Surface sterilization and sealing of cultures (surface sterilization of cultures

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

2. Practical 6: To study surface sterilization and sealing of cultures
(surface sterilization of cultures)
This Photo by Unknown Author is licensed under CC

3. Equipment Required: Laminar air flow, autoclave
Material Required: Tween-20, Beaker 70% ethanol, Non-absorbent
cotton, Muslin cloth and Culture vessel, HgCl2
Learning Objective: To prevent microbial contamination, that is
detrimental to culture growth.

4. Theory:
Plant tissue culture media being rich in sucrose and other organic nutrients, supports the growth
of many microorganisms (like bacteria and fungi).
Once in contact with the medium these microbes generally grow much faster than the cultured
tissue and finally kill it.
The contaminants may also give out metabolic wastes which are toxic to plant tissues.
It is, therefore, absolutely essential to maintain a completely aseptic environment inside the
culture vessels.
For this, two general precautions are:
(1) not to share the plant tissue culture working area with microbiologists and pathologists, and
(2) to remove contaminated cultures from the culture area as soon as detected.

5. Outline of the Procedure:
Explants Sterilization:
a. Rinse explants thoroughly under running tap water for 10-30 minutes.
b. Wash explants in a mild detergent such as Tween-20 before treatment
with the disinfecting solution. (Herbaceous material may not require this
step).
c. Submerge explants into the disinfectant solution. Seal bottle and gently
agitate.
d. Under sterile conditions, decant the solution and rinse explants several
times with sterile distilled water.

6. e. Sterilization procedures may be enhanced by placing the material in a 70% ethyl
alcohol solution prior to treatment with another disinfectant solution then rinsing
with 0.1% HgCl2 for 1.0-1.5 min is done to achieve complete sterilization.
The use of a two-step sterilization procedure has proven beneficial with certain species. Using a
wetting agent, such as Tween 20 or 80 the disinfectants to reduce surface tension and allow
better surface contact.
f. Sealing of cultures is done by making cotton plugs using non-absorbent cotton.
g. Cotton plugs are further covered with muslin cloth and tightly tied with a thread.

7. 5. Results: Sterilized explants will be placed onto the solid medium and
observed for growth and development without contamination.
6. Scope of the result: Less contamination will result in further experiments
to be done.
7. Required Results: Proper sterilization will increase success rate of tissue
culture experiment.

8. Cautions:
1. Explants should be sterilized properly.
2. Measurement should be accurate.
3. Sterilizing agent should be properly removed with water.

9. Autoclave for sterilization

10. Sterilization
• Culture medium supports the growth of microbes e.g bacteria, fungi, etc. these grow fast and kills
the plant cells.
• Microbes may come from glass vials, instruments, nutrient medium and also from the plant
material.
• Therefore, the surface of plant tissue and all non-living articles including nutrient medium must be
sterilized.
Sterilization of non-living articles
• The non-living articles viz. Nutrient medium, glassware, distilled water, instruments (wrapped with
brown paper) are sterilized by autoclaving under steam at a 15 lb/inc2 and temperature 121oC for 15
min.
• The glassware can also be sterilized by heating in oven at 150oC for 3-4 hrs.
• The thermolabile compounds are sterilized by passing through the bacterial filters.

11. 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.

12.  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

13. 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

14. THANKS A LOT 