Training Manual Application of Genetics and Biotechnology in ...

CHAPTER 3
MICROBIAL TECHNIQUES
M.S.Shekhar and S.Kannappan
1. STERILIZATION:
Sterilization is the complete elimination or destruction of all forms of
microbial life and is accomplished by either physical or chemical processes. Steam
under pressure, dry heat, and liquid chemical are the principal sterilizing agents used
in the laboratory.
1.a. Dry Heat: The only items for which dry heat sterilization is appropriate are
those that cannot be sterilized by steam because they cannot be penetrated, or will be
damaged by moisture. Dry-heat sterilization actually "bums up" microbial cells."
Glass wares are generally sterilized by dry heat in Hot air oven. A hot air oven is
electrically operated and should be equipped with a fan to ensure uniform
temperature inside. The required temperature for sterilization is generally 160°C for
one hour.
1. b. Moist heat : In moist heat (steam) sterilization bacteria die from the
coagu1,ation or denaturation of the protein constituents. Steam sterilization works
better than some forms of sterilization because steam destroys most resistant bacterial
spores in a brief exposure. Cotton, Filters, Instruments, Culture media are generally
sterilized by Autoclaving Autoclaves can sterilize anything that can withstand a
temperature of 121°C for 20 minutes.
2. BACTERIAL CULTURE MEDIA:
2.a. Luria-Bertani broth:
LB media formulations have been a laboratory standard for the cultivation of
Escherichia coli .These media have been widely used in molecular/microbiological
applications for the preparation of plasmid DNA and recombinant proteins. It