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Techniques
Natural microbial
populations
Microbial population in our environment large &
complex
Why important?
Pure cultures are important in microbiology for the following reasons
Once purified, the isolated species can then be cultivated with the
knowledge that only the desired microorganism is being grown.
HISTORY
A GREAT DISCOVERY
Agar was discovered around 1658 by Minoya Tarozaemon in
Japan
Agar was first used in microbiology in 1882 by the German
microbiologist Walther Hesse ,an assistant working in Robert
Kochs laboratory
STERILIZATION
BUT !
Sterilization continue
STERLIZATION
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Culture techniques
Selective method:
This method favour the growth of desired
species & discourage/kill the other organism
present in the mixed culture
(B)Physical methods of
selection
(a) Heat treatment
To select for endospore forming bacteria, mixed
culture can be heated to 80 C for 10 min before
incubate to culture media. Vegetative cells will
be killed, but endosperms will survive and
subsequently germinate and grow
(B)Physical methods of
selection
(b) Incubation temperature
To select psychrophilic or psychrotropic bacteria,
cultures are incubated at low temperature e.g: 0
- 5 C
(B)Physical methods of
selection
(c) pH of the medium
To select acid tolerant bacteria low pH medium
can be used
(B)Physical methods of
selection
(d) Cell size and motility
Membrane filter having pore size of 0.15m is
placed on the surface of an agar plate. During
inoculation, mixed culture placed on the surface
of the filter
Pure Cultures
Particular bacterial species comprises high
proportion in mixed population
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Paraffin Method
This is a simple and most economical method of maintaining pure cultures
of bacteria and fungi. In this method, sterile liquid paraffin in poured over the
slant (slope) of culture and stored upright at room temperature. The layer of
paraffin ensures anaerobic conditions and prevents dehydration of the
medium. This condition helps microorganisms or pure culture to remain in a
dormant state and, therefore, the culture is preserved for several years.
Cryopreservation
Cryopreservation (i.e., freezing in liquid nitrogen at -196C) helps survival of
pure cultures for long storage times. In this method, the microorganisms of
culture are rapidly frozen in liquid nitrogen at -196C in the presence of
stabilizing agents such as glycerol that prevent the formation of ice crystals
and promote cell survival.
Lyophilization (Freeze-Drying)
In this method, the culture is rapidly frozen at a very low temperature (70C) and then dehydrated by vacuum. Under these conditions, the
microbial cells are dehydrated and their metabolic activities are stopped; as
a result, the microbes go into dormant state and retain viability for years.
Lyophilized or freeze-dried pure cultures and then sealed and stored in the
dark at 4C in refrigerators.
Culture collections
In France Institute of Pasteur in Paris
England National Collection of Type Cultures in London
Federal Republic of Germany Deutsche Sammlung Von
Mikroorganismen in Darmstadt