The process of screening a pure culture by separating one type of microbes from a mixture is called isolation. A culture containing only one species of microbe is called pure culture. In a mixed culture, a particular species is present in small numbers in comparision to the numbers of others.
A single viable cell may be transferred on the culture medium to develop axenic pure culture by using micromanipulator which is used with a microscope for picking up a single colony from a mixed population.
By Exposure to Air
The nutrient agar slide or culture medium containing plate is exposed to the atmosphere for few minutes. After incubation, small colonies appear on the surface of medium which may be transferred on a fresh medium aseptically to obtain pure culture. Such technique is called sub- culturing. When the transfer is from solid medium (agar) to liquid medium (broth), the term picking off is used. In such cases the colour of the colony, their size, shape, appearance, form, consistency and optional properties are recorded.
By Streak Plate Technique
In this method the tip of a fine structure wire loop called inoculation needle consists of a wooden or glass handle with a nichrome wire the end of which is bend to form a loop is used to transfer microbes from culture broth. The straight wires are similar to wire loop except they do not have loop. These are used to transfer culture in colony formed on solid culture medium. In such case, the colony from solid medium is streaked on the surface of nutrient agar medium in a sterile petridish.
By Inoculating In Animals
If disease causing microbe is unable to grow on artificial culture media, the impure culture is injected into the susceptible animals such as guinea pigs or rabbits. These animals allow disease causing organism to grow while rest of the microorganism i.e. etiologic microbe can be isolated on pure form from blood or affected tissue.
By Enrichment Media
This medium is prepared by adding any number of growth factors. This addition enhances the growth and recovery of the desired microorganism. The differential media by virtue of their ingredients distinguish organism growing together. Thus, in EMB (eosin methylene blue complex), Escherichia coli imparts metallic sheen due to precipitation of eosin- methylene blue complex while other enrich bacteria generally do not show metallic sheen. On MacConkey agar medium, E.coli colonies are brick red in colour due to fermentation of lactose. On the other hand, Salmonella typhi does not give this appearance due to non- fermenter of lactose. The other media are selective media such as those which of their special composition promote the growth of one organism and inhibit the growth of others. The minimal medium lack certain growth factors. The medium supports the growth of those microorganisms whose nutritional requirements do not exceed those of the corresponding wild type of strain. Such media are helpful when auxotrophs are required .
The other methods to isolate microorganisms are: a. by controlling physical environment (especially temperature and pH), b. by culturing highly diluted microbial suspension by pour plate method in which isolated population of microorganisms growing from a single isolate can then be easily separated. In pour plate method, the sample is mixed with known quantity of distilled water. The suspension is plated (1 ml) on a presterilized petri dish, spread the sample with the help of loop, and then pour the melted agar medium after cooling it, over the sample containing plate. Incubate the petridish in an incubator at optimum temperature. If counting is not possible , then sample dilutions are made.
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