Pure Culture Methods

In the natural environments microorganisms exist in mixed cultures. To establish the role of microbial agent to a disease process, it is essential to demonstrate the organisms or its components in the diseased tissues. To accomplish this, the organism must be cultivated from the tissues. Similarly to know the kinds of organism present in the environment it is necessary to grow them in artificial media. Cultivation of the organism is also essential to obtain pure culture of clone of cells derived from a single cell to perform biochemical differentiation tests and susceptibility tests since mixed cultures give misleading results.

Artificial culture media
A medium is an environment which supplies the ingredients necessary for the growth of the organism. Various kinds of media have been prepared in the laboratory to isolate, grow and identify an organism. Depending on the need to isolate and identify an organism from a particular sample or environment, different kinds of media are formulated.

Kinds of media
Basal or supportive media
Basal medium is one that contains nutrients that allow the growth of most nonfastidious organism without affording growth advantage to any particular organism over others. Example is Nutrient agar, and Trypticase Soy agar.

Enrichment medium
Enrichment medium is a liquid medium which enhances thegrowth of certain bacterial species, while inhibiting the growth or prolonging the lag phase of unwanted organisms thus altering the ratio between the two in favor of the required bacterial species.

Example is Selenite F broth for the isolation of Salmonella from stool.

To get a pure culture of the organism, any one of the solid media mentioned above is used. In order to get discrete separate colonies, the surface of the medium must be dry. The material is inoculated on the surface by spreading with a sterile loop in such a way that bacteria are ultimately deposited singly. When the bacteria are at a sufficient distance from each other, the whole progeny of each accumulates locally during growth to form a discrete mass or colony which is readily visible to the naked eye. Each colony is presumed to be a pure culture, consisting exclusively of the descendants of a single cell. It may be picked up with a sterile wire to prepare a pure subculture in a fresh medium.

Growth and colony characteristics of Bacteria
The appearances of growths of bacteria in liquid media are generally not distinctive. There is a uniform turbidity in the liquid and little deposit at the bottom. Colony morphology of the isolated bacteria on the solid media has much more value. Attention is paid to the size of the colony (diameter in mm), their outline, whether circular and entire or indented, or wavy or rhizoid, their elevation low convex, high convex or flat plateau-like, umbonate or nodular, their translucency, whether transparent, translucent, or opaque, their pigmentation, colorless, white or otherwise pigmented, and whether they produce any change in the medium (haemolysis in a blood-containing medium).
Example: Colony characteristics of Staphylococcus aureus on Nutrient agar

After aerobic incubation at 37oC for 24 hours, colonies are 1-3 mm in diameter and have a smooth glistening surface, an entire edge, a soft butyrous consistency and an opaque, pigmented appearance.

Growth characteristics of yeasts
Yeasts are grown on Sabouraud Dextrose agar aerobically. Yeasts grow as typical pasty colonies and give out yeasty odor. The colony morphology varies with different yeasts.

Growth characteristics of filamentous fungi
The most common medium used for the isolation of fungi is Sabouraud Dextrose agar. While observing colony morphology, one must note the colors of the surface and the reverse of the colony, the texture of the surface (powdery, granular, woolly, cottony, velvety or glabrous), the topography (elevation, folding, margins, etc) and the rate of growth.

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