Identification of Streptococci

In smears from patient material, the microbiologist presumptively identifies gram-positive cocci in chains as streptococci (although the Gram-stain reaction and morphology only are reported to the physician). When culture growth is available, the type of hemolysis produced by colonies on blood agar plates leads to the next step(s) in identification. Alpha-hemolytic colonies from respiratory specimens are not identified further because they are considered normal flora. Beta-hemolytic colonies must be identified to determine whether or not they are group A (any specimen type) or group B (genital specimens from pregnant women).

Although serological testing (most commonly by the latex agglutination method, fig. 19.2) is the definitive method for grouping beta-hemolytic streptococci, rapid methods for grouping have become available only recently and they are expensive. Therefore, alternative, presumptive tests are commonly used in the laboratory for identifying groups A and B streptococci. For example, group A streptococci, but not other beta-hemolytic streptococci, are susceptible to low concentrations of the drug bacitracin. By using a bacitracin disk-diffusion assay such as you performed in Experiment 15.1, the susceptibility of suspected strains can be tested (see colorplate 29). Group B streptococci produce a substance called the CAMP factor (see Experiment 21.2 and colorplate 30) that enhances the effect of beta-hemolysins possessed by some strains of Staphylococcus aureus. All other groups of beta-hemolytic streptococci must be identified serologically, but in practice, determining the absence of group A and B strains is usually sufficient for clinical purposes.

In addition to using a rapid latex agglutination test for identifying group A streptococci (fig. 21.1), a rapid enzyme immunoassay test (see figs. 21.2 and 19.4) is available to detect the group A antigen directly from a throat swab, without first growing the organism in culture. This type of test is usually performed in clinics and in physicians' offices because it is rapid (10 to 30 minutes) and does not require culture expertise. However, for a positive test, a large number of organisms is needed on the swab. When negative results are obtained for patients with clinical evidence of pharyngitis, a throat swab for "strep" culture should always be sent to the clinical microbiology laboratory.

Morello-Mizer-Granato: I III. Diagnostic I 8. Microbiology of the I I © The McGraw-Hill

Laboratory Manual and Microbiology in Action Respiratory Tract Companies, 2003 Workbook in Microbiology, 7/e

Figure 21.1 A positive latex agglutination reaction for group A streptococci. The left-hand well shows a very fine, granular precipitate. In this well, the group A carbohydrate antigen has combined with latex beads coated with antibody against this specific antigen. The well on the right (group B antigen) remains negative, showing only the milky suspension of nonagglutinated latex particles. This antigen does not react with the anti-group A antibodies on the latex particles.

Figure 21.2 Diagram of a streptococcal enzyme immunoassay (EIA). (a) A throat swab from a patient with streptococcal pharyngitis is placed in a tube with extraction solution, which extracts the group A antigen. (b) The extraction solution is then passed through a filter where the group A antigen attaches to its surface. (c) After a wash step, an antibody against the group A Streptococcus, which is linked to an enzyme, is added to the filter where it attaches to the group A antigen. (d) After another wash, a colorless substrate specific for the enzyme is added and is split to a colored end product when it comes in contact with the antibody-bound enzyme. (e) If an antigen other than group A was present, no antibody would bind. Unbound antibody would be washed away, and no color reaction would be seen.

Figure 21.2 Diagram of a streptococcal enzyme immunoassay (EIA). (a) A throat swab from a patient with streptococcal pharyngitis is placed in a tube with extraction solution, which extracts the group A antigen. (b) The extraction solution is then passed through a filter where the group A antigen attaches to its surface. (c) After a wash step, an antibody against the group A Streptococcus, which is linked to an enzyme, is added to the filter where it attaches to the group A antigen. (d) After another wash, a colorless substrate specific for the enzyme is added and is split to a colored end product when it comes in contact with the antibody-bound enzyme. (e) If an antigen other than group A was present, no antibody would bind. Unbound antibody would be washed away, and no color reaction would be seen.

Procedure Microbiological SpecimensProcedure Microbiological Specimens

O Substrate O Substrate reacted with enzyme reacted with enzyme

Morello-Mizer-Granato: I III. Diagnostic I 8. Microbiology of the I I © The McGraw-Hill

Laboratory Manual and Microbiology in Action Respiratory Tract Companies, 2003 Workbook in Microbiology, 7/e

Table 21.1 summarizes the characteristics of streptococci and enterococci. In Experiment 21.1, we shall study some simple methods for isolating streptococci from clinical specimens and for presumptive and confirmatory identification of beta-hemolytic strains as group A. In Experiment 21.2, procedures for differentiating group B strains by the CAMP and serological tests will be followed.

Table 21.1 summarizes the characteristics of streptococci and enterococci. In Experiment 21.1, we shall study some simple methods for isolating streptococci from clinical specimens and for presumptive and confirmatory identification of beta-hemolytic strains as group A. In Experiment 21.2, procedures for differentiating group B strains by the CAMP and serological tests will be followed.

Purpose

To isolate and identify streptococci in culture

Materials

Sheep blood agar plate

Simulated throat culture from a 2-year-old child with acute tonsillitis

Demonstration blood agar plate showing alpha-hemolytic, beta-hemolytic, and nonhemolytic

strains of streptococci

Demonstration plate showing response of two strains of beta-hemolytic streptococci to bacitracin

disks (A disks)

Solution with extracted antigen of beta-hemolytic Streptococcus (prepared by instructor)

Latex test kit for serological typing

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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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