The below mentioned article provides a short-note on the isolation of bacteria in pure culture. The methods used to isolate the bacteria in pure culture are: 1. Streaking or Plating 2. Dilution and Plating 3. Use of Selective Medium 4. Differential Sterilisation by Chemicals 5. Differential Sterilisation by Heat and 6. Inoculation of a Susceptible Animal.
To determine accurately the specific causative agent of the disease by its diagnostic characters, the microbiologist must isolate a single bacterium, in pure form, from other bacteria with which it is mixed, as an alchemist purifies the metal from other metals or impurities. In clinical specimens (sputum, stool, urine etc.), the mixed microorganisms are common, may mask and confuse the diagnostic results.
To avoid this confusion, a pure culture is needed. The extraneous organisms may change the pH, may damage or kill the desired microorganisms. A pure culture is the one which contains only one kind of microorganisms whereas a mixed culture contains two or more kinds of microorganisms.
Pure culture may be compared to a bed of roses obtained in pure culture of roses. The methods, used to isolate the bacteria in pure culture, consist of separating a single living bacterial cell and allowing it to multiply on a suitable culture medium, usually in a solid medium, to form a pure colony.
Bacteria multiply very rapidly, so that a single bacterial cell deposited on a solid medium gives rise to a mass which is called a colony.
1. Streaking or Plating (Inoculation of Culture Media):
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. This procedure will thus give separate colonies of the organisms present in the material.
2. 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.
3. Use of Selective Medium:
Media which promote the growth of one type of organisms 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 widely used in the preparation of such media.
4. 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 an 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, alkalis and other chemicals.”Antiformin” (Sodium hydroxide) can also be used instead of a dilute alkali.
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.
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 pneumococci by inoculating a mouse. In the latter case, 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.