The process of bacterial cultivation involves the use of optimal artificial media and incubation conditions to isolate and identify the bacterial etiologies of an infection as rapidly and as accurately as possible.

ISOLATION OF BACTERIA FROM SPECIMENS

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 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.

Table1. Plating Media for Routine Bacteriology

Table1. Plating Media for Routine Bacteriology—cont’d

Table1. Plating Media for Routine Bacteriology—cont’d

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 semi quantitative analysis can be performed. A commonly used streaking technique is illustrated in Figure 1. 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 the streaking of each subsequent quadrant.

Fig1. A, Dilution streak technique for isolation and semiquantitation of bacterial colonies. B, Actual plates show sparse, or 1+ bacterial  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. 

Streaking plates inoculated with a measured amount of specimen, such as when a calibrated loop is used to quantify colony-forming units (CFUs) in urine cultures, is accomplished by spreading the inoculum down the center of the plate. Without flaming the loop, the plate is then streaked side to side across the initial inoculum to evenly distribute the growth on the plate (Figure 2). This facilitates counting colonies by ensuring that individual bacterial cells will be well dispersed over the agar surface. Typically a calibrated loop of 1 µL is used for urine cultures. However, in situations where a lower count of bacteria may be present such as a supra pubic aspiration, a 10 µL loop may be needed to identify the lower count of organisms. The number of colonies identified on the plate is multiplied by the dilution factor in order to determine the number of colony-forming units per millimeter in the original specimen (10³ for a 1 µL loop and 10² for a 10 µL loop). In addition, to standardize the interpretation of colony count, a laboratory should have guidelines for the reporting of organ isms based on the number and types of organisms present.

Fig2. 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.

Evaluation of Colony Morphologies

The initial evaluation of colony morphologies on the primary plating media is extremely important. Laboratorians can provide physicians with early preliminary information regarding the patient’s culture results. This information is also important for deciding how to proceed for definitive organism identification and characterization.

Type of Media Supporting Bacterial Growth. As previously discussed, different media are used to recover particular bacterial pathogens. In other words, the media selected for growth is a clue to the type of organism isolated (e.g., growth on MacConkey agar indicates the organism is most likely a gram-negative bacillus). Yeast and some gram-positive cocci are capable of limited growth on MacConkey agar. The incubation conditions that support growth may also be a preliminary indicator of which bacteria have been isolated (e.g., aerobic versus anaerobic bacteria).

Relative Quantities of Each Colony Type. 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. Several methods are used for semiquantitation of bacterial quantities including many, moderate, few or a numerical designation (4+, 3+, 2+) based on the number of colonies identified in each streak area (Table 2).

Table2. Semi-Quantitation Grading Procedure for Bacterial  Isolates on Growth Media

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 pinpoint, small, medium, large)

 • Colony pigmentation

• Colony shape (includes form, elevation, and margin of the colony [Figure 3])

 • Colony surface appearance (e.g., glistening, opaque, dull, dry, 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)

• Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification)

Fig3.  Colony morphologic features and descriptive terms for  commonly encountered bacterial colonies. 

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 bacterial 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. 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 on all different colony morphologies 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 Mac Conkey 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 4).

Fig4. Mixed bacterial culture on sheep blood agar (A) requires subculture of individually distinct colonies (arrows) to obtain pure  cultures of Staphylococcus aureus (beta hemolysis evident) (B) and Streptococcus pneumoniae (alpha hemolytic) (C). 

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 over inoculation 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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مواضيع ذات صلة

Principles of Bacterial Cultivation : Environmental Requirements

Structure and Function of the Bacterial Cell

Bacterial Metabolism

Bacterial Genetics: Nucleic Acid Structure and Organization

Detection of Bacteremia: Culture Techniques

Detection of Bacteremia: Types of Blood Culture Bottle

Detection of Bacteremia: Specimen Collection

The Study of the Intestinal Microbiota

Chlamydia psittaci and Psittacosis

Chlamydia Pneumonia and Respiratory Infections

Chlamydia trachomatis Genital Infections and Inclusion Conjunctivitis

Trachoma