Cultivation of Mycoplasma and Ureaplasma
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p531-533
2025-09-21
523
In general, the medium for mycoplasma isolation contains a beef or soybean protein with serum, fresh yeast extract, and other factors. As a result of the slow growth of these organisms, the medium must be selective to prevent overgrowth of faster-growing organisms that may be present in a clinical sample. Culture media and incubation conditions for these organisms are summarized in Table 1. Culture methods for M. pneumoniae, U. urealyticum, and M. hominis are provided on the Evolve site in Procedures 1, 2, and 3, respectively. The quality control of the growth media with a fastidious isolate is of great importance.
Table1. Cultivation of Mycoplasma pneumoniae, Ureaplasma spp., and M. hominis
PROCEDURE 1 Isolation of Mycoplasma Pneumoniae
PROCEDURE 2 Isolation of Ureaplasma urealyticum
PROCEDURE 3 Isolation of Mycoplasma hominis
For the most part, the different metabolic activity of the mycoplasmas for different substrates is used to detect their growth. Glucose (dextrose) is incorporated into media selective for M. pneumoniae, because this mycoplasma ferments glucose to lactic acid; the resulting pH change is then detected by a color change in a dye indica tor. Similarly, urea or arginine can be incorporated into media to detect U. urealyticum and M. hominis, respectively (Table 2). If a color change—that is, a pH change—is detected, a 0.1- to 0.2-mL aliquot is immediately subcultured to fresh broth and agar media.
Table2. Basic Biochemical Differentiation of the Major Mycoplasma spp. and Ureaplasma urealyticum
In some clinical situations, it may be necessary to provide quantitative information regarding the numbers of genital mycoplasmas in a clinical specimen. For example, quantitation of specimens taken at different stages during urination or after prostatic massage can help determine the location of mycoplasmal infection in the genitourinary tract.
APPROACH TO IDENTIFICATION
On agar, M. pneumoniae will appear as spherical, grainy, yellowish forms that are embedded in the agar, with a thin outer layer similar to those shown in Figure 1. The agar surface is examined under 20 to 60× magnification using a stereomicroscope daily for Ureaplasma spp., at 24 to 72 hours for M. hominis, and every 3 to 5 days for M. pneumoniae and other slow-growing species. Because only M. pneumoniae and one serovar of U. urealyticum hemadsorb, M. pneumoniae is definitively identified by overlaying suspicious colonies with 0.5% guinea pig erythrocytes in phosphate-buffered saline instead of water. After 20 to 30 minutes at room temperature, colonies are observed for adherence of red blood cells.
Fig2. Colonies of Mycoplasma pneumoniae visualized under 100× magnification. Note the variation in the size of the colonies (arrows). (Courtesy Clinical Microbiology Laboratory, SUNY Upstate Medical University, Syracuse, NY.)
Cultures for the genital mycoplasmas are handled in a similar fashion, including culture examination and the requirement for subculturing. Colonies may be definitively identified on A8 agar as U. urealyticum by urease production in the presence of a calcium chloride indica tor. U. urealyticum colonies (15 to 60 µm in diameter) will appear as dark brownish clumps. Colonies that are typical in appearance for U. urealyticum are shown in Figure 2. M. hominis are large (about 20 to 300 µm in diameter) and are urease negative (see Figure 2), with a characteristic “fried egg” appearance (Figure 3). On conventional blood agar, strains of M. hominis, but not of U. urealyticum, produce nonhemolytic, pinpoint colonies that do not stain with Gram stain. These colonies can be stained with the Dienes or acridine orange stains. Numerous transport and growth media systems for the detection, quantitation, identification, and antimicrobial susceptibility testing of the genital mycoplasmas are commercially available in the United States and Europe.
Fig2. Isolation of Mycoplasma hominis and Ureaplasma urealyticum (100× magnification). Note the “fried egg” appearance of the large M. hominis colony (arrow A) and the relatively small size of the U. urealyticum colony (arrow B). (Courtesy Clinical Microbiology Laboratory, SUNY Upstate Medical University, Syracuse, NY.)
Fig3. Colonial growth characteristics of Mycoplasma in agar medium.
SERODIAGNOSIS
Laboratory diagnosis of M. pneumoniae is usually made serologically. Nonspecific production of cold agglutinins occurs in approximately half of patients with atypical pneumonia caused by this organism. Antibodies to M. pneumoniae are typically detectable following approximately 1 week of illness, peaking between 3 to 6 weeks, followed by a gradual decline. The antibody response to M. pneumoniae varies greatly from patient to patient. Some patients fail to produce a detectable IgM level, whereas in others the IgM level will persist for months. The variability associated with the antibody response necessitates the comparison of paired sera for proper diagnosis. In addition, cold agglutinins form in association with M. pneumoniae infection. The most widely used serologic tests today are enzyme-linked immunosorbent assay (ELISA) tests, although newly developed indirect fluorescent antibody tests are being used with some success. IgM-specific tests such as the Immuno Card (Meridian Diagnostics, Cincinnati, Ohio) are commercially available, and a single positive result in children, adolescents, and young adults may be considered diagnostic in some cases. In addition, there is a commercially available, membrane-based assay that simultaneously detects IgM and IgG against M. pneumoniae (Remel EIA, Lenexa, Kansas) with good sensitivity and specificity compared to other tests. Several additional commercial assays are available that include EIA microtiter assays.
Although serologic tests such as indirect hemagglutination and metabolism inhibition for genital mycoplasmas are available, they are rarely used. Because of the antigenic complexity of the mycoplasmas, the development of a specific and useful serologic assay is a challenge.
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