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The following points highlight the top six methods for isolation of bacteria in pure culture. The methods are: 1. Streaking or Plating 2. Dilution and Plating 3. Use of Selective Medium 4. Differential Sterilisation by Chemicals 5. Differential Sterilisation by Heat 6. Inoculation of a Susceptible Animal.
Isolation of Bacteria: Method # 1.
Streaking or Plating:
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If the material containing bacteria is streaked several times on the surface of a solid medium with a platinum loop without recharging, fewer and fewer bacterial cells will remain on the loop as successive streaks are made and, finally, it may deposit a single bacterial cell on the surface.
Isolation of Bacteria: Method # 2.
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Dilution and Plating:
When the bacterial content of the material is expected to be high (e.g. feces), it is diluted with sterile nutrient broth or saline before plating.
Isolation of Bacteria: Method # 3.
Use of Selective Medium:
Media which promote the growth of one type of organism and inhibit or retard the growth of others are known as “Selective Media“. Inhibiting substances like brilliant green, gentian violet, bile salts, potassium tellurite and penicillin are willy used in the preparation of such media.
Isolation of Bacteria: Method # 4.
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Differential Sterilisation by Chemicals:
An example of this method is cultivation of tubercle bacilli from sputum (which contains numerous other organisms) after preliminary treatment with dilute mineral acid or alkali. In Petroff’s method, the specimen of sputum is mixed with three or four times its volume of 4 per cent sodium hydroxide solution, incubated for one half to hour at 37°C and centrifuged.
The deposit is neutralized, in the presence of an indicator, by dilute hydrochloric acid, and then seeded on a suitable medium. This treatment kills all other organisms except tubercle bacilli which are resistant to the action of dilute mineral acids, alkalies and other chemicals. “Antiformin” (Sodium hydroxide) can also be used instead of a dilute alkali.
Isolation of Bacteria: Method # 5.
Differential Sterilisation by Heat:
This method is employed where the organisms to be obtained in pure culture are more resistant to heat than others present in the material e.g. separation of spore bearing bacilli (which are more resistant to heat) from the non-spore bearers. The material is heated at 60°C for 30 minutes, cooled and then inoculated in a suitable medium. Only the spore bearers which have survived will grow.
Isolation of Bacteria: Method # 6.
Inoculation of a Susceptible Animal:
This method is employed to isolate pathogenic microorganisms which are not easily grown on culture media or which are readily overgrown by the contaminating organisms. As example, we may quote isolation of tubercle bacilli from pus, peritoneal and pleural fluids by inoculating a guinea pig and isolation of the pneumococci from inoculating a mouse.
The contaminating organisms are rapidly killed in the animal body whereas the pathogenic organisms multiply and can be recovered in pure culture from the tissues.
