Clinical Pathology(Micro)

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Slide coagulase test: This test is a rapid method for identifying S. aureus. Most strains of this species produce a cell-bound coagulase or “clumping factor” that is detected by mixing a suspension of the organism with EDTA-rabbit plasma. A saline control (left) must be included to assess autoagglutination. Not all S. aureus strains possess clumping factor, so negative slide coagulase tests must be confirmed with the tube coagulase test (see Plate 11-2C).

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Tube coagulase test: In this test, extracellular coagulase produced by S. aureus complexes with a component in plasma, called coagulase-reacting factor. This complex, in turn, reacts with fibrinogen to form fibrin and, consequently, the development of a visible clot. A positive test is shown in the botto,n of this fig­ure, and a negative test is shown in the top portion of the figure.

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Latex agglutination test for identification of S. aurcus: This alternative coagulase test procedure uses latex spheres that are coated with plasma. Fibrinogen bound to the latex detects clumping factor, and the immunoglobulin, also pres­ent on the latex, detects protein A on the surface of the S. aurcus cell. Mixing of colonial growth of S. aureus with the latex reagent results in rapid agglutination (left). A coagulase-negative staphylococcus, which produces a negative reac­tion, is also shown (right). (Courtesy of Murex Diagnostics, Norcross GA.)

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Passive hemagglutination test for identification of S. aureus. This alternative co­agulase test procedure uses sheep red blood cells that are coated with fibrino­gen. The mixing of colonial growth of S. aureus with the coated red blood cells results in visible clumping of the bacteria and red blood cells, as shown in the photograph. A nonsensitized red blood cell suspension must be included as a negative control (not shown).

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Disk tests for identification of staphylococci: Although furazolidone and baci­tracin disk tests are useful for differentiating micrococci from staphylococci, susceptibility to novobiocin is useful for presumptive identification of S. so pro­pliyticus, an important agent of urinary tract infections. In this figure, all three tests are shown for an isolate of S. saprophyticus. Susceptibility to furazolidone (FX, 100 pg) and resistance to bacitracin (Taxo A disk, 0.04 U bacitracin) indicate that the organism is a Staphylococcus, rather than a Micrococcus, species. Resistance to novobiocin (C—, 5 pg) presumptively identifies the isolate as S. saprophyticus.

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API STAPH (bioMerieux-Vitek, Inc., Hazelwood MO) for identification of staphylococci and micrococci: The strip is inoculated with an organism suspen­sion and is incubated overnight. Interpretation of the reactions generates a bio­type number that is used, along with a computer-assisted database, to identify the organism

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1D32 Staph (bioMerieux S.A., France) for identification of staphylococci and mi­crococci: This strip-format identification system, which is inoculated in a man­ner similar to the API STAPH (see Plate 11-2G) contains 26 biochemical tests to generate a biotype number that is used with a computerized database to iden­tify the organism. As of this writing, the 1D32 Staph system is not approved by the FDA for use in the United States.

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Surface of XLD agar illustrating yellow conversion of the medium from acid-producing colonies of E. coli.

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Non-lactose-fermenting colonies (no acid conversion of the medium) of Salmonella species growing on the surface of XLD agar. Note the black pigmentation of some of the colonies, indicating H2S production.

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Photograph illustrating an XLD agar plate inoculated with a 50/50 mixture of E. coli and Salmonella species. Note the preponderant growth of the Salmonella species (red colonies) compared with the few yellow, lactose-fermenting colonies of E. coli that have been effectively inhibited. The distinct pink halo around the Salmonella colonies indicates the decarboxylation of lysine, a helpful feature in differentiating Salmonella species (positive) from H2S-producing colonies of Proteus species.

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XLD agar plate inoculated with an H2S-producing strain of a Proteus species. Note the lack of a light pink halo around the colonies, indicating the lack of lysine decarboxylation (compare with the colonies shown in C).

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Surface of HE agar illustrating yellow acid production by colonies of E. coli.

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Surface of HE agar illustrating the faint green (colorless) colonies of non­lactose-fermenting members of the Enterobacteriaceae.

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Surface of MacConkey agar with 24-hour growth of red, lactose-fermenting colonies. The diffuse red color in the agar surrounding the colonies is produced by organisms that avidly ferment lactose, producing large quantities of mixed acids, and cause precipitation of the bile salts in the medium surrounding the colonies (eg, Escherichia coli).

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Surface of MacConkey agar illustrating both red, lactose-fermenting colonies and smaller, clear non-lactose-fermenting colonies

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Surface of EMB agar plates illustrating the green sheen produced by avid lactose-(or sucrose) fermenting members of the Enterobacteriaceae. Most strains of E. coli produce colonies with this appearance on EMB agar, and since E. coli is among the most frequent isolates from clinical specimens, the appearance of such colonies can often serve as presumptive identification of E. coli. However, characteristics other than the production of a green sheen on EMB must be assessed before an organism can be definitively identified as E. coli, since other lactose-fermenting Enterobacteriaceae can have a similar appearance.

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Surface of EMB agar plates illustrating a mixed culture of E. coli (green sheen colonies) and Shigella species. Most Shigella species do not ferment lactose and, thus produce nonpigmented, semitranslucent colonies on EMB agar. Other species incapable of fermenting lactose produce colonies that appear similar to those illustrated in these photographs.

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Surface of EMB agar plates illustrating a mixed culture of E. coli (green sheen colonies) and Shigella species. Most Shigella species do not ferment lactose and, thus produce nonpigmented, semitranslucent colonies on EMB agar. Other species incapable of fermenting lactose produce colonies that appear similar to those illustrated in these photographs.

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Gram stain of Campilobacter jejuni illustrating pleomorphic gram-negative bacilli, with short, curved, and spiral forms. Note that some cells connect to form gull-winged and “S” shapes.

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C. jejuni growing on nonselective Brucella agar plate following isolation from stool using the membrane filter technique (described in text). Note that growth has occurred only in the area of the plate underneath where the filter had been placed

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Close-up view of C. jejuni on blood agar illustrating raised, gray-white, and somewhat mucoid colonies.

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Growth of C. jejuni on Campy BAP agar illustrating the tendency of the organ­ism to grow along the streak lines.

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Tubes showing the rapid hippurate reaction. Purple color develops with the ad­dition of ninhydrin when hippurate has been hydrolyzed to form glycine and benzoic acid (positive tube on left compared with negative control on right). Of the Campylobacter species, only C. jejuni gives a positive hippurate reaction.

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Brucella blood agar plate showing growth of C. jejuni around cephalothin and nalidixic acid disks. Note that with C. jejuni a zone of inhibition forms around the nalidixic acid disk (rig/it), indicating that this species is susceptible to nalidixic acid but resistant to cephalothin. This test is easy to perform and al­lows presumptive identification of C. jejuni.

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Triple sugar iron (TSI) agar slant reactions illustrating the hydrogen sulfide (H25) reactions of several species. The tube to the extreme left illustrates the lack of H25, characteristic of C. jejuni, C. fetus subsp. fetus, and C. fetus subsp. venere­alis. Tubes 2, 4, and 5 (reading from left) illustrate a strong butt reaction, charac­teristic of C. sputorum biovar bubulus, C. sputorum biovar fecalis, or C. sputoru;n biovar sputorum. Tube 3 illustrates a strong slant reaction characteristic of C. mucosalis.

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Silver-stained tissue section of superficial gastric mucosa demonstrating clus­ters of blue-black staining bacilli along the epithelial lining, consistent with the bacillary forms of Helicobacter pylon. When observed in a Gram-stained prepa­ration, the individual cells are long, thick, and curved.

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Gram stain of streptococci growing in a broth culture: As their name suggests, strep­tococci characteristically grow in chains. These chain forms are most frequently seen when the organisms are grown in broth. On gram-stained smears prepared from growth on agar media, the organisms usually appear in pairs or in shorter chains

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Gram stain of S. pneumoniae: This photograph shows the typical appearance of pneumococci in blood culture broth. These bacteria characteristically grow in pairs in which the cells have a slightly elongated “lanceolate” morphology. With some cells in this frame, a clear area or “halo” may be observed surrounding the organ­ism pairs, indicating the presence of the polysaccharide capsule of S. pneumoniae.

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Alpha-Hemolytic streptococci on sheep blood agar: Streptococci initially may be classified on the basis of their hemolytic properties on sheep blood agar. Partial hemolysis of the erythrocytes results in a “greening” of the agar medium surrounding the colonies (cx-hemolysis). Streptococci that are a-hemolytic include S. pneumoniae, the viridans group of streptococci, and most Enterococcus (formerly Streptococcus) species.

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ß-Hemolytic streptococci on sheep blood agar: ß-Hemolytic streptococci pro­duce hemolysins that lyse sheep erythrocytes, resulting in a clearing of the medium surrounding the colonies. The group A streptococcus shown here demonstrates this type of hemolysis, as do group B, C, and G streptococci. The zones of ß-hemolysis surrounding colonies of group B streptococci, however, are not as large relative to the size of the colony as those seen with groups A, C, and G ß-hemolytic streptococci.

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Direct latex agglutination test for group A streptococci: Both latex agglutination and rapid enzyme immunoassays are currently available for the direct detec­tion of group A streptococci in throat swab specimens. With the latex aggluti­nation method shown here, the throat swab is extracted with nitrous acid. After the pH of the extract is adjusted, drops of the solution are reacted with a latex suspension coated with antigroup A antibodies, and with a control, noncoated latex suspension. Agglutination of the test (left), but not the control suspension (right), is a positive test. Although most of these tests are highly specific for the group A cell wall antigen, the sensitivities of these assays vary widely

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Strep A OIA (Optical Immunoassay; Biostar, Boulder CO): Strep OIA is an im­munoassay for direct detection of group A streptococci in throat swab speci­mens. The swab is extract with acetic acid, neutralized, and then mixed with antigroup A streptococcal antibodies bound to horseradish peroxidase (HRP). A drop of this mixture is placed on the surface of an OIA slide, which is coated with antigroup A streptococcal antibodies. After a 2-minute incubation, the slide is rinsed and HRP substrate is added and allowed to react for 4 minutes. After rinsing, the test is read by examining the hue of light reflected from the re­action area on the slide. If group A streptococcal antigen is present, the slide shows a purple spot (left). If no antigen is present, the slide surface retains its gold color, with or without a small blue dot, as shown on here (right).

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S. pneumoniae colonies on sheep blood agar: Two characteristics of S. pneumoniae can be used for presumptive identification. At left is shown a typical a-hemolytic mucoid strain of S. pneumoniae, an appearance that is due to large amounts of capsular polysaccharide. At right is a close-up photograph illustrating collapse of the central portion of the colonies owing to organism autolysis, resulting in the so-called checker piece and nail head colony morphologies shown here.