Pure Culture preservation strategies

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 4^o 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 -29^o 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      - 78^o 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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