Once a pure culture has been obtained from the mixture, the microbes may be maintained in the laboratory over long periods. Several techniques are used to preserve cultures, although refrigeration is the most common.
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By decreasing the environmental temperature to about 5°C, microbial metabolism will be slowed to the point that the rates of growth, reproduction, and death will be extended over very long periods.
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Living cells may be removed from refrigerator stored cultures over a period of several months in some cases. For long-term storage, alternative methods must be used. Some resistant microbes maybe preserved by freezing.
The culture tubes are placed in acetone dry-ice baths (-70°C) of liquid nitrogen (-200°C). However, the cells must be protected from ice crystal formation. If crystallization occurs inside the cells as the temperature drops, the sharp edges of the ice will slice organelles and membranes, which will lead to the death of the cell.
By adding such agents as glycerol or sucrose sugar to the culture, cell will be inhibited from forming harmful ice crystals, and the rate of survival will be greater.
Another preservative method that has found application in the food industry, is freeze-drying, or lyophilization. The culture to be preserved is placed in a special lypophilization chamber and the temperature is dropped to about -70°C as a high-vacuum pump draws off the water from the cell.
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This enables the culture to remain frozen as the cells are dried. Since the amount of water in the cell has been reduced to a minimum, the chance of ice crystal formation decreases, ensuring a relatively high survival rate, Skimmed milk, sucrose, and blood serum can be used to protect the microbes being lyophilized.
To rehydrate the cells for culturing, small amounts of culture medium is added to the preserved microbes and placed in a favourable growth environment.
Lyophilization is not a typical technique performed in medical laboratories but is used by research companies, industrial firms, and other organizations whose business is to provide pure microbial cultures for study. Most pure stock cultures are maintained by refrigeration in hospital and teaching laboratories.
Not all pure culture need be obtained from the environment by streaking or pour platting. Many biological supply companies keep stock culture of microbes that can be purchased at relatively low prices.
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These cultures are composed on only one species, although they may contain a variety of subspecies (strains). For many types of experimental work this genetic variation is acceptable.
However, some research demands that this subtle variation be eliminated. To develop and maintain such “super-pure” cultures, a great deal of control must be exercised, which results in higher costs.
One source of these pure cultures is the American Type Culture Collection (ATCC). The ATCC was organized in 1925 as a unique national resource for the acquisition, preservation, and distribution of authentic cultures of living microorganisms.
ATCC serves a broad spectrum of microbiology through its departments of Bacteriology, Cell Culture, Computer Science, Mycology, Protozoology, and Virology.
Currently, there are more than 30,000 different strains of authentic cultures of animal cells and microbes available. This is the most diverse collection of strains maintained at one facility anywhere in the world.
ATCC meets the demand of the scientific community for authenticated strains. More than 40,000 cultures were distributed to scientists around the world in 1981, and one-quarter million were distributed during the last 10 years.
ATCC has a history of successful preservation of strains by freezing and freezing-drying. In order to accomplish this, special handling techniques have been developed.