Obtaining pure cultures for identification is one of the most important laboratory procedures in microbiology. When samples from soil, water, food, or body are placed on nutrient-containing petri plates, it is quickly become clear that each sample contains a variety of microbes.
This is particularly true of specimens collected from the skin or mucous membranes of patients with infections. The clinical specimens contain both harmless microbes normally found on the body (normal flora) and the microbes causing the disease.
In order to accurately identify which organism is responsible for the illness, the many different types of microbes must be separated. After “paration, the individual microbes must be placed on a medium that will allow them to reproduce into a clearly visible population, or colony, composed of only that one species.
Samples from this colony can then be “picked” out and transferred to a separate culture for further growth and identification. The microbiologist will then be able to study such characteristics as morphology, metabolism, and antibiotic sensitivity.
There are several techniques used to separate mixed cultures; however, the streak plate method and the pour plate method are the two most common.
Skill is required to properly perform both methods, but the streak plate method is probably the more practical. A nutrient agar medium is prepared that is known to support the active growth of the organism isolated from the mixed specimen.
This medium is sterilized to remove all foreign microbes and ensure that only microbes from the specimen will grow on the petri plate. Special wires called inoculating needles or inoculating loops are used to transfer the specimen to the petri plate.
These must also be sterilized to prevent contamination. This is accomplished by placing the wire in an open Bunsen burner flame or other heating apparatus to destroy all microbes by incineration.
Inoculating needles are used to transfer microbes growing on solid media since the needle will carry fewer cells than the loop. The loop is used to transfer microbes from liquid since it will hold more of the microbes dispersed in broth culture.
The streaking patterns used to separate the microbes vary from laboratory to laboratory, but the object of each pattern is the same. The microbes are placed on the agar surface in a small concentrated area and the inoculating loop is flamed.
The loop is then used to spread sample, or inoculums into another section of the plate, thus dispersing fewer microbes into a larger area. This basic technique is repeated until individual cells have been moved so far apart that each cell may grow into a separated colony.
Since each colony has developed as a result of binary fission, all cells within it are of the same species. Each pure colony can then be “picked” with an inoculating needle and cells transferred to new, sterile media for further tests.
The pour plate method does not involve separation of microbes on the surface of agar plates, but separation within liquid, cooled media. Sterile agar in test tubes is kept about 48°-50°C. At this temperature, the agar will be cool enough to prevent most bacteria from being killed yet warm enough to remain in liquid form (agar begins to solidify at 42 °C).
The mixed inoculums is placed into the first of a series of agar-containing media tubes with an inoculating loop and shaken thoroughly. This disperses the cells throughout the medium.
The loop is then flame-sterilized and a sample is taken from the first tube for transfer into a second tube. This procedure is repeated a third, and possibly, a fourth time-a process called serial dilution. Each tube in the series contains fewer microbes than the previous tube.
If the technique is performed properly, the last tube in the series will contain the fewest separated cells, the tubes’ contents are then poured into sterile petri plates and allowed to solidify.
Since the cells have been dispersed throughout the medium, colonies may grow on the surface of the agar, within the agar, and under the agar. The surface colonies may be picked for isolation of pure cultures, but those within the agar are more difficult to reach for this purpose.
In addition to pure isolation, the pour plate method may be used to determine the number of viable (living) cells in the original specimen. The greatest accuracy in counting is obtained when plates have between 30 and 300 colonies. Plate with more than 300 colonies is likely to be inaccurate because of errors in technique or contamination.
The presence of a single colony on the surface of a pteri plate does not guarantee that the colony is pure. Several procedures must be carried out to determine that the pure culture techniques have been successful.
Microscopic analysis, and determination of physiological and colony characteristics must all be performed to confirm that the microbes have been successfully separated from the original mixture.
The Gram staining technique plays a vital role as a confirmation tests, as do other staining methods described later in this chapter. The physiological characteristics of a microbe are found by placing pure culture samples in different nutrients and biochemically appearance of a population of microbes as it grows on the solid medium surface.
Special terminology may be used by microbiologists to describe these features. For example, the amount of growth may be described as slight, moderate, or abundant. Colonies that grow very well may appear opaque, transparent, or translucent.
The form of colony takes on the surface of the agar may be said to be round, filamentous, rhizoid, or complex. Even through many bacteria may demonstrate any of these growth characteristics; no two bacterial species display the same combination of characteristics. This fact enables microbiologists to uniquely characterize a particular microbe with relative ease.