The following points highlight the six main techniques which are used to develop for the isolation of pure culture from mixed culture. The techniques are: 1. Use of Micromanipulator 2. Isolation by Exposure to Air 3. Isolation by Streaking or Streak Plate Technique 4. Isolation by Inoculating in Animals 5. Isolation by Using Selective or Enrichment Media 6. Other Methods to Isolate Microorganisms.
Technique # 1. Use of Micromanipulator:
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.
Technique # 2. Isolation by Exposure to Air:
The nutrient agar slide or culture medium containing plate is exposed to the atmosphere for few minutes. After incubation (overnight or more), 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 optical properties are recorded.
Technique # 3. Isolation by Streaking or 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 cases, the colony from solid medium is streaked on the surface of nutrient agar medium in a sterile Petri dish.
This technique consists of the following steps:
(i) Hold the broth culture containing tube in left hand and shake it.
(ii) Sterilize the wire loop of the inoculation needle on burner flame.
(iii) Remove the cotton plug of the broth culture tube by little finger of right hand.
(iv) Flame the mouth of the test tube immediately.
(v) Keep the test tube in such a way as given in figure; insert the wire loop to form a thin film and replace the cotton plug
(vi) The thin film in the loop is streaked in either a zigzag manner by removing the loop backwards or forwards firmly. Care should be taken that loop should not be firmly pressed against the agar surface.
(vii) Incubate the Petri dish in incubator at a required temperature.
(viii) Growth of the bacteria will be visible (after an overnight incubation) on the streaked marks.
Technique # 4. Isolation 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 fail to grow. The etiologic microbe can be isolated in pure form from blood or affected tissue.
Technique # 5. Isolation by Using Selective or Enrichment Media:
Certain media are selective because these have chemicals or dyes which enrich the media. They have growth inhibitory effect on some microorganism. Such media separate the dominant species Chemical dyes, such as malachite green and crystal violet, are used to inhibit the growth of bacteria and yeast. Sodium azide is a metal-binding agent that inhibits the growth of anaerobic bacteria, but does not affect the anaerobic lactic acid bacteria.
Enrichment Medium:
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 which is due to precipitation of eosin-methylene blue complex while other enteric bacteria generally do not show metallic sheen.
Similarly, 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 are of their special composition promote the growth of one organism and inhibit the growth of others.
The minimal medium lacks 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 (in case of those microorganisms.
Technique # 6. Other Methods to Isolate Microorganisms:
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 Petri dish in an incubator at optimum temperature (27 ± 1 °C). If counting is not possible, then sample dilutions are made as given in Fig. 3.5.
