This article throws light upon the top five techniques used to obtain pure cultures of organism. The techniques are: 1. Direct Transfer Technique 2. Single-Cell Isolation Technique 3. Streak-Plate Technique 4. Dilution Technique 5. Pour-Plate Technique.
1. Direct Transfer Technique:
Sometimes bacteria, yeasts, and molds may be found in pure culture under natural condition. The transfer of such cultures can be made directly to a suitable medium. Several molds produce aerial conidia which are comparatively free from contaminations.
In certain disease of man and animals the causal organism is found in the blood e.g. typhoid fever and anthrax. A pure culture can be obtained by the transfer of infected blood with proper precautions to avoid contamination.
2. Single-Cell Isolation Technique:
The best way to secure a pure culture from a mixed one would be to pick out a single cell of the desired type. This can be done by the use of micro-manipulator in combination with a microscope. Micro-manipulator has a micropipette with a very fine capillary point.
The micropipette can be moved as desired. A single cell can be picked with it help of a micropipette and transferred to a suitable medium for growth. But this technique requires a skilled operator, is cumbersome to operate and involves greater chances of contamination during operation.
3. Streak-Plate Technique:
The inoculum is streaked or spread over the surface of a solid medium. The streaking process accomplishes a thinning out of the population. There is no uniform distribution of the organisms. Where most thickly populated, they will grow in mass. But where only few are present, they develop distinct colonies.
These isolated colonies are the progenies of single cells and whether they are so, may be verified from microscopic examination. Pure culture may be secured by the transfer of a part of such colonies to a container with suitable culture medium.
4. Dilution Technique:
Lister and other workers of his time employed serial dilution technique to separate a single species from a mixture in which it was a predominating type.
A small amount of the material which contains mixture of bacteria is added to a test tube containing sterile medium of known volume; one ml of liquid-bacteria mixture is then diluted to the desired dilution by transfer through a series of test-tubes containing similar volume of sterilised medium.
It is evident that a stage will be reached when higher dilutions will contain no organisms and will show no growth upon incubation. Some of the tubes of higher dilution showing growth will be found to contain only one colony of a species which can be ascertained by microscopic examination.
5. Pour-Plate Technique:
The specimen containing bacteria is first diluted in tubes of an agar medium. As one is not sure about the magnitude of the bacteria population in the original specimen, dilution through more than one tube is essential to get well isolated colonies. The medium is maintained in liquid state at about 42Â°C to enable a thorough distribution of inoculum. The inoculated material is poured into petridishes.
The medium is then uniformly distributed over the bottom of petridish by gently tilting the dish and allowed to cool and set. Some of the bacterial cells are separated from one another and their movement is prevented due to the solidification of medium. In due time colonies develop and in most cases they originate from a single cell and are pure cultures. They may then be transferred directly to suitable media.
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