Biotechnology

DESIGN AND Laboratory Design

BASIC REQUIREMENTS and it’s
OF A TISSUE Requirements
CULTURE LABORATORY

Any laboratory designed for plant tissue culture or biotechnology must focus on cleanliness & maintaining of aseptic
condition. The essential 7 fundamental matter is the contamination free condition in all steps of the procedure. Any
laboratory, in which tissue culture techniques are performed, regardless of the specific purpose, must contain a
number of basic requirements. These are:

a. A general washing area

b. A media preparation, sterilization & storage area
c. Environmentally controlled incubators or culture rooms
d. An observation/data collection area
e. Acclimatization area

Location:
Location of the laboratory depends on the requirements of an individual & the space available in the existing building.
However, the following points need to be considered for the site selection of plant tissue culture lab establishment.
 The selected area should be relatively free from dust, smoke, molds, spores & chemicals.
 The laboratory should be far away from a plant pathology laboratory.
 Access to the lab should be through an outer hall room.
 Location should have well maintained lawn & disease free shrubs.

a. Washing area:
The washing area should contain good quality basin, large sink & well drainage facilities. It should have access to
dematerialized water & double distilled water. Space for drying ovens or racks, automated dishwashers, acid baths,
pipette washers & driers & storage cabinets should also be available in the washing area.

General guidelines for washing area:

1. Reusable glassware for tissue culture should be emptied immediately & need to be soaked in water. Media or
agar must never be allowed to dry on the glassware
2. All glassware containing corrosive chemicals or fixatives should be separated from the rest of the tissue
culture glassware.
3. All glasswares contaminated or coming into contact with microorganisms should be autoclaved before
washing.
4. The contents of any containers should be discarded immediately after completion of an experiment.
5. Flaks or beakers used for agar based media should be rinsed immediately after dispensing the media into
culture vassels so as to prevent drying of the residual agar in the beaker prior to washing.

Precaution at the time of acid cleaning:

1. Personnel concerned with acid clean must wear a full face mask & acid resistant apron & gloves.
2. Handling of acid should be done in a fume hood.
3. One should never add water to acid.
4. Acid should be added slowly to water for dilution.
5. After acid cleaning, if the solution in the acid becomes dark colored, it should be discarded.

b. Media Preparation Area:

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 1
This area comprises the central section of the laboratory, home to most of the activities. This area should have ample
storage space for the chemicals, culture vessels & glassware required for media preparation & dispensing. The general
laboratory section includes the area for media preparation for autoclaving the media & also for many of the activities
that relate to the handling of tissue culture materials. Laboratory equipments required for media preparation room are
as follows
1. Gas, water & electric supplies & compressed air & vacuum line.
2. Water heater
3. Different types of glasswares
4. Hot plate with magnetic stirrer
5. Coarse & sensitive balance
6. Spatula for use during weighing
7. Microwave oven for rapid heating media & agar mixture
8. pH meter
9. Distillation apparatus
10. De-ionizer
11. Kitchen timer for timing of sterilization
12. Metal racks for holding test tubes in the autoclave
13. Test tubes, flasks, plastic containers
14. Autoclave or cooker
15. Storage tank for distilled and /or de-ionized water.

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 2
 Fig. 1 A hypothetical layout of a tissue culture laboratory

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 3
Transfer Area/Inoculation room:
All the activities of sterile transfers are performed in this room. There must be a laminar air flow cabinet where all the
precautions should be taken to prevent entry of any contaminant into the culture vial during the process of inoculation
or subculture. Laminar air flow hoods are usually sterilized by switching on the hood and wipping the working
sueface with 70% ethyl alcohol for 15 minute before initiating any operation under the hood. Ultraviolet light (UV) is
sometimes installed to disinfect the area; this light should only be used when people and plant materials are not in the
room. This room is provided with:
1. Laminar air flow cabinet: Inoculation @ subculture by maintaining aseptic condition.
2. Steribed sterilizer, Sprit lamp/Bunsen burner: Sterilization of the knives, scalpels, forceps etc.
3. Stereo-microscope: Observe for specific part.
4. Etyl alcohol: Sterilization and flaming of small instruments.
5. Tiles/glass plates: Use during sterile cutting.
6. Hypochloride solution: Sterilization of plant material
7. Kitchen timer: Timing for sterilization.

c. Incubation Room/Culture Room:

This is the room where light, temperature, humidity are maintained. All of these environmental considerations will
vary depending on the size of the growth room.
Temperature: is an important consideration for the tissue culture and other factors like light, relative humidity, and
shelving depend on it. Generally temp. of the growth room remain in the range of 25± 2 oC. Temp. in the primary
growth room can be maintained by air conditioner.
Lighting facility: Intensity of light in the room can easily be maintained by using fluorescent light with timer.
However, most culture rooms are lighted at the 1000 lux (for 1000cft) with some going up 5000-10000 lux.
Light duration: 16-18 h/day.
Light quality: Spectral quality of light received by in vitro cultures is very important.
Relative humidity: Relative humidity (RH) is very difficult to control inside the room but humidifier can be used to
control humidity. Humidity inside the room should be 70-75%
Shelves: Shelving with primary growth rooms can vary depending upon the situations & explants grown. Wood is
recommended for the inexpensive easy to build shelves.
This room is provided with
1.Temperature control (25± 2oC)
2.Electricity supply essential for lighting, cooling and heating
3.Shelves for culture racks
4.Fluorescent tubes for lighting
5.Timer for regulating day length
6.Racks for culture vials
7.Rotary shaker for suspension cultures
8.Observations table.

d. Data collection Area:

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 4
Culture room is prepared by glsswall. Qualititative data could be collected from out side of the culture room through
the glasswall. The quantitative data could be collected from inside the cultureroom by following aseptic rules and
regulation.

e. Acclimatization area:
Plants regenerated from in vitro tissue cultures are transplanted to vermiculite pots. The potted plants are ultimately
transferred to greenhouses or growth cabinets and maintained for further observations under controlled conditions of
light, temperature and humidity.

Chemicals for culture media:

A. Inorganic elements:
a. Macro nutrient:
The need of macro nutrients is higher in tissue culture media. It provides both anion & cation for the plant cell. The
name of each element with available form & important functions are given below:

Sl. Name of the Available form Function

No macro nutrient
.
1. Nitrogen (N) KNO3 ,NH4 NO3 Both structural & functional role in protein synthesis
2. Phosphorus (P) KH2PO4 Activation in nucleotide synthesis
3. Potassium (K) KNO3 Essential for activation of many enzymes, maintenance of ionic
balance of the cell.
4. Calcium (Ca) CaCl2.2H2O Acts as a cofactor & largely bound to the cell wall & cell
membrane, Essential for cation-anion balance by counteracting
organic inorganic anions.
5. Magnessium (Mg) MgSO4.7H2O Essential for photosynthesis & many other enzymatic reactions.
6. Sulphur (S) MgSO4.7H2O,K2SO4 Functional role in protein synthesis.

b. Micro nutrients
Micro nutrient is essential for plant cell tissue growth. The name of elements, available salt combination & function
are given below:

Sl. Name Available form Function

No.
1. Zinc (Zn) ZnSO4.7H2O Act as a component of a number of enzymes, plays active role in
protein synthesis, specially in the synthesis of tryptophan
2. Manganese (Mn) MnSO4.4H2O Help in photosynthesis
3. Copper (Cu) CuSO4.5H2O Plays an important role in electron transport chain at the time of
photosynthesis
4. Molybdenum (Mo) It participates in the conversion of nitrate to ammonium
5. Boron (B) H3BO3 It is required for the synthesis of cell wall & cell membrane
6. Iron (Fe) FeSO4.5H2O Formation of protein, important for biosynthesis of chlorophyll
7. Cobalt (Co) CoCl2. 6H2O Helpful for nitrogen fixation
8. Chlorine (Cl) CaCl2. 2H2O To control the osmoregulation of cell development.

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 5
 B. Organic Components
a. Vitamins: Normally plants synthesis vitamins endogenously. When plant cells & tissues are grown on in
vitro condition some essential vitamins are absolutely required.
Sl.
Name of the vitamin Function
No.
Thiamine (vitamin-
1.
B1)
2. Nicotinic acid B20
3. Pyridoxin-HCl (B6) Promotion of cell growth &
4. Folic acid development.
5. Biotin
6. Riboflavin
7. Retinol (vitamin-A)

b. Myo-inositol: It has several functions like sugar transport, carbohydrate metabolism, membrane structure
& cell wall formation.

c. Sugar: It can be supplied in the form of sucrose, glucose, and fructose. It is a source of carbon.

d. Amino acid: Cultured tissues are normally capable of synthesis of amino acid. In spite of this, the
addition of amino acids to the media is important for stimulating cell growth. Unlike inorganic nitrogen,
amino acids are taken up more rapidly by plant cells. Glycine is the most common amino acid used in
different tissue culture media. Some of the other amino acids like glutamine, asparagines, cystine etc. are also
required for cell culture.

e. Plant growth regulators: Plant growth regulators are the organic molecules which have different
regulatory effects on growth & development in whole plants & plant tissues. It is the most critical component
of any culture media accepted that without regulators, in vitro culture is often impossible. Plant growth
regulators which are often used in plant tissue culture are the following.

i. Auxin: The major functions of auxin are cell division, cell elongation, organogenesis. It is frequently
used as a rooting hormone. The most frequently employed auxins are IAA (Indole-3-acetic acid), IBA
(Indole-3-butyric acid), NAA (Napthalene acetic acid) @ 2, 4-D (2 ,4-Dichlorophenoxy acetic acid). IAA
is a naturally occurring auxin is added in concentration of 0.01-10 mg/l. The most effective auxin of callus
proliferation for most cultures is 2, 4-D, but unfortunately it strongly suppresses organogenesis & should
not be used in experiments involving root & shoot initiation.

ii. Cytokinin: Cytokinins are derivatives of adenine, which promote cell division, regulate growth and
development in plant tissues. It is known as shooting hormone essential for induction of auxillary
branching and adventitious shoot formation. The most widely used cytokinins are kinetin, zeatin, BAP
(Benzyladenine), 2iP (2- isopentenyladenine).

iii. Other regulators: Other types of hormones which may be used in plant tissue culture include
gibberellins (GA3), which promotes shoot elongation, and internodal elongation, ethylene and abscisic
acid.

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 6
Major equipments and their function:
Sl.
Name of the equipments Function
No.
1. Autoclave machine, Pressure cooker Sterilization of media, glassware &small instrument.
2. Balance Measurement of chemical from the range of µgm to Kg
3. Hot plate magnetic stirrer To mix the chemical & other ingredient of media
4. pH meter To determine the pH of various chemicals & media
5. Refrigerator To store all sorts of temperature-sensitive chemical & stock solution.
6. Micro oven To melt agar, agarose & other gelling agents.
7. Hot air oven For dry heat sterilization of cell & suspension culture
8. Shaker Use for gentle rotation of cell& suspension culture
9. Filter sterilization unit with vacuum Filtration of thermoliable compound like growth regulator, vitamin,
pump amino acid etc.
10. To study the cell & tissue culture material at different stages of
Microscope
development
11. Luxmeter To measure the light intensity of the culture room
12. Thermometer To record the temperature reading of laboratory & culture room
13. Centrifuge machine To sediment cell & clean supernatant
14. Laminar air flow cabinet To avoid air remaining contaminant

General rules to be followed in a tissue culture laboratory:

1. A laboratory should have an inventory & a complete up-to-date record of all the equipments along with their
operating manual.
2. A laboratory should have an inventory & a complete up-to-date record of all the chemicals including the name
of manufacturer & grade.
3. All chemicals should be assigned to specific areas preferably by their alphabetical order.
4. Strong acid & bases should be stored separately.
5. Special handling or storage procedure should be posted in the records so that retrieving of chemical is easy,
because chemicals need storage at different temperatures ( for example room temperature 4 o, -20o C)
6. Chloroform, alcohol, phenol, which are volatile or toxic in nature, must be stored in a fume hood.
7. Chemicals which are hygroscopic in nature must be stored in desiccators in order to avoid caking.
8. Chemicals kept in refrigerator or freezers should be arranged either alphabetically or in small baskets.

Safety rules:
1. Eating, smoking and drinking is strictly prohibited in the tissue culture laboratory.
2. Toxic chemical must be handled with appropriate precautions and should be discarded into separate labeled
containers. e.g. Organic compounds, halogens etc.
3. Broken glass and scalpel blades must be disposed into individual marked containers.
4. Pipettes, tips, Pasteur pipettes and other things used in the lab should be first collected in autoclavable bags
and then it should be finally autoclaved and disposed in safe place.
5. Pipetting any solution should not be conducted without using any pipette.
6. First aid kits should be placed in every laboratory and every individual working in the laboratory should know
its location and how to use its contents.
7. Fire extinguishers should be provided in each laboratory.

Sourav Debnath
Associate Professor, Dept. of Biochemistry and Food Analysis, PSTU.
Email: [email protected] Page 7