Cleaning and sterilization during tissue culture

1. Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPS

2. Practical 2: Cleaning and sterilization during tissue culture
 Glasswares
 Surface
 Media
This Photo by Unknown Author is licensed under CC

3. Equipment Required: Glassware’s, Autoclave or pressure cooker, Electronic Balance.
Materials Required: Spatula, Balance and weighing boat, Boiling tubes, Aluminium
foil, Hot plate, Deionized tissue culture grade water.
Learning Objective: To prepare and to sterilize glassware for further experiments

4. Theory:
• Nutritional requirements for optimal growth of a tissue in vitro may vary with the species.
• Even tissues from different parts of a plant may have different requirements for satisfactory growth
(Murashige and Skoog, 1962).
• As such, no single medium can be suggested as being entirely satisfactory for all types of plant
tissues and organs.
• When starting with a new system, it is essential to work out a medium that will fulfill the specific
requirements of that tissue.

5. Outline of the Procedure:
a) Measure out approximately 90% of the final required volume of tissue culture
grade water (e.g. 900 ml water for a final volume of 1000 ml). Select a container
twice the size of the final volume.
b) Add the powdered medium and stir until completely dissolved. Heating may be
required to bring powders into solution.
c) Rinse the original container with a small volume of tissue culture grade water to
remove traces of the powder.
d) Add desired heat stable or heat sensitive supplements [e.g. Sucrose, vitamins,
Growth regulators (auxins, cytokinin’s)]

6. e) Adjust the final pH (5.7) of media using IN NaOH or IN HCl. Add the gelling
agent (Agar) in medium (for the preparation of solid MS media only). If a
gelling agent is used, heat until the solution is clear.
f) Dispense the medium into the culture vessels before (or after) autoclaving.
Add heat labile constituents after autoclaving.
g) Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C,
for the time period described under Sterilization of Media Protocol. Allow
medium to cool prior to use.

7. • Sterilization of glassware tools/vessels- All the glassware should be of Pyrex or corning. All the glassware
should be kept overnight dipped in sodium dichromate-sulphuric acid solution. Next morning, glassware
should be washed with fresh running tap water, followed by distilled water and placed in inverted position in
plastic bucket or trays to remove the extra water. For drying the glassware, it is placed in hot air oven at high
temperature about 1200c for ½- 1 h.
• In the case of plastic lab ware, washing should be carried out with a mild nonabrasive detergent followed by
washing under tap water or the plastic ware after general washing with dilute sodium bicarbonate and water
followed by drainage of extra water, rinsed with an organic solvent such as alcohol, acetone and chloroform.
Washed and dried glassware or plastic ware should be stored in dust proof cupboards.
• To prevent reinfection following sterilization, empty containers are wrapped with aluminium foil. Stainless
steel, metal tools (knives, scalpels, forceps, etc) are also wrapped with aluminium foil and pads o cotton
wool are stuffed into the opening of the pipettes, which are either also wrapped in aluminium or placed in
an aluminium or stainless steel box. The period of sterilization usually ranges between 1 and 4 hour.

8. Cleaning of glassware
• Graduated measuring cylinders
• Conical flasks
• Beakers
• Petridishes,
• Centrifuge tubes
• Pipettes (2 ml, 5 ml and 10 ml)
• Culture vials
• Bottles
• Glass rods
• Culture tubes

9. Procedure for cleaning of glassware
Soak glassware in 10% soap water (teepol) for 1 hour.
Transfer glassware to conc. HCl and keep for 2 hours.
Rinse glassware in tap water.
Wash the glassware at least twice with distilled water.
Keep glassware for drying in oven at 100 o
C for 1 hour.
keep glassware in oven at 140-160 o
C for 2 hours.

10. Autoclave for sterilization

12. Sterilization of the plant material
(Surface) sterilization
● The plant material should be surface sterilized to remove the surface
borne micro-organisms.
Water
10% v/v solution of liquid detergent (Teepol) for 10-15 min.
70% ethyl alcohol for 1 min. in front of laminar air flow.
Treatment with 0.1% HgCl2 (W/V) or 5-10% sodium hypochlorite.

13.  SURFACE STERILISATION OF EXPLANT
For surface sterilization chromic acid, Hgcl(0.11%),calcium
hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are used.
Process depends on the type of explant.
SEED : absolute ethyl alcohol calcium hypochlorite bromine
water sterile water
FRUIT : ethyl alcohol
STEM : running water
LEAF : surface clean
sodium hypochlorite
sodium hypochlorite
Hgcl2 sterile water
sterile water
sterile water
dried
explant

14. Books/Journal
Genetic Engineering and Biotechnology –Concepts, Methods and Applications by Chopra VL & Nasim
A. 1990; Oxford & IBH.
Web links:
http://www.biologydiscussion.com/plant-tissues/culture/plant-tissue-culture-laboratory-with-
diagram/14561

15. THANKS A LOT 