A pure culture of an organism is a valuable asset and it deserves to be preserved for future. Normally, the cultures which are of daily use are grown in agar slants and kept in the refrigerator. When required, a loopful of growth is transferred to fresh agar or broth and the organisms are allowed to grow in an incubator. For short-term preservation, the procedure proves adequate.
But periodical transfer of large number of culture is cumbersome. There is also another risk. During the course of repeated passages an organism may change genetically by spontaneous mutation, so that it is no longer identical to the original pure culture.
Therefore, for more efficient management of pure culture maintenance, various methods of long term preservation have been developed. The underlying principle of these methods is to slow down the metabolic rate to an extreme minimum level. This can be achieved by exclusion of water which is essential for optimal functioning of life process, or by lowering the temperature to a level where normal enzyme functions are practically nil. Exclusion of oxygen in case of aerobic organisms is also helpful in slowing down their metabolic activities
One of the best methods of long-term preservation of bacterial cultures is by lyophilization which involves freeze-drying of a small volume of the suspension of an organism taken in a small ampoule. The content of the ampoule is frozen by rotating it in a super-cooled bath of alcohol or acetone (-80°C) as a thin film. Several such ampoules are then evacuated till the contents become dry and powdery.
The neck of the ampoules is then sealed under vacuum. Such ampoules are then preserved in the refrigerator (4°C). The viability of lyophilized cultures is variable from species to species and, therefore, has to be tested at reasonable intervals, though a majority of bacterial species remains viable for years. For reviving a lyophilized culture, the ampoule is broken the contents are aseptically transferred to fresh medium and incubated till growth occurs.
Another effective means is cryopreservation of cultures at the temperature of liquid nitrogen (-196°C) where all enzyme-catalysed biochemical reactions are practically shunted off. A suspension of the organism is mixed with a cryoprotectant like dimethyl sulfoxide and dispensed in small volumes in specially prepared ampoules.
These are sealed and immersed in a bath of liquid nitrogen in a special container. They are kept there until they are required to be revived. The ampoule is taken out and passed through a temperature gradient to bring down to room temperature before transferring the contents of the ampoule to fresh culture medium. In liquid nitrogen the organisms remain in a state of suspended animation and they retain their original characters for a long time.
For lyophilization and cryopreservation sophisticated equipment’s are necessary. Such facilities may not be available in all laboratories. Some simpler methods of long term preservation are also available, though they are not as efficient. In one such method, a suspension of the organism in sterilized skimmed milk is dispensed in 0.1 ml portions in sterilized cotton plugged Durham’s tubes containing a few filter-paper discs.
Several such tubes are then inserted in a hard-glass larger tube which is placed in a vacuum desiccator containing dry CaCl2 evacuated by a vacuum pump. The tubes are kept in the desiccator till the filter paper discs are completely dry. Next, the neck of the larger tube is connected to a vacuum pump and sealed.
The larger tubes containing the smaller Durham’s tubes with the organism adsorbed to filter paper discs can be preserved at 4°C. When the culture has to be revived, the neck of the larger tube is cut open, one of the small tubes can be taken out and the contents may be added to fresh liquid medium. This method is a simplified version of lyophilization without requiring freezing of the culture.
Some soil-borne organisms can be successfully preserved for fairly long time in soil itself. For this purpose, good dry garden soil is distributed one gram each in 6 cm screw cap tubes. The tubes are plugged with cotton wool and caps are wrapped by brown paper. They are then sterilized by steaming at 100°C for 1 hr on three successive days. The tubes are next dried in desiccator over dry CaCl2 for 3 days and each tube is inoculated with 0.1 ml of the culture to be preserved. They are again dried for 24 hr in a desiccator, capped tightly and preserved at 4°C.
A very simple method of preservation, though not suitable for all organisms, consists of covering completely a full-grown slant or stab culture with a sterilized non-volatile mineral oil or liquid paraffin. The cultures so prepared can be stored at 4°C. The basis of this method is to shut-off metabolism by exclusion of air. Some organisms are unable to sustain viability for long time under such conditions. Viability has to be tested periodically by transferring a loopful of growth to fresh medium.