosomal RNA (rRNA)-targeted oligonucleotide probes for the discrimination
of different phylogenetic groups of microbes and the principles of combinatorial labeling. As a
result, by the combined application of seven DNA probes, each labeled with up to three
fluorochromes, seven kinds of microbial strains can be distinguished simultaneously. The specific
Rainbow-FISH digital procedure developed by Sunamura and Maruymama consists of systematic
background noise reduction and target signal equalization to discriminate between microbes and
nonmicrobial particles, and aims at facilitating image processing and analysis, in particular the
normalization steps that are usually very laborious when imaging natural environmental microbes,
since their cellular rRNA content can vary between cells, even within the same sample.

Raman-FISH

Raman-FISH is a new technique that combines FISH technology with Raman microspectroscopy
for ecophysiological investigations of complex microbial communities. The procedure exploits the
so-called red shift phenomenon or the significant change in the resonance spectra, as visualized by
Raman microscopy that follows anabolic incorporation of 13C isotope, when compared with
normal 12C, into microbial cells. Metabolic labeling through stableisotope incorporation is
combined with microbial species identification by in situ hybridization with specific 16S rRNA
probe for structural and functional interrelated analyses of microbial communities at a single-cell
resolution.

ReD-FISH

ReD-FISH, which stands for replicative detargeting FISH, is similar to CO-FISH, whereby BrdU
is added to the culture medium during DNA replication for incorporation into the newly
synthesized strand. ReD-FISH allows the replication timing of specific sequences to be

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determined. If BrdU has been incorporated in the sequence of interest, the newly formed DNA
strand will be detargeted (as in CO-FISH, see above), and each oligonucleotide probe will only be
able to hybridize to one of the parental strands, and only one chromatid will be display a signal.
However, if the sequence of interest has not been replicated and not incorporated BrdU, then a
FISH analysis will reveal the standard double signal on both chromatids of the metaphase
chromosome. The ReD-FISH technique has been instrumental in ascertaining the replication
timing of telomeres, leading to the observation that telomeres replicate throughout S-phase and
that telomeres from p and q arms of the same chromosome replicate asynchronously.

Reverse-FISH

First demonstrated in 1990, Reverse-FISH is the process whereby the FISH probe comprises DNA
from the material of interest. This can be a chromosome of a specific species in the cellular
background of another species (i.e., a somatic cell hybrid). It can also be chromosomal material
obtained in other ways such as by flow sorting or microdissection. The reverse terminology refers
to the probe being the material of interest, usually aberrant, and being painted onto control or
reference metaphase chromosomes to identify what sequences/chromosomal regions the probe
contains or is missing. Reverse-FISH has been useful for characterizing marker chromosomes and
chromosome amplifications in cancer.

RING-FISH

In situ hybridization of oligonucleotide probes to high copy number nucleic acid targets, such as
rRNA, is a standard method for the identification of microorganisms in enviromental samples.
RING-FISH is a modified version of this technique that relies on the use of high concentrations of
polynucleotide probes for an increase in sensitivity and visualization of any part of the genetic
material of a bacterial cell, regardless of copy number. The signal amplification achieved with
these polynucleotides, the length of which ranges between one to several hundred base pairs, is
mediated by their secondary structures and intermolecule interactions, resulting in a conspicuous
network of polynucleotides that builds upon the initial hybridization site. RING stands for
recognition of individual genes, but also refers to the characteristic ring-shaped or halo appearance
of the fluorescence signals at the bacterial cell periphery.

RNA-FISH

RNA-FISH is a method that allows detection of RNA within cells. Transcripts can be visualized
either in the nucleus or in the cytoplasm. The technique, also known as expression-FISH, has been
used to analyze the transcriptional activity of endogenous genes as well as exogenous genes such
as those belonging to integrated viral genomes and transgenes. The technique permits investigation
into allelic-specific expression on a per cell basis and is expected to provide a platform for gene
expression profiling studies in single cells. RNA-FISH has also been instrumental in studying
different functional aspects of genome organization and nuclear architecture. Further, as a
technique, it is being examined as a prenatal diagnosis tool for myotonic dystrophy type 1.

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RxFISH

RxFISH is a color banding technique that is also described as chromosome bar-coding. The method
relies on sequence homologies between human and the apes, such as gibbon (98%), to produce, by
cross-species hybridization, a specific banding pattern on human metaphase chromosomes. This
is possible because of the intrachromosomal rearrangements that have occurred during primate
evolution. If the probes are labeled with a number of fluorochromes, usually three, this allows a
colorful and reproducible banding to be observed and analyzed. Due to the color bands, it is easier
than G-banding to see chromosomal rearrangements, especially intrachromosomal
rearrangements. However, in combination with G-banding, RxFISH can provide very detailed
information about chromosomal breakpoints, for example in cancer.

Split-Signal FISH

Split-Signal FISH is a fast and sensitive dual-color FISH assay for the detection of frequently
occurring chromosome translocations affecting specific genes in hematopoietic malignancies. The
technique is equally suitable for metaphase and interphase analysis and has been increasingly used
for both diagnostic and prognostic purposes. In its current form, the assay involves the design and
differential labeling of two probes from the flanking regions of the translocation breakpoint. The
signals normally co-localize and appear fused, but as a result of the translocative event, they will
split. Split-Signal FISH was initially introduced as an innovative and simple experimental
approach for the detection of all types of MLL gene translocations in ALL and AML, using only
a single FISH test. However, a prototypic version of the protocol with differentially labeled probes
for genes spanning or adjacent to the translocation breakpoints, on the two chromosomes involved,
had been previously used for the detection of the Burkitt translocation t(8;14) in B cell lymphomas
and the detection of t(11;14) in mantle cell lymphomas.

T-FISH

The T in T-FISH can stand for tyramide, tissue, or telomere. The three versions of T-FISH are
discussed in the order of their arrival in the field. Tyramide-FISH: tyramide is a compound that
binds to peroxidase easily and thus has been used to increase the sensitivity greatly in FISH
experiments, with the use of only one or two layers of reagents for visualization. The first layer
uses a peroxidase-conjugated antihapten antibody or a compound such as strepavidin to bind to
the labeled probe. Fluorochromes or haptens, such as biotin, are conjugated to tyramine
derivatives. This leads to a massive build-up or towers of fluorochromes or moieties that can be
visualized by fluorochromes, making detection ultrasensitive. The chemistry behind tyramide
signal amplification (TSA) is now licensed, and kits can be purchased from various companies.
The technology has been used to map gene loci and look for specific transcripts in cells. Tissue-
FISH: tissue samples are collected frequently from patients or experimental animals. These
samples can be frozen, fixed, or embedded in paraffin wax. A number of research groups have
developed methods of delineating sequences in tissues by FISH, for example, in paraffin sections

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and frozen sections. However, it was Nomura and collaborators, who, in 2003, coined the term T-
FISH to denote FISH on tissue sections. Telomere-FISH: some groups have called FISH using
telomeric probe.

3-D FISH

3-D FISH was developed in Germany by the groups of Peter Lichter and Thomas Cremer to
analyze spatial positioning and relative organization of chromosomes and subchromosomal
regions within cell nuclei. The methodology relies on using cross-linking fixation reagents, such
as paraformaldehyde, to preserve nuclear architecture and large-scale chromatin organization. Due
to the resulting crosslinking of proteins, an effective permeabilization step is required to allow the
probes to penetrate the sample. This is often performed using detergents and freeze and thaw cycles
at −180°C. To visualize the 3-D signals, operators will need to use confocal laser-scanning
microscopy or use accurate deconvolution tools. One of the most significant achievements in the
field was the publication of a paper displaying the simultaneous painting of all human
chromosomes in the nuclei of three-dimensionally preserved cells. The introduction of 3-D FISH
protocols and their wide application in the field of chromosome biology has significantly
propelled, over the last few years, our understanding of chromosome structure and function.

Zoo-FISH

Zoo-FISH, also known as crossspecies chromosome painting, consists of hybridizing libraries of

DNA sequences, also known as chromosome paints, from one species to the chromosomes of
another species, to identify regions of synteny. From a methodological point of view, the protocol
does not differ significantly from standard FISH protocols, but there can be some issues with signal
intensity and background noise, as pointed out by Solovei (www.biologie.uni-
muenchen.de/ou/humbio/pdf/solovei/protocol/11_zoofish.pdf). Solovei discusses that good
metaphase spreads are absolutely fundamental, that suppression of repetitve sequences may be
unnecessary, and that higher concentrations of probe are usually required. A critical step to be
borne in mind is that the more divergent the organisms are from each other, the lower the
stringency should be within the protocol. The first study using Zoo-FISH used human and mouse
whole chromosome painting probes on primates, rodents, even-toed ungulates, and whales. Since
then, this type of chromosomal analysis has been widely used, permitting the establishment of
important chromosome homology maps, revealing interesting insights into chromosome
rearrangements during evolution, and allowing us to infer information on ancestral karyotypes.

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 CHAPTER: 19
Principles and Design of experiments

Experimental design
An experiment is a type of research method in which you manipulate one or more independent
variables and measure their effect on one or more dependent variables. Experimental design means
creating a set of procedures to test a hypothesis. A good experimental design requires a strong
understanding of the system you are studying. By first considering the variables and how they are
related (Step 1), you can make predictions that are specific and testable (Step 2).

How widely and finely you vary your independent variable (Step 3) will determine the level of
detail and the external validity of your results. Your decisions about randomization, experimental
controls, and independent vs repeated-measures designs (Step 4) will determine the internal
validity of your experiment.

Step 1: Define your research question and variables

You should begin with a specific research question in mind. You may need to spend time reading
about your field of study to identify knowledge gaps and to find questions that interest you.We
will work with two research question examples throughout this guide, one from health sciences
and one from ecology:

Example question 1: Phone use and sleep

You want to know how phone use before bedtime affects sleep patterns. Specifically, you ask how
the number of minutes a person uses their phone before sleep affects the number of hours they
sleep.

Example question 2: Temperature and soil respiration

You want to know how temperature affects soil respiration. Specifically, you ask how increased
air temperature near the soil surface affects the amount of carbon dioxide (CO2) respired from the
soil.

To translate your research question into an experimental hypothesis, you need to define the main
variables and make predictions about how they are related. Start by simply listing the independent
and dependent variables.

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 Research question Independent variable Dependent variable
Phone use and sleep Minutes of phone use before Hours of sleep per night
sleep
Temperature and soil Air temperature just above the CO2 respired from soil
respiration soil surface

Then you need to think about possible confounding variables and consider how you might control
for them in your experiment. Confounding variable How to control for it

Phone use and sleep Natural variation in sleep patterns among individuals. Control statistically:
measure the average difference between sleep with phone use and sleep with phone use rather than
the average amount of sleep per treatment group.

Temperature and soil respiration Soil moisture also affects respiration, and moisture can decrease
with increasing temperature. Control experimentally: monitor soil moisture and add water to
make sure that soil moisture is consistent across all treatment plots.

Finally, put these variables together into a diagram. Use arrows to show the possible relationships
between variables and include signs to show the expected direction of the relationships.

Here we predict that increasing phone use is negatively correlated with hours of sleep, and predict
an unknown influence of natural variation on hours of sleep.

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Here we predict a positive correlation between temperature and soil respiration and a negative
correlation between temperature and soil moisture, and predict that decreasing soil moisture will
lead to decreased soil respiration.

Step 2: Write your hypothesis

Now that you have a strong conceptual understanding of the system you are studying, you should
be able to write a specific, testable hypothesis that addresses your research question.

Null (H0) hypothesis Alternate (Ha) hypothesis

Phone use and sleep Phone use before sleep does Increasing phone use before
not correlate with the amount sleep leads to a decrease in
of sleep a person gets. sleep.
Temperature and soil Air temperature does not Increased air temperature
respiration correlate with soil respiration. leads to increased soil
respiration.

The next steps will describe how to design a controlled experiment. In a controlled experiment,
you must be able to:

1-Systematically and precisely manipulate the independent variable(s).

2-Precisely measure the dependent variable(s).

3-Control any potential confounding variables.

If your study system doesn’t match these criteria, there are other types of research you can use to
answer your research question.

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References:

1 "A Taste of ESPRESSO". Retrieved 15 September 2015.

2 "Media advisory: Press Conference to Announce Major Result from Brazilian
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 Important Terms and definitions
Research Methodology: The systematic process of planning, conducting, and analysing research
to answer scientific questions or solve problems in the field of life sciences.
Hypothesis: A testable statement or prediction that proposes an explanation for a phenomenon
being studied in research.
Experiment: A controlled procedure carried out to test a hypothesis and gather empirical
evidence.
Variable: Any characteristic or factor that can be measured, manipulated, or controlled in a
research study.
Control Group: A group in an experiment that does not receive the treatment being tested, used
as a standard of comparison for evaluating the effects of the treatment.
Experimental Group: A group in an experiment that receives the treatment or intervention being
tested, used to assess the effects of the treatment.
Data: Information collected or observed during a research study, often in the form of numerical
measurements or qualitative descriptions.
Quantitative Research: Research that focuses on collecting and analysing numerical data to
describe or explain phenomena in the life sciences.
Qualitative Research: Research that focuses on collecting and analysing non-numerical data to
understand the underlying meanings, patterns, or relationships in the life sciences.
Literature Review: A critical analysis of existing research and scholarly articles relevant to a
particular topic or research question.
Sampling: The process of selecting a subset of individuals or items from a larger population to be
included in a research study.
Random Sampling: A sampling technique in which every member of the population has an equal
chance of being selected for inclusion in the study.
Systematic error or deviation from the true value in research results, often influenced by factors
such as sampling methods, researcher preferences, or participant characteristics.
Validity: The extent to which a research study accurately measures or assesses what it claims to
measure or assess.
Reliability: The consistency or stability of research findings over time or across different
conditions, indicating the degree to which results can be trusted and replicated.

271
Ethics: Principles and guidelines governing the conduct of research involving human participants,
animals, or other subjects, ensuring their rights, welfare, and confidentiality are protected.
Informed Consent: Voluntary agreement by individuals to participate in a research study after
being fully informed about the purpose, procedures, risks, and benefits involved.
Institutional Review Board (IRB): A committee responsible for reviewing and approving research
protocols to ensure compliance with ethical standards and regulations.
Confidentiality: The protection of sensitive information obtained from research participants,
preventing unauthorized access or disclosure.
Peer Review: The process of evaluating the quality and validity of research manuscripts by experts
in the field before publication in scholarly journals.
Theory: A well-substantiated explanation or framework that organizes and explains a wide range
of observations or phenomena in the life sciences.
Deductive Reasoning: A logical process in which specific conclusions are drawn from general
principles or premises, often used in hypothesis testing and theory development.
Inductive Reasoning: A logical process in which general conclusions are inferred from specific
observations or evidence, often used in data analysis and interpretation.
Population: The entire group of individuals, items, or phenomena that meet specific criteria and
are the focus of a research study.
Sample: A subset of the population selected for inclusion in a research study, intended to
represent the larger population.
Replication: The process of repeating a research study or experiment to confirm or extend
previous findings, enhancing the reliability and validity of results.
Case Study: An in-depth analysis of a particular individual, group, or phenomenon, often used to
explore complex issues or provide detailed descriptions in the life sciences.
Correlation: A statistical relationship between two or more variables, indicating the degree and
direction of their association but not necessarily implying causation.
Causation: The relationship between cause and effect, where changes in one variable lead to
changes in another variable, often established through experimental research designs.
Experiment Design: The overall plan or structure of an experiment, including the selection of
variables, manipulation of treatments, and control of potential confounding factors.
Dependent Variable: The variable that is measured or observed in a research study, often
influenced by changes in the independent variable.

272
Independent Variable: The variable that is manipulated or controlled by the researcher in an
experiment, hypothesized to cause changes in the dependent variable.
Null Hypothesis (H0): A hypothesis that states there is no significant difference or effect between
groups or conditions in a research study, subject to rejection based on empirical evidence.
Alternative Hypothesis (H1): A hypothesis that states there is a significant difference or effect
between groups or conditions in a research study, supported when the null hypothesis is rejected.
Statistical Significance: The likelihood that an observed difference or relationship in research
results is not due to chance alone, typically assessed using inferential statistics.
Type I Error: A statistical error that occurs when the null hypothesis is incorrectly rejected, leading
to the false conclusion that a significant effect or difference exists when it does not.
Type II Error: A statistical error that occurs when the null hypothesis is incorrectly retained,
leading to the false conclusion that no significant effect or difference exists when it does.
P-value: A probability value that indicates the likelihood of obtaining the observed results in a
research study if the null hypothesis were true, typically compared to a predetermined
significance level.
Confidence Interval: A range of values within which the true population parameter is estimated
to lie with a certain level of confidence, based on sample data and statistical analysis.
Descriptive Statistics: Statistical methods used to summarize and describe the characteristics or
properties of data, such as measures of central tendency, variability, or distribution.
Inferential Statistics: Statistical methods used to make inferences or generalizations about
populations based on sample data, including hypothesis testing and estimation.
Parametric Test: A statistical test that makes certain assumptions about the distribution of data
and population parameters, typically used with interval or ratio data.
Nonparametric Test: A statistical test that does not make assumptions about the distribution of
data or population parameters, often used with ordinal or nominal data.
ANOVA (Analysis of Variance): A statistical technique used to analyse differences among group
means in a research study, assessing the variability within and between groups.
Regression Analysis: A statistical method used to model and analyse the relationship between
one or more independent variables and a dependent variable, predicting outcomes or estimating
effects.
Chi-Square Test: A statistical test used to determine whether there is a significant association
between two categorical variables in a research study, based on observed and expected
frequencies.

273
T-test: A statistical test used to compare the means of two independent or related groups in a
research study, determining whether there is a significant difference between them.
Meta-Analysis: A statistical technique used to systematically combine and analyse the results of
multiple independent studies on a specific research question or topic.
Systematic Review: A comprehensive and structured synthesis of existing research evidence on a
particular topic or research question, conducted using predefined methods and criteria.
Research Design: The overall plan or strategy for conducting a research study, including the
selection of methods, procedures, and techniques for data collection and analysis.

274
 LIST OF ABBREVIATIONS

Abbreviation Description
etc. (et cetera) and the others
i.e (Id est) that is Square
viz. (Videlicet) namely
et al. And co workers
BOD Biological Oxygen Demand
CM Centimetre
CTAB Cetyl trimethyl ammonium bromide
Cl Chloride
CFU Colony forming unit
DAPI Days After Pathogen Inoculation
DAS Days After Sowing
DAT Days After Transplanting
0C Degree Celsius
DMRT Duncan’s multiple Range test
EDTA Ethylene diamine tetraacetic acid
Fig. Figure
FW Fresh weight
G Gram
> Greater than
≥ Greater than equal to
Ha Hectare
H Hour
Hrs Hours
HCl Hydrochloric acid
< Less than
m° Metre
µl Micro litre
µm Micro meter
Mg Milligram

275
mL Millilitre
ml 1 ' Millilitre per Litre
Mm Millimetre
mM Millimole
Mt Million Tonnes
NBT Nitroblue tetrazolium chloride
No. Number
NB Nutrient Broth
O.D Optical Density
ml ' Per millilitres
min ' Per minute
% Percentage
PO Peroxidase
PAL Phenylalanine ammonia lyase
± Plus Minus
PPO Polyphenol oxidase
PDA Potato Dextrose Agar
PDB Potato Dextrose Broth
p.s.i Pound per square inch
ROS Reactive oxygen species
R Replication
Sr. No Serial number
Na Sodium
NaOH Sodium Hydroxide
NaOCl Sodium Hypochlorite
SOD Superoxide dismutase
TPC Total Phenol content
T Treatment
UV Ultra violet
w'v Weight'Volume
w'w Weight'weight
WP Wettable powder

276
 SCIENTIFIC SYMBOLS

Symbol Description
α (alpha) Significance level (usually 0.05)
β (beta) Type II error probability
μ Population mean
σ Population standard deviation
x̄ Sample mean
s Sample standard deviation
p Population proportion
q 1-p
n Sample size
Z Standard normal deviate
t Student's t-distribution
χ² Chi-square statistic
F F-statistic
r Pearson correlation coefficient
p-value Probability value
DNA Deoxyribonucleic acid
RNA Ribonucleic acid
mRNA Messenger RNA
tRNA Transfer RNA
rRNA Ribosomal RNA
cDNA Complementary DNA
g Gene
G Genotype
P Phenotype
A, T, C, G Nucleotides (Adenine, Thymine, Cytosine, Guanine)
Enzymes E
S Substrate
P Product
Km Michaelis constant

277
Vmax Maximum velocity
ATP Adenosine triphosphate
ADP Adenosine diphosphate
AMP Adenosine monophosphate
NAD⁺ Nicotinamide adenine dinucleotide (oxidized)
NADH Nicotinamide adenine dinucleotide (reduced)
FAD Flavin adenine dinucleotide
FADH₂ Flavin adenine dinucleotide (reduced)
pH Potential of hydrogen
kDa Kilodalton (unit of mass)
mg/mL Milligrams per millilitre (concentration)
µM Micromolar (concentration)
N Nitrogen
P Phosphorus
K Potassium
DO Dissolved oxygen
BOD Biological oxygen demand
COD Chemical oxygen demand
x Independent variable
y Dependent variable
± Plus or minus (standard error)
* Statistically significant difference

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