Methods Of Isolating Pure Culture

In clinical microbiology isolation of pure culture is a daily routine task. It is done to confirm the presence of certain microbe in the mixed culture so that it can be treated specifically.

Isolation of pure culture is also important to identify particular bacterial species or strain. Because in isolated or on pure culture we can perform characteristic tests and get confirmation of the presence of particular species or strain of bacteria.

There are number of methods used for the isolation of pure culture from a mixed culture, few main are discussed here.

• Surface plating is the method routinely employed in clinical bacteriology and enables the isolation of distinct colonies which may be picked out, if necessary for further purification and study.

• Enrichment, selective and indicator media are widely used for the isolation of pathogens from specimens such as feces, with varied flora.

• If we treat the mixed culture with appropriate bactericidal agents those kill rest of the bacteria in a mixed culture then pure culture of the desired bacteria can be obtained. This is frequently done for the isolation of tubercle bacilli from sputum and other clinical specimens, by treatment with alkali, acid or other substances to which most commensalism are susceptible but tubercle bacilli are resistant.

• Obligate aerobes and anaerobes may be separated by cultivation under aerobic or anaerobic conditions.

• While separating the pure culture from the mixed culture we can incubate culture on different temperature. This is useful when the optimum temperature requirement of the desired bacteria is different from the temperature requirement of other bacterias in the culture. Only thermophilic bacteria grow at 60 C. A mixture containing N. meningitides and N. catarrhalis can be purified by incubation at 22 C when only the later grows.

• Some time we can heat up the entire mixed culture this is about giving extreme temperature conditions artificially. This can kill vegetative bacteria from the culture however, spore forming bacteria can sustain. So it is easy to isolate spore forming bacterias from the culture of mixed bacteria. This method can be used to isolate tetanus bacilli from dust and similar sources.

• Separation of motile from nonmotile bacteria can be effected using Craigie’s tube. This consist of a tube of semisolid agar, with a narrow open at both ends placed in the centre of the medium in such a way that it projects above the level of the medium. The mixture is inoculated into the central tube. On incubation, the motile bacteria alone traverse the agar and appear at the top of the medium outside the central tube.

• To isolate pathogenic bacteria from a mixed culture of non pathogenic bacteria, mixed culture is inoculated into appropriate animal. Where pathogenic bacteria develop disease and can be isolated as pure culture from the diseased animal. This method can be used to isolate anthrax bacilli from other aerobic sporulating bacilli by inoculating into mice or guinea pigs. Anthrax bacilli produce a fatal septicemia and may be cultured pure from the heart blood.