Microorganism for the production of industrially important products are useful only if they can be maintained indefinitely in healthy, pure and genetically stable form. Industrial culture collection consists of stock culture. Stock culture may be simply defined as a culture which serves as source of inoculum. Stock cultures are of two types.
1. Working stock culture: Working stock culture: This culture is maintained at vigorous and uncontaminated condition. Since this culture is used frequently, it must be routinely checked for characteristic feature and contamination.
2. Primary stock culture: This culture is kept for long term storage and are maintained at low physiological activity condition. The cultures are used to produce new working stock culture as per need.
Method of preservation of primary stock:
Periodic transfer: This method is also called active transfer, sub culturing or serial transfer. Here, the temperature of storage and type of medium chosen should support slow rather than rapid growth of culture. Slants (agar slopes) are most widely used as culture medium. Culture after maturation can be preserved in refrigerator and it can be maintained for 2 – 6 months. This is the least desirable method because mutation cannot be prevented and frequent transfer increase chance of contamination and genetic changes.
Oil overlay: Many bacteria and other microorganism can be preserved by this method for 1 to 2 years. In this method, agar slant growth is overlayed with sterile and inert mineral oil up to half inch above the tip of slant surface and stored cold. Advantage of oil overlay method are as below
1. Some of the growth can be removed to inoculate fresh medium without contaminating the stock. Therefore, the stock can be used for number of times which is not possible in subculture medium.
2. Control of water and gas exchange in the medium
However, this method causes loss of sporulation and sometimes biochemical activity. Non – sporulating molds cab be well preserved by this method.
Preservation in soil: Soil can be used either as carrier or growth medium. If used as carrier, abundant spores of bacterial or fungal species must be prepared in advance. The spores are then placed in sterile soil and resulting preparation dried in air or under vacuum. For many fungi, maintenance by use of soil is very useful. Most soil may be inoculated with the fungus and may be allowed to grow until sporulation has completed. Preparation of ‘Murcha’ is similar to this except for the use of starchy material in place of soil.
Desiccation or drying: This preservation technique is preserving yeast cells. Cells are kept directly in contact with silica gel as desiccant. Silica gel is kept in screw capped container (depth of 1 cm, sterilized in hot air oven). The culture is grown to stationary phase in suitable medium and then suspended in 5 % skimmed milk medium. The latter is finally transferred to gel without saturating it. The heat developed should be dissipated quickly. Gel is dried at room temperature for 2 weeks and finally put in air – tight container for storage at 4°C. Culture preserved this way is suitable for several years.
Lyophilization (freeze drying): Most satisfactory method for long term preservation of those microorganism which can withstand rigor of the process. In this process, dense cell suspension (stationary phase) is placed in small vial (ampules) and frozen under vacuum (200 μm Hg) at – 60 to – 70°C and vials are sealed using oxygen; natural gas flame.
This suspension is prepared in various protective media such as bovine serum media containing sugar or skimmed milk. It is also advantageous to include inositol (5%). The whole is stored under refrigeration in dark. Organism in the protective media remain viable for as long as 30 years. Advantages of Lyophilization are as follows
- Requires no sub culturing
- Ease of transportation
- Genetic stability
- Low cost
- Less storage space
The only disadvantage of this method is, not all microorganism can be subjected to this method because not all microorganism can withstand the rigor of the process.
Use of low temperature: In this technique, cells are prepared as dense suspension in a medium containing a cryoprotectant such as glycerol or dimethyl sulfoxide. Ampules are prepared and frozen at controlled rate to – 150°C. Initially, temperature is allowed to go down approximately at the rate of 1°C/min up to – 20°C. Then after, temperature is brought down as rapidly as possible. Electrolytes should be kept at low concentration as possible. Finally ampules are stored in liquid nitrogen refrigeration either by immersing (- 196°C) or in gas phase nitrogen (- 150°C). This method has been successful with many species including human cell which cannot be preserved by lyophilization. By using this technique, most species remain viable for 20 – 30 years. However, this method is expensive due to nitrogen needed for replenishment.