1. Introduction.
Maintenance of pure
cultures of bacteria requires frequent attention so that the organisms are not
lost or contaminated. They have to be transferred at regular intervals to
suitable media before they are destroyed by their own waste products. But
repeated sub culturing will lead to the loss of biological, immunological and
cultural characteristics of the organism. Usually, more than 5 subcultures are
not preferred.
In microbiology laboratories
we maintain culture of different strains of microorganisms as they are required
for laboratory classes, research works, or for other particular procedures.
They are called Stock culture collection.
Microbial cultures are also maintained as reference
strains for taxonomic studies. American
Type Culture Collection (ATCC) in USA maintains 1 million ampules of
different strains of microorganisms. Microbial Type Culture Collection &
Gene Bank (MTCC) in Chandigarh is another central collection unit in India.
Preservation is the
maintenance of microorganisms for extended period of time in viable condition
without contamination or lose of genetic and morphological characteristics.
Preservation methods either stop or slow down the metabolism and multiplication
of microbes. Different methods are used to maintain the pure culture of
microorganisms. However, no single
method is complete for preserving all types of microorganisms.
2.
Types
of culture preservation methods
Based on the period of storage, culture preservation
is divided into Short term methods
and Long term methods.
Short term methods
|
Long term methods
|
·
Agar slant cultures (Sub
culturing) & Refrigeration
|
Mineral
Oil or Liquid Paraffin Method
|
|
Saline
suspension storage
|
|
Drying
in Vacuum
|
|
Storage
at low temperatures (Cryopreservation)
|
|
Lyophilization
(Freeze drying)
|
3.
Short term methods (1 Type)
3.1.
Agar slant cultures
(Sub-culturing) and Refrigeration
Pure culture can be
maintained by periodic aseptic sub-culturing on to the suitable media. Type of
media, storage temperature and interval between sub-culturing depends on the
bacterial species. These are referred as stock cultures. Solid media is usually
preferred as the toxic material produced by organisms diffuse into slant and
thus away from microbial growth. Most heterotrophs can be easily maintained on
nutrient agar. Nutrient agar slants in screw capped bottles are preferred as it
prevent drying of the media. Disadvantage is that repeated sub-culturing leads
to mutational changes in the strain.
After preparation of
stock cultures, they are incubated for 24 hrs or more to get sufficient growth
and stored in cool dry place or refrigerator till use. The temperature inside
the refrigerator is usually 4o C which can slow down the growth and
metabolism of microorganisms. Thus nutrient use from media is considerably reduced
which is useful in the maintenance of organisms. Here also periodic
sub-culturing is needed as waste product accumulation adversely affect the
microbial growth.
4.
Long term methods (5 Types)
4.1.
Mineral Oil or Liquid Paraffin
Method
Agar slants in screw capped tubes are inoculated and incubated till
good growth is obtained. Then it is covered with sterile mineral oil or liquid
paraffin to a depth of 1 cm above the slant. This method can be used to preserve
bacteria and fungi in viable condition at room temperature from months to years.
Paraffin layer prevents the dehydration of the medium and also produce
anaerobic condition so that microorganisms remain in a dormant state.
This method has many
advantages.
The unique advantage of the method is
that sub-culturing can be done without affecting the stock culture.
Sub-culturing is done by taking loopful of growth under the oil from these
slants, touching the loop to the glass surface of tube to drain off excess oil
and then inoculating to fresh medium.
- Many species remain viable for longer periods under oil than in tubes without oil.
- Sub-culturing can be done as and when required without affecting the stock cultures.
- Useful for strains susceptible to mutations on repeated sub-culturing
- Simple and economical method as equipment like dessicator, vaccum pump etc are not needed.
4.2.
Saline suspension storage
Sodium chloride in high
concentration is inhibitory to the microbes. But 1% salt solution is useful for
culture preservation and is considered as sub lethal concentration. In this
method bacteria are suspended in salt solutions in screw capped tubes to avoid
evaporation. Tubes can be stored at room temperature and when required, can be
sub-cultured to agar slants. This is an easy method for storing bacterial
cultures for 2-3 years and particularly useful in labs with limited equipment
facility.
4.3.
Drying in Vacuum
Organisms
are found to survive longer if they are dried in a desiccator than when air
dried. Different methods are employed for drying in vacuum. Organisms are suspended in liquid media and
dried in a desiccator in vacuum over dehydrating agents such as H2SO4
or P2O5 (Phosphorus
pentoxide). It is called Liquid drying or L –Drying. This method is
particularly useful for microbes which are sensitive to freeze drying as this method
involves
vacuum-drying of samples from the liquid state without freezing. .
The microbial suspensions can be also dried on sterile coverslips, filter
paper, in small test-tubes, or more conveniently in sterile ampoules which can
subsequently be sealed off in vacuo.
4.4.
Freezing and Cryopreservation
The
organisms suspended in nutrient broth containing 15% glycerol can be frozen and
can be stored at -29o C for up to 2 years. The availability of liquid nitrogen provided
another useful means for preserving stock culture. In this method dense cell
suspensions are prepared in the medium containing cryopreservative agents like
glycerol or Dimethyl sulfoxide (DMSO). These agents prevent ice crystal
formation during freezing process which may damage microbial cells. This
suspension is sealed into small ampoules or vials and then frozen at controlled
rate to -150 o C. Then the ampoules or vials stored in a liquid
nitrogen refrigerator either by immersion in a liquid nitrogen (-196°C) or by
storage in a gas phase above liquid nitrogen (-150 o C). This method
is useful for preservation of organisms that cannot withstand freeze
drying. Cultures can be maintained for
10-30 years or more without any change in the characteristics of microbes.
However, this method is expensive as liquid nitrogen has to be replenished at
regular intervals to replace the loss due to evaporation.
4.5.
Lyophilisation (Freeze drying)
This
method is mainly used to preserve organisms that would be killed by ordinary
drying process. In this method, dense cell suspension is prepared in small
vials and frozen at -60 to - 78o
C. Vials are connected to high vacuum so that ice present in frozen culture
undergo sublimation under vacuum. Sublimation is evaporation from solid to
gaseous stage without passing through liquid phase. This causes dehydration of
bacteria with minimum damage to delicate cell structure.
Vials
are then sealed under vaccum and stored in refrigerator. Many species of
bacteria can be preserved by this method for more than 30 years in viable
condition without any change in characteristics. Minimal storage place is
required for lyophilised cultures, 100s of cultures can be stored in a small
area. Also these vial are convenient to mail in sealed containers to other
laboratories. Lyophilised cultures are revived by rehydrating the opened vials
by adding liquid medium and then transferring it to the suitable media.
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