The JEMBEC system should be incubated at 35° to 37° C as soon as the plate is received in the laboratory. Body lu s (e.g., joint or cerebrospinal fluid [CSF]) should be Jsept at room temperature or placed at 37° C before Mturing because both the gonococci and meningococci are sensitive to cold.
Any volume of clear fluid greater than 1 mL suspected of containing either of these pathogens should t - centrifuged at room temperature at 1500x g for 15 minutes. The supernatant fluid should then be removed and the sediment should be vortexed and inoculated onto the appropriate media (described later).
Any specimens or cultures in which N. meningitidis 'i a consideration should be handled in a biological fSafety cabinet to avoid laboratory-acquired infections.
Members of the genus Neisseria discussed in this chapter Moraxella catarrhalis appear as gram-negative $plococd (Figure 42-2) with adjacent sides flattened. The direct Gram stain of urethral discharge from symptomatic males with urethritis is an important test for gonococcal disease. The appearance of gramnegative diplococd inside polymorphonuclear leukocytes is diagnostic in this situation. However, because the normal vaginal and rectal flora are composed of gram-negative coccobacilli, which can resemble Neisseria spp., direct examination of endocervical secretions in symptomatic women is still only presumptive evidence of gonorrhea and the diagnosis must be confirmed by culture.
The direct Gram stain of body fluids for either N. gonorrhoeae or N. meningitidis is best accomplished using a cytocentrifuge, which can concentrate small numbers of organisms 100-fold.
Molecular assays have replaced old enzyme-linked immunosorbent assay systems for rapid diagnosis of Neisseria gonorrhoeae. The Food and Drug Administration has deared a number of amplified and nonamplified tests. (For a discussion of molecular technology, see Chapter 8.) The nonamplified DNA probe assay (GenProbe, San Diego, Calif) has a chemiluminescent detection system for direct detection of gonococcal ribosomal RNA in genital and conjunctiva] specimens. This test performs well in high-risk patients, is rapid (results are available in 2 hours), and is suitable for screening many patients simultaneously.
Amplified assays are more sensitive than the nonamplified assay and are commercially available from Roche Diagnostic Systems (Branchburg, NJ), Gen-Probe (San Diego, Calif), and Becton Dickinson and Company (Sparks, Md). These tests are suitable for large-scale screening programs, but none are admissible as evidence in medicolegal cases. An advantage of all molecular assays is the ability to test for Chlamydia trachomatis from the same specimen at the same time. Neisseria gonorrhoeae DNA can be found in a specimen for up to 3 weeks after successful treatment, so amplified molecular assays should not be used to assess test of cure.
The detection of Neisseria meningitidis capsular polysaccharide antigen in body fluids (e.g., urine, serum, CSF) is no longer recommended in the United States.
N. meningitidis, M. catarrhalis, and saprophytic Neisseria spp. grow well on 5% sheep blood and chocolate agars; N. gonorrhoeae is more fastidious and requires an enriched chocolate agar for growth on primary culture. Because gonococo, and sometimes meningococci, must be isolated from sites that contain large' numbers of normal flora (e.g., genital or upper respiratory tracts), selective media have been developed to facilitate their recovery. The first of these was Thayer-Martin medium, a chocolate agar with an enrichment supplement (IsoVitaleX) and the antimicrobials colistin (to inhibit gramnegative bacilli), nystatin (to inhibit yeast), and vancomycin (to inhibit gram-positive bacteria). This original medium was subsequentiy modified to include trimethoprim (to inhibit swarming Proteus), and its name was changed to MTM (modified Thayer-Martin) medium. Martin Lewis (ML) medium is similar to MTM except that anisomycin, an antifungal agent, is substituted for nystatin and the concentration of vancomycin is increased.
A transparent medium containing lysed horse blood, horse plasma, yeast dialysate, and the same antibiotics as MTM, called New York City (NYC) medium, also has been used. The advantage of NYC medium is that genital mycoplasma (.Mycoplasma homi-nis and Ureaplasma urealyticum; see Chapter 47 for more information regarding these organisms) will also grow on this agar. Some strains of N. gonorrhoeae are inhibited by the concentration of vancomycin in the selective media, so the addition of nonselective chocolate agar is recommended, especially in suspect cases that are culture-negative or for sterile specimens (e.g., joint fluid).
Unlike the pathogenic species, some of the saprophytic Neisseria spp. (N.flavescens, N. mucosa, N. sicca, and N. subflava) may grow on MacConkey agar, although poorly. N. gonorrhoeae and N. meningitidis will grow in most broth blood culture media but grow poorly in common nutrient broths such as thioglycollate and brain-heart infusion. M. catarrhalis and the other Neisseria spp. grow well in almost any broth medium.
Incubation Conditions and Duration
Agar plates should be incubated at 35° to 37° C for 72 hours in a C02-enriched, humid atmosphere. The pathogenic neisseriae and M. catarrhalis grow best under conditions of increased C02 (3% to 7%). This atmosphere can be achieved using a candle jar, C02-generating pouch, or C02 incubator. Only white, unscented candles should be used in candle jars because other types may be toxic to N. gonorrhoeae and N. meningitidis.
Humidity can be provided by placing a pan with water in the bottom of a C02 incubator or by placing a sterile gauze pad soaked with sterile water in the bottom of a candle jar (Figure 42-3).
Table 42-3 describes the colonial appearance and other distinguishing characteristics (e.g., pigment) on chocolate agar.
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