Setting up of a Tissue Culture Laboratory | Biotechnology

In this article we will discuss about the setting up of a tissue culture laboratory that needs proper planning and depends upon space availability, volume of work to be carried out and funds. The available laboratory space is divided into two distinct laboratory areas i.e., media preparation area and aseptic technique.

1. Media Preparation Area:

Laboratory tables or benches and revolving stools with adjustable height, Hot plate and magnetic stirrers, analytical loading single pan balance-with precision of 0.001 g, weighing range 0.1 mg- 80g, digital read out, Top pan loading balance for quick weighing, range 100 mg- 500g capacity, sensitivity 0.1 g, Refrigerator and freezer, Water purification and storage system, Glassware washing facility with proper drainage, Gas outlet, Electric hot-air oven range upto 250 °C and microwave oven, Digital pH meter, Range 0-14 pH, accuracy 0.1 pH, temperature compensation 0- 100°C, Autoclave preferably horizontal., Continuous supply of single and double distilled water.

Storing glasswares, plastic wares, chemicals, plugs and appliances required for media preparation are as follows:

Requirements:

Culture tubes/conical flasks/petri dishes of various capacities, Measuring cylinders- 25 ml, 100 ml, 500 ml, 1000 ml, Cotton for plugs/plastic caps (autoclavable), General glasses/plastic wares of various capacities such as volumetric flasks, beakers, reagent bottles, pipittes, vacuum filtration system and glass rods.

Washing Powder/Liquid Detergent, Disinfectants:

Powder or liquid detergents or wetting agents such as Tween-20, 70% alcohol and absolute alcohol). Glass distilled water, Stock solutions of nutrients of tissue culture media or readymade/ prepared powdered media, Sucrose, Agar (tissue culture grade), and sterile culture vessels with distilled water.

Chemicals of analytical grades (Inorganic, organic salts, vitamins, amino acids, growth tors/hormones and activated charcoal), Coconut milk, yeast extract, malt extract, casein lysate and extracts of potato, carrot and tomato.

Spatulas, weighing butter paper/boats, stirring bars (magnetic) for magnetic stirrers and stirring rods, Brush (flask and test tubes), gloves (disposable 23 cm), mop (household), scoop, towels Centrifuge (low speed): Variable electronic speed control speed indicator, Amp. meter, timer, dynamic break, starting switch, 230V 50Hz. Electric Autoclaves-Vertical: with safety valves, pressure gauge, steam-release cock.

Horizontal:

Mounted to tubular stand heavy hings.

Steamers:

With immersion heaters of ejection safety device, size: 31 cm × 13cm × 10cm to 61cm × 20cm × 15cm.

Filter Sterilisation Equipment:

Syringe filter holder: 2.5cm -Pressure Filter Holder: 4.7cm Double Distilled Water Equipment: Built-in energy regulator all glass water stills, heat re-sistant boiling flask with heater.

Environmental Growth Cabinets:

Cabinet with controlled temperature, light and humidity, temperature range 4° to 45°C with timer to regulate photoperiods. Gyratory Shakers: Capacity: 50 to 1000 ml flasks speed: 80 to 200 rpm.

Laminar Air-flow Cabinets:

Constant flow of purified air, sizes: 0.6m, 1.2 m and 1.8m. The other miscellaneous equipment’s which are required for tissue culture are: air conditioners, arrow heads, bunsen bummers, deep-freeze, dissecting needles, glasswares, forceps, fluorescent lamps/ tubes, heaters, hot plates with magnetic stirrer, inoculation cabinets, metal trays and bowls (for transport of cultures), tubes, refrigerators, UV germicidal lamps, wooden or metal racks, etc.

2. Aseptic Technique:

In in vitro condition plant cells and microbes have basically same requirements. When the culture medium contains sugar (as carbon source) it attracts a variety of microorganisms which grow fast than that of the cultured tissue in medium and they ultimately kill the plant cells. It is, therefore, necessary to have complete aseptic condition around the culture equipment’s which prevents contamination of the culture medium.

Following are the main, sources of contamination of the medium and the subsequent methods to check them:

1. The microorganisms may be present in the nutrient medium at the time of its preparation. These microorganisms can be destroyed by proper plugging and autoclaving the culture tubes/flask. The medium can be completely sterilised by maintaining it at 120° C for about 20 minutes at 15 Ib pressure in the autoclave.

2. The explant (plant part to be cultured) may carry microorganisms with it; therefore, the excised part should be surface-sterilised by mercuric chloride (1 to 2%) or by sodium hypochlorite solution for 30 minutes.

Precautions must be observed to prevent the entry of microorganisms when the plug of the culture is removed during transfer of the plant material to the medium or from one medium to another. The inoculation chamber may be sterilised by UV -radiations. Correct pH of the medium is important. Highly alkaline or acidic pH affects the nutrient up-take in culture tissues. Therefore, the tissue culture medium is adjusted to a pH of 5.6 to 6.0, before autoclaving.

Semi-solid and liquid media are most commonly used for growing plant cells. A high concentration of gelling agent (agar-agar, gelatin, silica gel) makes the medium very hard and decreases the nutrient uptake by the tissues. Agar at 0.8% to 1.0% concentration is widely used.

It is essential to remove dirt and debris from the plant tissue and should be washed in a weak detergent solution and rinsed several times with distilled water prior to sterilisation, Some woody trees, such as buds arid twigs, are cleaned by immersing them briefly in a 70% ethanol solution, mulch wets and spreads over the tissue surfaces more effectively than a higher concentration alcohol.

Sodium Hypochlorite (NaOCI):

It is the most common chemical agent used to sterilise plant tissues (0.025%-0.25% NaOCI). Diluted house hold bleach can also be used for this purpose, which normally contains 5,25% NaOCI. It is equally effective and considerably less expensive, Calcium hypochlorite (CaOCI): It can be used as a substitute for NaOCI.

CaOCI causes slightly less damage to plant tissues but tends to precipitate out of solution. To avoid the accumulation of CaOCI on the plant tissue surfaces, sterilisation solutions should be filtered or decanted prior to use.

Hydrogen Peroxide (H20V Solution: Plant tissues can also be surface sterilised using a H₂O₂ (3%-10%).

It is much easier to remove from tissues than NaOCI and CaOCI:

Other Substances:

Plant tissue can also be surface- sterilised by bromine water (1%-2%), silver nitrate (AgNO₃ I %) and mercuric chloride (MgCI₂ 0.1 %-I %),

Cleaning:

Clean the glasswares/plastic wares in 10% commercial detergent liquid or powder for 1 hr and then in HCI for 2 hr. Remove the traces of detergent and acid by thorough washing with tap water. Rinse vessels with double distilled water and allow them to dry over night at room temperature.

Plug glasswares such as conical flasks or test tubes with non-absorbent cotton or cover by the plastic caps, Wrap the petri dishes with aluminium foil. Place forceps and scalpels in test tubes, plug the tubes with cotton or cover with aluminium foil. Plug the mouth end of the pipettes with cotton. Wrap them individually in aluminium foil.