Listar and his coworkers employed this 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. 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 will be found to contain only one colony of a species, which can be ascertained by microscopic examination.
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