A number of methods are available for the isolation of bacteria. The following are a few important methods:
1. Surface plating:
It is also called as streak culture method. The specimen to be cultured is taken in a platinum loop. One lapful of the specimen is transferred on to the surface of a well dried plate. Then it is spread over a small area at the periphery. The inoculum is then distributed thinly over the plate. This is done by streaking with the loop in a series of parallel lines in different segments of the plate. The loop should be flamed and cooled between different sets of streaks. After incubating the plate, it can be seen that growth is confluent at the original site of inoculation. But it becomes progressively thinner. In the final series of streaks, well separated individual colonies of bacteria can be obtained.
2. Enrichment and selective media:
Bacteria can be isolated by growing in enrichment or selective media. In these media, substances which inhibit the growth of unwanted bacteria is added. So there is a growth of only the bacteria which is wanted.
3. Aerobic and anaerobic conditions:
Aerobic and anaerobic bacteria can be separated by cultivation under aerobic or anaerobic conditions. The most widely used medium for anaerobic bacteria is Robertson’s cooked meat medium. It contains fat-free minced cooked meat in broth. It is covered with a layer of sterile Vaseline.
4. Isolation by difference in temperature:
Thermophile bacteria grow at 60° C. Some bacteria like N. magnitudes grow at 22° C. By incubation at different temperatures, bacteria can be selectively isolated.
5. Separation of vegetative and spore forming bacteria:
Vegetative bacteria are killed at 80° C. But spore forming bacteria like tetanus bacilli survive at this temperature. So by heating at 80° C vegetative bacteria can be eliminated and spore forming bacteria can be isolated.
6. Separation of motile and non-motile bacteria:
This can be achieved by using Craig’s tube or a U tube. In the U tube, the organisms are introduced in one limb and the motile organism can be isolated at the other limb.
7. Animal inoculation:
Pathogenic bacteria can be isolated by inoculation into appropriate animals, e.g. Anthrax bacilli can be isolated by inoculation into mice or guinea pigs.
Bacteria of different sizes may be separated by using selective filters.
By means of micromanipulation, single bacterium can be separated and cultured.