BTE-308: Plant Biotechnology and

Genetic Engineering

Lecture-3
Tissue Culture: Part II
Different Sterilization Techniques

mmh.akash@bracu.ac.bd
 Steps for Plant Tissue Culture
Preparation/Sterilization of instruments

Preparation and Sterilization of culture medium

Preparation of explant

Inoculation of Explant

Incubation for growth

• Callus formation
• Shoot induction
• Root induction

Acclimatization of plantlets (TCPs)

Transfer to pots
Tissue culture is carried out in a completely sterile condition!

What are the different sterilization techniques that are used

at different stages of plant tissue culture?
 Different Sterilization Techniques
In general, the methods of elimination of those sources of infection can be
grouped under SIX different categories of sterilization procedures-
Types of Sterilization Objectives Equipment used
Wet/Steam sterilization Sterilization of different media Autoclave, pressure cooker
Dry sterilization Sterilization of glassware, small Drying oven
instrument (e.g. forceps)

Filter sterilization Sterilization of thermo/heat-labile Membrane filters

compounds, e.g. vitamins,
hormones, etc.

Alcohol sterilization Sterilization of worker hand and Chemicals used, e.g. 70%
laminar airflow alcohol

Flame sterilization Sterilization of small instruments Bunsen burner or spirit lamp

(e.g. forceps)

Chemical sterilization Sterilization of explants Different sterilants, e.g.

detergents (Tween-20), NaOCl,
fungicide, antibiotics, etc.
 Steam (wet) Sterilization
Most of the nutrient media are sterilized by the use of an AUTOCLAVE!

The standard condition for autoclaving media are 1210C with a pressure of
15psi.

The following guidelines are usually followed for the duration of sterilization-
Volume of culture media in Time
the culture tube or flask
20-50 mL 20 minutes
50-500 mL 25 minutes
500-5000 mL 35 minutes

Advantages of autoclaving are-

 Quick method
 Simple and easy handling
 Virus and other microorganisms are destroyed easily
 Dry Sterilization
The sterilization of glassware and metallic instruments can be carried out on
dry heat for 3 hours at 160-1800C.

An exposure of 1600C dry heat for 2 hours is regarded as equivalent to moist

heat sterilization at 1210C for 15 minutes.

This technique is used when the material to be sterilized is totally dry and
contains no liquid material.

Now a day, both dry and wet sterilization programmable autoclave machines
are available.
 Filter Sterilization
Some growth regulators, amino acid and vitamins that are HEAT-LABILE, and
get destroyed during the extreme conditions of autoclaving, are sterilized by
filtration.

This can be done using a sieve or filter membranes of 0.45 to 0.22 µm.

A range of bacteria-proof membrane filters (Millipore-Millipore Co.) are

available in the market.

1-200 mL of solution can be sterilized by applying these filter assembles.

 Other Sterilization Techniques
Alcohol Sterilization
Sterilization of laminar airflow, worker hands during work.

Flame Sterilization
Sterilization of instruments which are continuously used during manipulation
work.

Instruments are soaked in 70-80% alcohol followed by flaming on a burner in

the laminar airflow hood.

Chemical Sterilization
Sterilization of explants using alcohol, detergents (e.g. Tween-20), surface
sterilant (e.g. Bromine water) etc.