Isolation Of Bacteria From Specimens

As discussed in detail throughout Part VII, the cultivation of bacteria from infections at various body sites is accomplished by inoculating processed specimens directly onto artificial media. The media are summarized in Table 7-1 and incubation conditions are selected for their ability to support the growth of the bacteria most likely to be involved in the infectious process.

To enhance the growth, isolation, and selection of etiologic agents, specimen inocula are usually spread over the surface of plates in a standard pattern so that individual bacterial colonies are obtained and semiquantitative analysis can be performed. A commonly used streaking technique is illustrated in Figure 7-9. Using this method, the relative numbers of organisms in the original specimen can be estimated based on the growth of colonies past the original area of inoculation. To enhance isolation of bacterial colonies, the loop should be flamed for sterilization between streaking each subsequent quadrant.

Streaking plates inoculated with a measured amount of specimen, such as when a calibrated loop is used to quantify colony-foiming units (CFUs) in urine cultures, is accomplished by spreading the inoculum evenly over the entire agar surface (Figure 7-10). This facilitates counting colonies by ensuring that individual bacterial cells will be well dispersed over the agar surface.

Evaluation of Colony Morphologies

Initial evaluation of colony morphologies on the primary plating media is extremely important. Labora-

torians can provide physicians with early preliminary information regarding the patient's culture results. This information also is important for deciding which subsequent steps to take for definitive organism identification and characterization.

Type of Media Supporting Bacterial Growth. As previously discussed, different media are used to recover particular bacterial pathogens so that determining which media support growth is a clue to the type of organism isolated (e.g., growth on MacConkey agar indicates the organism is a gram-negative bacillus). The incubation conditions that support growth may also be a preliminary indicator of which bacteria have been isolated (e.g., aerobic vs. anaerobic bacteria).

Relative Quantities of Each Colony lype. The predominance of a bacterial isolate is often used as one of the criteria, along with direct smear results, organism virulence, and the body site from which the culture was obtained, for establishing the organism's clinical significance.

Colony Characteristics. Noting key features of a bacterial colony is important for any bacterial identification; success or failure of subsequent identification procedures often depends on the accuracy of these observations. Criteria frequently used to characterize bacterial growth include the following:

  • Colony size (usually measured in millimeters or described in relative terms such as pin-* point, small, medium, large)
  • Colony pigmentation
  • Colony shape (includes form, elevation, and margin of the colony [Figure 7-11])
  • Colony surface appearance (e.g., glistening, opaque, dull, transparent)
  • Changes in agar media resulting from bacterial growth (e.g., hemolytic pattern on blood agar, changes in color of pH indicators, pitting of the agar surface; for examples see Figures 7-3 through 7-8)
  • Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification)

Many of these criteria are somewhat subjective, and the adjectives and descriptive terms used may vary among different laboratories. Regardless of the terminology used, nearly every laboratory's protocol for bacteria] identification begins with some agreed-upon colony description of the commonly encountered pathogens.

Although careful determination of colony appearance is important, it is unwise to place total confidence on colony morphology for preliminary identification.

L igure 7-9 A, Dilution streak technique for isolation and semiquantitation of bacterial colonies. B, Actual plates show sparse, or 1+ bacteria] growth that is limited to the first quadrant. C, Moderate, or 2+ bacterial growth that extends to the second quadrant. D, Heavy, or 3+ to 4+ bacterial growth that extends to the fourth quadrant.

Streak pattern

Streak pattern

Liquid specimen of inoculum

Figure 7-10 A, Streaking pattern using a calibrated loop for enumeration of bacterial colonies grown from a liquid specimen such as urine. B, Actual plate shows well-isolated and dispersed bacterial colonies for enumeration obtained with the calibrated loop streaking technique.

Liquid specimen of inoculum

Figure 7-10 A, Streaking pattern using a calibrated loop for enumeration of bacterial colonies grown from a liquid specimen such as urine. B, Actual plate shows well-isolated and dispersed bacterial colonies for enumeration obtained with the calibrated loop streaking technique.

Bacteria of one species often exhibit colony characteristics that are nearly indistinguishable from those of many other species. Additionally, bacteria of the same species exhibit morphologic diversity. For example, certain colony characteristics may be typical of a given species, but different strains of that species may have different morphologies.

Gram Stain and Subcultures. Isolation of individual colonies during cultivation not only is important for examining morphologies and characteristics but also is necessary for timely performance of Gram stains and subcultures.

The Gram stain and microscopic evaluation of cultured bacteria are used with colony morphology to decide which identification steps are needed. To avoid confusion, organisms from a single colony are stained. In many instances, staining must be performed with each different colony morphology that is observed on the primary plate. In other cases, staining may not be necessary because growth on a particular selective agar provides dependable evidence of the organism's Gram stain morphology (e.g., gram-negative bacilli essentially are the only clinically relevant bacteria that grow well on MacConkey agar).

Following characterization of growth on primary plating media, all subsequent procedures for definitive identification require the use of pure cultures (i.e., cultures containing one strain of a single species). If sufficient inocula for testing can be obtained from the primary media, then a subculture is not necessary, except as a precaution to obtain more of the etiologic agent if needed and to ensure that a pure inoculum has been used for subsequent tests (i.e., a "purity" check). However, frequently the primary media do not yield sufficient amounts of bacteria in pure culture and a subculture step is required (Figure 7-12).

Using a sterile loop, a portion of an isolated colony is taken and transferred to the surface of a suitable enrichment medium that is then incubated under conditions optimal for the organism. When making transfers for subculture it is beneficial to flame the inoculating loop between streaks to each area on the agar surface. This avoids overinoculation of the subculture media and ensures individual colonies will be obtained. Once a pure culture is available in a sufficient amount, an inoculum for subsequent identification procedures can be prepared.

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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  • fre-swera
    Why the primary negative was not spreading the inoculum evenly over the agar surface?
    4 years ago

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