Fluorescence Microscope- Principle, Instrumentation, Applications, Advantages, Limitations

Fluorescence microscope definition

Florescence microscopy is a special type of light microscopy that uses flourescence. A fluorescence
microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in
addition to, reflection and absorption to study properties of organic or inorganic substances. The
“fluorescence microscope” refers to any microscope that uses fluorescence to generate an image,
whether it is a more simple set up like an epifluorescence microscope, or a more complicated design
such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent
image.

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic
radiation while phosphorescence is a specific type of photoluminescence related to fluorescence.Unlike
fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs.

The fluorescence microscope was devised in the early part of the twentieth century by August Köhler,
Carl Reichert, and Heinrich Lehmann, among others.Most fluorescence microscopes in use are
epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are
done through the same light path (i.e. through the objective).Some materials (known as ‘auto-
fluorescent’ materials) exhibit fluorescence naturally, and do not need to be specially prepared for
fluorescence microscopy. Detection of auto-fluorescent materials relies mainly on selecting the
appropriate filters matching the natural excitation and emission wavelengths. For most practical
specimens, fluorescent dyes known as fluorophores which exhibit relatively intense fluorescence at
specific wavelengths, are used to tag selected parts of the specimen. This is similar to the way that
normal optical dyes or stains are used in light microscopy. These fluorophores can be extremely
selective, to the point where, in some cases, single molecules are detected.Due to the high degree of
contrast available by using fluorophores, fluorescence microscopy has become an important tool in the
life sciences. Here, specimens can be extremely complicated in both composition and structure, so their
analysis is aided by the additional contrast provided by fluorescence tagging. In the second half of the
20th century, two techniques were developed that greatly added to the capabilities of fluorescence
microscopy:

Immunofluorescence: engineered fluorescent antibodies are used to tag specific antigens/proteins

through their specific chemical interactions.

Expressible fluorescent proteins: the DNA of a cell is modified so that a specific fluorescent tag is added
to a protein expressed by the cell. This protein can then be tracked using fluorescence microscopy.

The fluorophores themselves are usually organic molecules with conjugated aromatic groups as part of
their structure. A common example of a fluorophore is fluorescein isothiocyanate, or FITC. Some
fluorophores can also change their fluorescence properties in the presence of other compounds,
allowing those other compounds to be detected indirectly. An example of this is calcium imaging, where
fluorophores having an aminopolycarboxylic acid structure bind specifically to Ca2+ ions and change
their fluorescence properties

Principle of Fluorescence Microscopy

Most cellular components are colorless and cannot be clearly distinguished under a microscope. The
basic premise of fluorescence microscopy is to stain the components with dyes.

Fluorescent dyes, also known as fluorophores or fluorochromes, are molecules that absorb excitation
light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The
delay between absorption and emission is negligible, generally on the order of nanoseconds.

The emission light can then be filtered from the excitation light to reveal the location of the
fluorophores.

Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites
fluorescence species in the sample, which then emit light of a longer wavelength.

The image produced is based on the second light source or the emission wavelength of the fluorescent
species rather than from the light originally used to illuminate, and excite, the sample.
Light of the excitation wavelength is focused on the specimen through the objective lens. The
fluorescence emitted by the specimen is focused on the detector by the objective. Since most of the
excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective
together with the emitted light.

Parts of Fluorescence
Microscope

Fluorescence Microscope from LEAM Solution Inc

Typical components of a fluorescence microscope are:

1.Fluorescent dyes (Fluorophore)

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.

Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with
several π bonds.

Many fluorescent stains have been designed for a range of biological molecules.

Some of these are small molecules that are intrinsically fluorescent and bind a biological molecule of
interest. Major examples of these are nucleic acid stains like DAPI and Hoechst, phalloidin which is used
to stain actin fibers in mammalian cells.

2. light source

Four main types of light sources are used, including xenon arc lamps or mercury-vapor lamps with an
excitation filter, lasers, and high- power LEDs.

Lasers are mostly used for complex fluorescence microscopy techniques, while xenon lamps, and
mercury lamps, and LEDs with a dichroic excitation filter are commonly used for wide-field
epifluorescence microscopes.

3. The excitation filter

The exciter is typically a bandpass filter that passes only the wavelengths absorbed by the fluorophore,
thus minimizing the excitation of other sources of fluorescence and blocking excitation light in the
fluorescence emission band.

4. The dichroic mirror

A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small
range of colors while reflecting other colors.

5. The emission filter.

The emitter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore
and blocks all undesired light outside this band – especially the excitation light.

By blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence,
optical filters ensure the darkest background.

Types of Fluorescence Microscopes

While all fluorescence microscopes work by roughly the same core principles, there are several different
subtypes, with varying levels of complexity and specialization.

Wide-field epifluorescence microscopy

In this method, the entire specimen is illuminated by the light of a single wavelength (the excitation
wavelength) at one time. The parts of the specimen that fluoresce at this wavelength will absorb it, then
re-emit it at another wavelength (the emission wavelength). The excitation light can also reflect off the
sample, or pass through it. The basic elements of this microscope are very similar to a normal reflected
light microscope. However, fluorescence microscopes contain additional optics that ensure the sample is
illuminated by only the excitation wavelength, and that allows only light of the emission wavelength to
reach the detector or observer. Wide-field epifluorescence microscopes are the simplest and most
common of the fluorescence microscopes. Often, epifluorescence microscopy is a simple add-on to an
existing optical microscope.

Confocal fluorescence microscopy

In a standard confocal microscope, an additional set of optics is used so that only light from a small
point, in a narrow focal plane in the specimen reaches the observer, and any out of focus light is
rejected. A set of electronically controlled elements quickly scans the location of this point across the
entire specimen. The resulting image consists of a thin slice through the specimen, which can be
assembled into a three-dimensional representation if the depth of the focal plane is also scanned. A
confocal fluorescence microscope works using the same mechanism, but with the addition of optical
elements like those used in wide-field epifluorescence microscopy. In addition, some confocal
fluorescence microscopes use a laser as the source, giving the additional advantage of very high
brightness and a narrow excitation wavelength band. Confocal imaging is particularly useful in
fluorescence microscopy, since stray fluorescence emission from locations in front of and behind the
focal plane are rejected.

Total internal reflection fluorescence microscopy (TIRF)

When a beam of light reflects off of an interface, for example the interface of a glass coverslip and a
water-based medium below it, an evanescent wave of light is formed on the water side of the coverslip.
This wave, which is the same wavelength as the reflected light, is extinguished within 100-200 nm of the
interface. So when using this evanescent wave to excite fluorescent molecules on the water side of the
cover glass, only molecules within 100-200 nm of the glass are illuminated.This illumination technique,
known as total internal reflection (or TIRF), therefore is extremely selective in the sense that it only
detects molecules that are very close to the cover glass. As an example, this technique can be used to
image cells which are attached to the cover glass, without stray fluorescence emission coming from the
background medium.

Applications of Fluorescence Microscope

To identify structures in fixed and live biological samples.

Fluorescence microscopy is a common tool for today’s life science research because it allows the use of
multicolor staining, labeling of structures within cells, and the measurement of the physiological state of
a cell.

Flourescence Tagging

This is the sample preparation procedure on how to fluorescently label the structures of interest.Once
fluorescent tagging is accomplished, analysis by fluorescence microscopy is done similarly to analysis
using a normal optical microscope.

In general, these steps are:

After selecting a fluorophore and method of tagging, select microscope optics that are matched for the
fluorophore. A wide variety of filters are available, and microscope manufacturers have created guides
for matching filters to fluorophores.

Clean the microscope slide and cover slip (after first ensuring that these elements are not
autofluorescent in the wavelength range of interest).

Place a droplet of the sample on the slide, then gently place a cover slip over the droplet.

Start at low magnification, adjusting the focus, illumination, and x-y position as needed.

Increase magnification to the desired level and continue to adjust focus and x-y position.

Collect digital images of the areas of interest, making sure to name the files with descriptive titles, or
make note of the file names and descriptions, for later retrieval.

While performing the analysis, it is important to keep in mind that fluorophores can be sensitive to
photobleaching. This process, in which fluorophores permanently lose their fluorescence properties
after a certain amount of exposure to the excitation light, is caused by chemical reactions within the
fluorophore, or between it and the surrounding medium.

Fluorophores have varying levels of susceptibility to photobleaching, so in some cases, the problem can
be avoided by switching to a more stable dye. If not, photobleaching can also be mitigated by reducing
the exposure time or the exposure intensity, by using attenuating filters or a less intense light source.
For quantitative analysis, when the absolute intensity of emitted light needs to be measured, a curve of
photobleaching vs. time can be generated, then the measured data can be corrected based on this
curve.

Advantages of Fluorescence Microscope

Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in
live-cell imaging.

This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-
fluorescing material.

The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.

Different molecules can now be stained with different colors, allowing multiple types of the molecule to
be tracked simultaneously.

These factors combine to give fluorescence microscopy a clear advantage over other optical imaging
techniques, for both in vitro and in vivo imaging.

Limitations of Fluorescence Microscope

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching.
Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons
excited during fluorescence.

Cells are susceptible to phototoxicity, particularly with short-wavelength light. Furthermore, fluorescent
molecules have a tendency to generate reactive chemical species when under illumination which
enhances the phototoxic effect.

Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows
observation of the specific structures which have been labeled for fluorescence.

Care of a fluorescence microscope

The guidelines for caring for a fluorescence microscope are very similar to those for an optical
microscope:
Keep dust and other contamination away from the microscope by using a dust cover when it is not in
use.

Avoid touching the optical components with bare hands, and instead use non-powdered gloves when
handling these components. The optics in the filter cube are generally very sensitive to scratches and
contamination, so should be handled with care.

When replacing or installing optical components, be mindful that they may have a correct orientation
relative to the light path. (For example, the side of the dichroic mirror that should be facing the light
path is usually marked in some way.)

If any optical components get dirty, clean them only using the methods recommended by the
manufacturer.

Avoid bumping the microscope, since the optical elements in it can be knocked out of alignment.

Some alignment and maintenance should only be done by trained technicians.

Membrane Filtration

The Membrane filter is also known as the molecular or biological filter. It is an effective, accepted
technique for the filtration of fluid samples.The quality of water and the quantity of microorganisms can
be determined by using this membrane filtration method. The main purpose of the Membrane filtration
technique is to ascertain the residence or absence of a particular coliform group that is usually existing
in wastewater and groundwater. A membrane filter is 150μm thick and contains about millions of
microscopic pores. The diameter of these spores is uniform. Based on the requirement the size of these
pores is adjusted, during the process of polymerization.The most widely accepted membrane filter
possesses a pore size of 0.22μm and 0.45μm.High tensile strength polymers such as cellulose acetate,
cellulose nitrate, or polysulfone are the main constituents of the membrane filter. The contaminants
which are larger than the pore size are trapped on the surface of the membrane filter, that’s how they
filtrate the liquids. If the desired particles are larger than the contaminant then we can trap the desired
particles or product on the membrane filter by decreasing the pore size. While the contaminants will
pass through the membrane.

Membrane filtration mechanism

During membrane filtration, a part of fluid termed as permeate (filtrate) moves within the membrane,
while other components are expelled by the membrane and clutched in the retentate (concentrate)
steam.

Membrane filtration unit also known as the “Membrane filtration assembly”.

It consists of a funnel, locking ring, carbon disc, stainless base, rubber stopper, filter flask, and Vacuum
pump.

The funnel is located at the top, which is used to pass the water sample.

A locking ring or clamp is located at the end of this stainless funnel. It controls the flow of the essay
liquid sample.

Over the carbon disk, a membrane filter is located. This carbon disc and the membrane filter is held by
the stainless base.

A rubber stopper is located at the base that controls the water flow to the filter flask.

A Vacuum motor is connected with the filtration unit by a connector which is located at one end of the
filter flask.

The Vacuum produces a negative force which allows the suction of filtrate within the membrane filter.
Types of membrane filtration

There are present mainly four types of membrane filter. These filters are classified based on their pore
size such as;

Microfiltration

Ultrafiltration

Nanofiltration

Reverse osmosis (RO)

Microfiltration

It separates those particles that have a size range of 0.1 to 10 μm.

From the approximate molecular weight, it separates those macromolecules which has a molecular
weight of less than 100,000 g/mol.

It is designed to separate sediment, algae, protozoa or large bacteria from the supplied liquid sample.

It is a pre-treatment process of ultrafiltration and a post-treatment process for granular media filtration.

Application of Microfiltration

In the water treatment plant, it is used to separate pathogens such as the protozoa Cryptosporidium
and Giardia lamblia, etc.

In industries, it is used for the cold sterilization of beverages and pharmaceuticals. It basically eliminates
bacteria and other undesired suspensions from liquids such as juice, wine, and beer in particular.

It is also used for petroleum refining. It removes particulates from flue gases.

In the dairy industry, it is used to remove bacteria and the associated spores from milk.

Microfiltration is also used for the Clarification and purification of cell broths. It separates the
macromolecules from proteins, large molecules, or cell debris.

It is used for clarification of dextrose.

Paints and Adhesives industries also use Microfiltration for their production.
Ultrafiltration

The pore size of an Ultrafiltration membrane ranges from 0.1 μm to 0.01 μm.

It is designed to eliminate proteins, endotoxins, viruses, and silica.

Generally, it retains those Suspended solids and solutes which possess a high molecular weight and pass
those solutes that have a low molecular weight such as water.

Application of Ultrafiltration (UF)

UF is used for the production of potable water. It removes the particulates and macromolecules from
raw water.

In dairy industries, it is used for the production of Protein concentration. During the cheese whey
processing, it obtains the whey protein concentrate (WPC) and lactose-rich permeate.

It separates effluent from the paper pulp mill.

Used in the production of Cheese.

It helps in the removal of bacteria from the milk.

It is also used in wastewater treatment plants.

Helps in Enzyme recovery.

Used for the clarification and concentration of Fruit juice.

In medical laboratories used for Dialysis and other blood treatments.

Used for Desalting and solvent-exchange of proteins.

Nanofiltration

The pore size of a Nanofiltration membrane ranges from 0.001 μm to 0.01 μm. The pore size of the
Nanofiltration membrane is smaller than microfiltration and ultrafiltration.

It is designed to separate multivalent ions, synthetic dyes, sugars and specific salts.

During the development, the dimensions of pores can be controlled by pH, temperature, and time.

The density of pores ranges from 1 to 106 pores per cm2.

It is made of polyethylene terephthalate and other related stuff.

Application of Nanofiltration

In fine chemistry and Pharmaceuticals industries, it is used for the recovery and management of Non-
thermal solvents, Room temperature solvent exchange.

In Oil and Petroleum chemistry, it is used to remove tar components in feed and for the Purification of
gas condensates.

In Bulk Chemistry, it is used to polish the Product, and for the Continuous recovery of homogeneous
catalysts.

In the Natural Essential Oils industries, it is used for the Fractionation of crude extracts and for the
Enrichment of natural compounds Gentle Separations.

In the Medicine industries, it is used to extract the amino acids and lipids from blood and other cell
culture.

Reverse osmosis (RO)

The pore size of a Reverse osmosis membrane ranges from 0.0001 μm to 0.001 μm. It has the finest
separation membrane.

It can retain all molecules except for water.

It required osmotic pressure due to the small size of the pores.

Application of Reverse osmosis

In households, it is used in drinking water purification systems.

It is also used in solar-powered desalination unit to produce potable water from saline water.

Used in reverse osmosis water purification unit (ROWPU) which is a self-contained water treatment
plant.

It is also used to purify the rainwater which is collected from storm drains.

In power plants, it is used to remove minerals from boiler water.

RO also used to produce deionized water.

In food industries, it is also used for the desalination of different products.

In Maple syrup production it is used to remove water from sap.

Use for the production of Low alcoholic beer.

Used in small-scale hydrogen production. It prevents the formation of mineral deposits on the surface of
electrodes.
Membrane Filtration Method

First, collect the sample and dilute it.

Select the suitable nutrient medium for isolation and enumeration of different pathogenic
microorganisms.

Transfer the medium into an absorbent pad containing a Petri plate. Immerse the absorbent pad within
a liquid broth. The saturated absorbent pad will promote microbial growth.

Use a sterilized forceps to place the membrane filter over the carbon disk.

Sterilize the opening of the sample bottles by flame and pour the sample through the funnel.

To draw the sample through the filter turn on the vacuum.

After that remove the filter from the funnel by using sterile forceps and placed it over the prepared petri
dish.

Incubate the Petri plate for an appropriate period of time and at the desired temperature.

Perform Quantitative analysis, use a colony counter to count the numbers of colonies on the Petri plate.

Perform Qualitative analysis, to identify the isolated colony and for water quality check. Subculture the
isolated colony, stain them, observe under microscope, and perform further biochemical tests to
distinguish the distinct type.

Advantages of Membrane Filtration

It allows the filtration of any volumes of non-turbid water through the disk.

This method is inexpensive.

No requirement for chemicals.

Can remove 90–100% pathogens from the water sample.

This method is more energy efficient.

It doesn’t denature the proteins.

Heat sensitive media can be sterilized by using this method.

It allows the isolation and enumeration of bacterial colonies by transferring the disk from one medium
to another.

As compared to the conventional MPN standard methods, membrane filtration provides a more rapid
result. It takes 24 hours to provide the result.

It takes less time as compared to the MPN method.

It provides relevant and reliable results.

Disadvantages of Membrane Filtration

The turbid water can not be used in membrane filtration.

There may be a risk of bacterial abundance, as the water carries numerous microorganisms.

Glass filters are breakable and can break quickly.

The membrane filters can crack easily.

Only liquids are sterilized by this method.

Filters are costly to repair, mainly nano-filters.

Constitutional restrictions of supplies used in filters alter the effectiveness of this process such as
damage of glass filters, fracture of the membrane filter, and consumption of the filtrate by Sietz filter.

Require a high differential pressure.

Clogging can occur.

Application of Membrane Filtration

In industries and laboratories, it is used to sterilize the heat-labile fluid materials.

Most effective and acceptable method for filtration of drinking water.

In the pharmaceutical, cosmetics, electronics, and food and beverage industries is is used to monitor the
bacterial cells.

Used in wastewater treatment.

Used in cold sterilization of beverages and pharmaceuticals.

Used for separation of milk fraction.

Used to concentrating the proteins.

Used for defeating skimmed milk and whey.

Used for the partial demineralization of whey.

Anaerobic bacteria are bacteria that cannot grow in the presence of oxygen. They are the
common causes of infection and special precautions are taken for their isolation and culture in
clinical diagnosis. Cultivation of the microorganism causes a challenge when the specimen
comes in contact with air

Anaerobic Incubation
Main Principle: reduce the O2 content of culture medium and remove any oxygen already present inside
the system or in the medium .

Oxygen is ubiquitous in the air so special methods are needed to culture anaerobic microorganisms. A
number of procedure are available for reducing the O2 content of cultures; some simple but suitable
mainly for less sensitive organisms, others more complex but necessary for growth of strict anaerobes.

Bottles or tubes filled completely to the top with culture medium and provided with tightly fitting
stopper. Suitable for organisms not too sensitive to small amounts of oxygen.

Addition of a reducing agent that reacts with oxygen and reduces it to water e.g., Thioglycolate in
thioglycolate broth. After thioglycolate reacts with oxygen throughout the tube, oxygen can penetrate
only near the top of the tube where the medium contacts air.

Obligate aerobes grow only at the top of such tubes.

Facultative organisms grow throughout the tube but best near the top.

Microaerophiles grow near the top but not right at the top.

Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.

Stringent anaerobes
can be grown only by
taking special
precautions to
exclude all atmospheric oxygen from the medium. Such an environment can be established by using one
of the following methods:

1. Pre-reduced media

During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved
oxygen. A reducing agent e.g., cysteine, is added to further lower the oxygen content. Oxygen free N2 is
bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes which are
being flushed with oxygen – free nitrogen, stoppered tightly, and sterilized by autoclaving. Such tubes
are continuously flushed with oxygen free CO2 by means of a cannula, restoppered, and incubated.

2. Anaerobic Chambers

This refers to a plastic anaerobic glove box that contains an atmosphere of H2, CO2, and N2. Culture
media are placed within the chamber by means of an air lock which can be evacuated and refilled with
N2. Any oxygen in the media is slowly removed by reaction with hydrogren, forming water; this reaction
is aided by a palladium catalyst. After being rendered oxygen free, the media are inoculated within the
chamber (by means of the glove ports) and incubated (also within the chamber).

3. Anaerobic Jar

Anaerobic jar is a heavy- walled jar with a gas tight seal within which tubes, plates, or other containers
to be incubated are placed along with H2 and CO2 generating system (GasPak system) . After the jar is
sealed oxygen present in the atmosphere inside jar and dissolved in the culture medium, is gradually
used up through reaction with the hydrogen in the presence of catalyst. The air in the jar is replaced
with a mixture of H2 and CO2, thus leading to anoxic conditions. At present, it is the most commonly
used method for anaerobiosis; they are of two types:

GasPak Anaerobic System:

Components of GasPak Anaerobic System

1. polycarbonate jar (or anaerobic container)

2. a lid with a gasket to prevent airflow (for jar system)

3 Indicator strip (a strip impregnated with an oxidation-reduction indicator such as methylene blue or
resazurin)

4. disposable gas generating pouch (a pouch containing sodium borohydride and sodium bicarbonate)

5. a palladium catalyst

Principle
Inoculated plates or tubes are placed inside the polycarbonate jar or anaerobic container along with gas
generator sachet and indicator strip and is sealed completely. In the presence of water, chemicals
present inside the sachet i.e. sodium bicarbonate (NaHCO3) and sodium borohydride (NaBH4) react
chemically producing hydrogen and carbon dioxide gas.

The
hydrogen
thus
produced
reacts with oxygen present inside the jar producing water (which forms as condensation on the inside of
the jar).

2H2 + O₂ + catalyst = 2H2O

This reaction is catalyzed by the element palladium, which is attached to the underside of the lid of the
jar. The carbon dioxide replaces the removed oxygen, creating a completely anaerobic environment.
McIntosh and Fildes’ Anaerobic Jar

The anaerobic jar consists of a 8*5 inch (20*12.5cm) jar of stout glass or metal with a tight
fitting metal lid. The lid can be clamped airtight with a screw and is fitted with two tubes with
taps, one for introduction of gas inside (inlet) and the other as outlet for vacuum valve. A
thermometer is used to measure its internal temperature. A pressure gauge is used to measure the
internal pressure. The lid also contains two terminals that can be connected to an electric supply.
A capsule containing alumina pellets coated with palladium (palladinised alumina) is suspended
under the lid by stout wires which are connected with the terminals to heat the catalyst for its
activity. Nowadays, catalyst active at room temperature is also available.

Working Principle of McIntosh and Fildes’ Anaerobic Jar

It works on physical evacuation and replacement where the air inside the chamber is evacuated
and replaced with their mixture of gases 5% CO2, 10% H and 85% N. It is practically impossible
to evacuate all the air so some amount of O2will still be left behind. The residual oxygen is
converted to water using spongy palladium or platinum catalyst. The catalyst act as a catalyzing
agent causing slow combination of hydrogen and oxygen to form water. Reduced methylene blue
is generally used ad indicator(it is a mixture of NaOH, methylene blue and glucose). It becomes
colourless anaerobically but becomes blue when exposed to O2.

Procedure of McIntosh and Fields Jar

The inoculated culture plates are kept inside the jar along with an indicator.
The lid is screw tightened.
The inlet tube is closed and the outlet tube is connected to avacuum pump (at least three
quarters of the air of the jar can be removed).
The outlet tap is tightly closed when the pressure on the vacuum gauge is reduced to
100mmHG
 The inlet tap is connected to hydrogen supply and opened. Hydrogen is passed through a
small wash bottle.
The reduced pressure is increased to 760mmHG (i.e., atmospheric) by monitoring on the
vacuum gauze as 0. The electric terminals for heating the paladinized crystals are
switched on (when the room temperature is used, heat is not required).
The catalyst helps the combination of hydrogen and oxygen to form water. This process is
allowed to continue for 20minutes.

The McIntosh and Fields jar are incubated at 37oC for 48hrs.
Reduced methylene blue indicator is used to check the efficacy of anaerobiasis. A tube
containing reduced methylene blue solution had to be kept inside the jar along with the
culture plates. Methylene blue is colorless in reduced conditions and turns blue when
oxidized.

The function of the catalyst is to stimulate the reaction of H2 with O2 to H2O. The cold catalyst
is used to create low oxygen atmospheres within anaerobic gas jar.

Benefits include:

1. Safety in use because the maximum operating temperature is lower than the flash point of
hydrogen gas.

2. Acts as an efficient heat sink.

3. Helps to create anaerobic conditions quickly.

Advantages of GasPak over the conventional equipment

Gaspak jar is much simpler technique than the McIntosh and Filde’s anaerobic jar where one
needs to pump gases in and out.
MICROBIAL UNITS OF MEASUREMENTS

Microorganisms are often very small even when compared to microscopic cells from animals.
They are about 1/10th the size of a typical human cell.

To put a numerical value an microbial size, most measurements of microbes are done with the
unit of measure of micrometer(µm) which is one millionth of a meter (one 2500th of an inch).
Most microbes are around 1 micrometer in size. Viruses are typically 1/10th that size.

However, length is not the only measurement that pertains to microbes. Microbial genomes are
also measured. DNA is measured in base pairs of DNA. For example the common intestinal
bacteria, such as Escherichia coli is 4.6 million base pair (bp).

Also, microbes are usually not weighed individually, but can be as an aggregate for various
experiments. An estimate of the weight of an individual microbe can be made by estimating the
number of microbes. This is especially important for biomass studies where the units of
measurement are in units like pico, 10-12 of a kilogram (kg), nanogram 10-9 of a kg and
microgram 106 of a kg.

Microbial growth is an important measure in understanding microbes. Microbial growth is the

division of one microbe into two daughter cells in a process called binary fission. As a result,
doubling of the microbial population occurs. The microbial population undergoes exponential
growth if the number surviving from the division exceeds unity on average. The measurement of
an exponential microbial growth curve in batch culture requires bacterial enumeration (cell
counting) by direct and individual (microscopic, flow cytometry) or direct and bulk (biomass),
indirect and individual (colony counting) or indirect and bulk (most probable number, turbidity,
nutrient uptake) methods.

The unit for the measurements listed above excluding microscopic is colony forming unit (cfu).
Colony-forming unit is used to estimate the number of viable cells in a sample. Counting with
colony-forming units requires culturing the microbes and counting only viable cells which is in
contrast with microscopic examination which counts all cells, living or dead
BIOASSAY/ BIOLOGICAL STANDARDIZATION

Bioassay

Bioassay is an assay designed to analyze any compoundby use of a suitable biological system
like animal tissues, microbes, etc. Bioassay can be defined as estimation or determination of
concentration or potency of a physical, chemical or biological substance (agent) by means of
measuring and comparing the magnitude of the response of the test with that of standard over a
suitable biological system under standard set of conditions. Biological assays or biological
standardizations or simply bioassays are methods used for estimation of the potency of
substances by observing their pharmacological effects on living organisms (in vivo) or isolated
tissues (in vitro) and comparing the effect of these substances of unknown potency to the effect
of a standard. In this analysis, the response produced by the test compound is compared with that
of standard sample the way similar to other analytical methods but here the biological system is
involved in the determination.

Bioassay is assessment of a biological substance. It is a type of scientific experiment typically

conducted to measure the effects of a substance on a living organism and is essential in the
development of new drugs and in monitoring environmental pollutants. Bioassays are based
upon the use of biological responses as detection system for biologically active substances. In the
simplest form, it is used to assay the presence and concentration of a particular substance by
comparison with a known amount of the same substance. Both are procedures by which the
potency or the nature of a substance is estimated by studying its effects on living matter.
Bioassay is a procedure for the determination of the concentration of a particular constitution of
a mixture.

An assay is an investigative (analytic) procedure in laboratory medicine, pharmacology,

environmental biology, molecular biology and biotechnologyfor qualitatively assessing or
quantitatively measuringthe presence, amount or functional activity of a target entity (the
analyte).The analyte can be a drug, an enzyme, a biochemical substance or a cell in an organism
or organic sample. The measured entity is generally called the analyte, the measurand or the
target of the assay.The assay usually aims to measure an intensive property of the analyte and
express it in the relevant measurement unit (e.g molarity, density, functional activity in enzyme
international units, degree of some effect in comparison to a standard, etc)

Principle of Bioassay

The bioassay compares the test sample with a same internationally applicable standard
substance.It determines the quantity of test sample required to produce an equivalent biological
response to that of standard substance.

Advantages of Bioassay

i. They help to determine the concentration and also the potency of the sample.Potency is a term
which denotes activity of the compound per molecule basisi.e if a compound shows better
activity at minute concentration, greater is the potency and if its activity is low at lower
concentrations, lesser is the potency.

ii. It is especially used to standardize drugs, vaccines, toxins or poisons, disinfectants, antiseptics
etc,as these are all used over biological system in some or other form.

iii. These also help determine the specificity of a compound to be usede.g Penicillin is effective
against gram positive but not on gram negative.Testing of infected patients’ sputum helps
determine which antibiotic be given for quick recovery.

iv. Certain complex compounds like Vitamin B-12 which cannot be analysed by simple assay
techniques but can be effectively estimated by bioassays.

v. Sometimes the chemical composition of samples is different but has some biological activity.

vi. They are used to analyse samples where no other methods of assays are available.

Types of bioassay

Direct Assay

Indirect Assay based on quantitative response

 Indirect Assay based onQuantal responses

Direct Assay

In this type of bioassay, the standard sufficiently produces measurable and specific
response. The response must be clear, easily recognized and directly measured
Indirect assay

For quantitative response, the relationship between the dose and the response is first
ascertained. Then the dose corresponding to a given response is obtained from the
relation for each preparation separately.
Quantal

For quantal response, the assay involves all or none response. These can be bioassayed by
end point method. Quantal response are population response based on all or nothing
response such as death.

Various Bioassay methods

Graded Assay

Matching method

Bracketing method

Interpolation method

Three point Bioassay

Four point Bioassay

Seed Bioassay

Antimicrobial Assay

Antifungal Assay –cylinder plate method and paper disc method

 Antimitotic Assay

Introduction to Antibiotics disc preparation

Antibiotics are compounds synthesized naturally and artificially that have an inhibitory action on
other microorganisms. Penicillin was the first identified antibiotics from the fungus known as
Penicillium notatum. Since then many antibiotics have been identified and tested. A good antibiotic
should be effective against wide range of microbes, have less side effects, it should be highly stable
and should be readily absorbed by the body tissues. The antibiotics are classified on basis of mode of
action as,

Cell wall inhibitors

Cell membrane inhibitors

Nucleic acid synthesis inhibitors

Protein synthesis inhibitors

Metabolic inhibitors

Antibiotic sensitivity test: With the introduction of a variety of antimicrobial it became necessary to
perform the antimicrobial susceptibility test as a routine. For this, the antimicrobial is allowed to
diffuse out into the medium and allowed to interact in a plate freshly seeded with the test organism.

Methods of antibiotic susceptibility testing: The methods of antimicrobial susceptibility testing are
divided into types based on the principle of testing involved,

1) Diffusion: Strokes method, Kirby-Bauer method

2) Dilution: Broth dilution, Agar dilution.

3) Diffusion and dilution: Epsilometer test (E-test) method.

Antimicrobial resistance: Antimicrobial resistance is the resistance of a microorganism to an

antimicrobial medicine to which it was previously sensitive. Resistant organisms are able to
withstand attack by antimicrobial medicines, so the standard treatments become ineffective and
infection persists even after treatment. Antimicrobial resistance is the cause of the use, particularly
misuse, of antimicrobial medicines and it develops when the microorganism mutates or acquires a
resistance gene.

Procedure for the preparation of Antibiotic discs

1. Preparation of filter paper discs

Filter paper used for antibiotic discs include Whatman filter paper No.3. Others are
chromatographic paper and blotting paper. The preparation involves punching of holes of
approximately 6mm diameter in the Whatman filter paper which are placed in a petri dish and
sterilized in autoclave.

2. Preparation of Antibiotic solutions

Antibiotics may be received as powders or tablets. It is recommended to obtain pure antibiotics

from commercial sources, and not use injectable solutions. Powders must be accurately weighed
and dissolved in the appropriate diluents to yield the required concentration known as stock
solutions, using sterile glassware.

3. Impregnation of paper disc

Using a mechanical pipette, a fixed volume of antibiotic solution is loaded on each sterile disc
one by onewhere the disc is already placed in petri dish approximately 5mm apart. Precautions
are taken that the tip gets slight contact with the disc. Another method of impregnating is
immersion method where blank discs are soaked in known concentration of antibiotic solution
and then allowed to dry.

4. Drying and Storage

Without covering the petri dishes, the discs are allowed to dry in a clean incubator at 37oC for
10-15 minutes. The disc is ready to use in sensitivity test. After drying, discs can be placed in a
sterile air-tight labeled container with desiccant at the bottom. A layer of sterile cotton is placed
over the desiccant to avoid contact with the discs. The discs can be stored in freezer.