CH4. Sterilization Techniques

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CH4. Sterilization Techniques
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In general different sterilization procedures can
be grouped under three categories

• Preparation of sterile media, containers and
  small instruments.
• Maintenance of aseptic conditions.
• Preparation of sterilized explant material.

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Preparation of sterile media, containers and
small instruments

• The most popular method of sterilizing culture
  media and apparatus is by autoclaving the
  material using steam/dry sterilization.
• Steam sterilization
• Test tubes/flasks (20-50mL media)-- 121?, 15 psi
  for 20 min.
• Flasks (50-500 mL media)-- 121?, 15 psi for 25
  min.
• Flasks (500-5000 mL media) 35 min
• Bottle should not be tightly and tops should be
  loose.
• After autoclaving, there are kept in the laminar
  airflow and tops of bottles are tightened.

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• During and after autoclaving, the following
  should be consider.
• The pH of the media is lowered by 0.3-0.5 units.
• Autoclaving at too high temperature can
  caramelize sugar, which may be toxic.
• Volatile substances can be destroyed by the use
  of an autoclave.
• Autoclaving for too long periods can precipitate
  salts, and depolymerize the agar.

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Dry sterilization

• The sterilization of glassware and metallic
  instruments can be carried out in dry heat for 3
  h at 160-180 ?.

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Filter sterilization

• Some chemicals, enzyme, antibiotics are
  sterilized by filtration through a sieve
  membranes of 0.45 to 0.22 µm.
• Most filters are of cellulose acetate and/or
  cellulose nitrate, and are available in
  pre-sterilized, plastic disposable units.

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Ultra violet sterilization

• Sterilization of nutrient media by irradiation
  (?ray) is hardly ever used for tissue culture
  media preparation because it is extremely
  expensive as compared to the usual method of
  autoclaving.
• However, the pre-sterilized disposable plastic
  wares are generally UV-radiation sterilized.

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Maitenance of Aseptic Conditions

• The following procedures are employed during
  aseptic transfer
• Alcohol sterilization
• The laminar airflow cabinets should also be
  sprayed with 70 ethanol before use.
• Flame sterilization

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Sterilization of Explant
Table A comparison of the effectiveness and
properties of common surface sterilants for
explants.
Sterling agent Conc. Ease of removal Treatment time (min) Remarks
NaClO 1 1.4 5 30 Very effective
Ca(ClO)2 9 10 5 30 Very effective
H2O2 10 12 5 15 Effective
Bromine water 1 2 2 10 Very effective
AgNO3 1 5 30 Effective
Mercuric chloride 0.01 1 2 10 Satisfactory
Antibiotics 4 50mg/L 30 60 Effective