Preservation of Microorganisms

PRESERVATION OF MICROORGANISMS

In addition to their uses in the manufacture of medicines, microorganisms are employed in a variety of tests and assays, particularly those to measure the activity of antimicrobial chemicals. Useful organisms, therefore, need to be correctly preserved in order to ensure that their desirable properties are not changed during storage or, worse, the culture dies completely and is irreplaceable. Many bacteria and fungi can conveniently be stored for a few days, or possibly weeks, in the form of liquid cultures in tubes, or as colonies on Petri dishes. Organisms that readily form spores—Bacillus and Clostridium species of bacteria and most fungi—can be stored for months or even years in this way provided that the culture medium does not evaporate to dryness, but non-sporing organisms vary substantially in their survival capacities. Gram-positive bacteria generally tend to survive better than Gram negative ones: species like Pseudomonas aeruginosa, for example, may die in a few weeks, even at refrigeration temperatures, if maintained as colonies on unsealed Petri dishes. Even if a culture that is to be preserved does not die completely when stored in the refrigerator, there is a risk that the cells that do survive are not typical of the population as a whole; they may, for example, be mutants that have increased resistance to adverse conditions in general, and so fail to give the expected results when used in tests on antibiotic activity. The dual aims of a culture preservation procedure therefore are to maintain the viability of the highest possible percentage of cells and to minimize the risk of selecting atypical mutants.

The most common procedures for long term storage are by freezing at −80 °C (or lower) in refrigerators, by storage in liquid nitrogen at −196 °C in special vessels, or by freeze-drying (also called lyophilization). In each case, cryoprotectant chemicals—compounds like glycerol or dimethyl-sulphoxide—are incorporated at a concentration of about 10% v/v in the liquid culture of the organism in order to minimize both the formation of damaging ice crystals and osmotic stresses that can accelerate cell death during freezing and thawing.

Reference cultures, those with well defined biosynthetic capabilities or resistance properties, can be obtained in a freeze-dried form from internationally accessible culture collections like the American Type Culture Collection (cultures having the designation ATCC before a reference number) or the UK National Collection of Industrial and Marine Bacteria (NICMB). Increasingly. pharmacopoeias and regulatory agencies are requiring tests that employ microorganisms to be conducted with cultures or test suspensions of cells that are no more than five subcultures from the reference material obtained from the designated culture collection.