Laboratory Requirements and General Techniques
Requirements:
A tissue culture laboratory should have facilities for
- (i) Washing and storage of glasswares and other labwares
- (ii) Preparation, sterilization and storage of nutrient media
- (iii) Aseptic manipulation of plant material
- (iv) Maintenance of cultures under controlled conditions
- (v) Observation of cultures
- (vi) Acclimatisation of plants developed in vitro
Laboratory
Media Room: Usual facilities required
- Benches at a height suitable to work while standing
- A deep freeze to store stock solutions, enzymes, coconut milk etc.
- Refrigerator to store various chemicals
- Plastic carboys for storing distilled water
- Weighing balance
- Hot plate-cum-magnetic stirrer
- pH meter
- Vacuum pump for filter sterilization
- Autoclave
Culture vessels
Growth Room
- Air-conditioners, heaters attached to temperature regulator/controller
- For higher/lower temp. other incubators to be used
- Light: Generally a diffused light of 1 klx; with provision of getting 5-10 klx light.
- Relative humidity of about 50%; if it goes below there has to be some mechanism.
- Shelves to store cultures……..on flasks/jars/petri-dishes
- Shaking machine: for suspension cultures
Greenhouse
Techniques:
Glassware and plasticware washing – use detergents – domestic or industrial dish washer – hot
air cabinet
Sterilisation:
- Two obvious general precautions; (i) Not to share the plant tissue culture working area
wit microbiologists or pathologists (ii) To remove contaminated cultures from the culture
area as soon as detected.
- The possible sources of contamination of the medium:
- (a) the culture vessel
- (b) the medium itself
- © the explants
- (d) environment of the transfer area
- (e) instruments used to handle plant material
- (f) environment of the culture room
- (g) the researcher and other workers
Medium:
Microbial contaminants are present there right from the start; to destroy them the media is
autoclaved; at 121°C and 10-15 pounds/sq. in for 20-10 min (sterilization).
Some growth regulators (hormones, urea, vitamins, antibiotics etc.) are thermolabile and they
cannot be autoclaved. They are filter-sterilised.
Glassware and Plasticware:
They are usually sterilized along with media.
Instruments:
The instruments used for aseptic manipulation such as forceps, scalpels, needles, spatula etc. are
normally sterilized by dipping into 95% alcohol and then flaming and cooling; done several
times during operation.
Recently glass bead sterilizer (steripot - 250°C) and infra-red sterilizers (700°C) are available.
Plant Materials:
Plant parts would carry wide range of microbes – they must be thoroughly sterilized before they
are planted on the media.
Sterilsing agent Concentration (%) Duration Effectiveness
(min)
Calcium hypochlorite 9-10 5-30 Very good
Sodium hypochlorite 2 5-30 Very good
Hydrogen peroxide 10-12 5-15 Good
Bromine water 1-2 2-10 Very good
Silver nitrate 1 5-30 Good
Mercuric chloride 0.1-1 2-10 Satisfactory
Antibiotics 4-50 mg/l 30-60 Fairly good
Ethyl and isopropyl alcohol have also been used to surface sterilize some plant tissues.
Transfer area:
All precautions have to be taken to make sure that no contaminants get entry into the culture.
Laminar air-flow cabinet: coarse filter, ‘high efficiency particulate air’ (HEPA) filter (removes
particles larger than 0.3 µm), ultraclean air with velocity of 30 m/min.
Mode of action of some antimicrobial agents:
Antimicrobial compound Mode of action Comments
Aminoglycosides Inhibit protein synthesis by Bactericidal
Sterptomycin interaction with 30S and 50S
Kanamycin ribosomes
Neomycin
Gentamycin
Tobramycin
Amikacin
Spectinomycin
Quinolones Interfere with DNA Bactericidal
Nalidixic acid replication by inhibition of
Ofloxacin DNA gyrase
Norfloxacin
Enoxacin
Ciprofloxacin
Β-lactams Inhibit bacterial cell wall Bactericidal
Penicillin synthesis
Ampicillin
Carbenicillin
Cephradine
Cephamenadole
Cefuroxime
Ceftazidime
Sublactum
Imipenem
Aztreonam
Tetracyclines Inhibit protein synthesis by Bacteriostatic
acting on 30S ribosome
Chloramphenicol Inhibit protein synthesis by Bacteriostatic
acting on 50S ribosome
Macrolides and lincosamides Inhibit protein synthesis by Bacteriostatic
Erythromycin acting on 50S ribosome
Lincomycin
Glycopepetides Interferes with bacterial cell Bactericidal for gram positives
Vancomycin wall synthesis
Polymixins (B/E) Attach to cell membrane and Bactericidal for gram
modify ion flux resulting in negatives
cell lysis
