The process of screening a pure culture by separating only one type of microbes from a mixture is called isolation. Various techniques have been developed in the laboratory for the isolation of a pure culture from mixed cultures.
In this technique, diluted suspension of the microbes is smeared on the surface of an appropriate agar medium and single cells are then isolated by manipulation under microscope. These single cells are then allowed to grow in new sterilized media. The colonies are purified following their growth.
A culture can be purified by serially diluting the mixed culture sample in a sterile liquid medium. The final dilution will contain one or none of the microorganisms.
Growth in the last tube of the dilution series may be presumed to have originated from a single cell. This method is useful in the isolation and purification of Yeasts and bacteria.
The principle is based upon the stepwise dilution of a known quantity of a sample in a known quantity of sterile water or buffer. For example, if l ml of a sample is diluted in 99 ml sterile water, the dilution will – or 10~2. Again, if l ml of 100 10 diluted sample is mixed with another flask containing 99 ml distilled water, then the dilution will be or 104. Following is procedure; a sample can be diluted from 10″‘to 10 numbers of times. But, when this is being done, density of the sample gets diluted and individual organisms get separated.
Pour plate technique
This technique involves plating of an appropriately diluted culture suspension mixed with molten agar medium. An aliquot of appropriately diluted inoculums is added to the agar medium held at 40-45° C, mixed and the content is then poured into sterile Petri dishes.
After the medium solidifies, the plates are incubated. Pour plates show both surface and sub-surface growth. This method is useful to isolate and purify cultures of aerobic bacteria. Following incubation, the plates are examined for the presence of individual colonies growing throughout the medium.
The pure colonies which are different in size, shape and colour may be isolated and transferred to fresh media to make pure cultures. Pour plates are used as a means of determining the number of viable organisms in a liquid such as water, milk, urine or broth culture.
It is also used to determine the hemolytic activity of deep colonies of some bacteria like streptococci.
In this method a small amount of the diluted sample is transferred to the surface of a suitable solid medium with the help of a sterilized wire loop and streaked at right angle and then incubated. Growth decreases along the streak and ultimately isolated colonies only appear. This is the most practical method of obtaining discrete colonies and pure cultures.
In the spread plate technique, an aliquot of the diluted sample is transferred to the agar surface and is spread uniformly with a sterile bent glass rod or metal. Colonies arise on the surface of the medium after appropriate incubation period.
Following the completion of incubation in streak plate, pour-plate or spread plate techniques discrete and well separated colonies appear.
The next step is to sub-culture some of the cells from one of the colonies to fresh agar plates or nutrient agar slants with a sterilized loop for further examination. Each of this isolated new culture represents the growth of a single species and is known as pure or stock culture.
Sub-culturing is the term used to describe the procedure of transferring of the microorganisms from their parent growth source to a fresh medium or from one indium to another
Isolation by exposure to air
The nutrient agar slide or the culture medium containing plate is exposed the atmosphere for few minutes. After incubation for at least 12 hours, small colonies appear on the surface of the medium, which may be transferred on a fresh medium aseptically to obtain pure culture.
By using selective or enrichment media
Certain media are called selective because these have chemicals or dyes, which enrich the media. They have growth inhibitory effects on some microorganisms, whereas no effects on others. Such media separate the dominant species. Chemical dyes, such as malachite green and crystal violet are used to inhibit the growth of anaerobic bacteria but does not affect anaerobic lactic acid bacteria.
The other methods for isolating microorganisms are a) by controlling the physical environment especially temperature and pH and b) by inoculating certain test animals.