this step by diluting the conjugate in Evans blue. The optimal dilution was found to be 1/1,500. This modified method was used to serotype about 200.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 191-192
Vol. 25, No. 1
0095-1137/87/010191-02$02.00/0 Copyright © 1987, American Society for Microbiology
Modified Indirect Immunofluorescence Test for Serotyping Large Numbers of Ureaplasma urealyticum Clinical Isolates ANNE NAESSENS* AND SABINE LAUWERS
Department of Microbiology, Akademisch Ziekenhuis, Vrije Universiteit Brussel, 1090 Brussels, Belgium Received 2 June 1986/Accepted 30 September 1986
A technical modification of the indirect immunofluorescence test for serotyping Ureaplasma urealyticum clinical isolates is described. The use of a tissue culture plate filled with ureaplasma agar made the serotyping easier to perform and proved to be very handy, especially for performance of large series.
Ureaplasma urealyticum has been implicated in nongonococcal urethritis (23) and has been associated with spontaneous abortion, low birth weight, and infertility (4, 6, 8, 12). Although theories about the role of this organism in these pathologies are still conflicting, there are reports establishing a causal relationship between U. urealyticum infection and unfavorable fetal outcome (3, 7, 22). The interpretation of isolation rates in different patient populations, however, is difficult, because U. urealyticum can be isolated frequently from genital tract specimens in asymptomatic persons and from the cervices of patients with normally developing pregnancies. Because U. urealyticum exhibits an antigenic heterogeneity, it has been postulated that serotyping of the U. urealyticum isolates may be helpful in distinguishing between more pathogenic and less pathogenic isolates. An initial typing scheme for U. urealyticum has been established with eight different specificities (20) and has been expanded further (10, 16). Nevertheless, additional serotypes may exist, necessitating adjustment of this classification. Different serotyping studies have already been performed, however, with conflicting results. Some authors did find an association between disease and the prevalence of a certain serotype (5, 9, 19), whereas others did not (14). Further large-scale studies, with similar typing methods, are warranted. Different methods for serotyping of U. urealyticum have been described: the metabolic inhibition test (15), the agar growth inhibition method (1), the indirect hemagglutination test (2), the mycoplasmacidal test (11), and the indirect immunofluorescence test (17). Because of the possibility of using highly diluted antisera and detecting mixed serotypes in a single specimen, the indirect immunofluorescence test is most commonly performed. Blocks of agar containing unfixed U. urealyticum colonies are cut from the isolation plate. Appropriately diluted antisera are added to the blocks of agar. After incubation at room temperature, the agar blocks are washed, and diluted fluorescein-conjugated rabbit antiserum is added. The agar blocks are washed and incubated as before and examined for fluorescence. Since the initial description of the test, technical modifications have been proposed to make the test easier to perform. The technique developed by Panangala and Lein (13) uses a template of Plexiglas (Rohm & Haas Co.) that holds the blocks of agar throughout the staining process. The amounts of antisera and conjugate needed are reduced, and the washing procedure is greatly simplified. Although much easier to perform, this method is still labor intensive and thus *
not suitable for
serotyping large numbers of U. urealyticum isolates. Stemke and Robertson (21) described a technique in which alternate wells of a microtiter plate are filled with agar. Onto the agar, plugs with U. urealyticum colonies (cut from the original isolation plate) are transferred. On these plugs in the microtiter plate, the indirect immunofluorescence test is performed. The latter test has several disadvantages: plugs containing U. urealyticum colonies may be inverted on the agar wells of the microtiter plate, which eventually may lead to false-negative results; only a small surface (diameter, 6 mm) can be screened for fluorescent U. urealyticum colonies; and washing is still laborious. The aims of our modification of the immunofluorescence technique were: (i) obtaining a more rapid and yet careful washing; (ii) screening of a relatively large surface for U. urealyticum colonies; and (iii) the use of small amounts of antisera and conjugate. U. urealyticum antisera were prepared by immunization of albino rabbits with U. urealyticum antigens. Antigens and antisera were prepared as previously described (19). Dilutions for the antisera were chosen which gave strong fluorescence in the homologous reactions with minimal fluorescence in the heterologous reactions and fell within the range of 1/300 to 1/800. Wells of a sterile 24-well tissue culture plate (diameter, 15.6 mm) were filled with 2.7 ml of A7 agar (18) each, leaving approximately a 1-mm border (Fig. 1). Each well was inoculated with 2 drops of a fresh 18-h primary U. urealyticum culture in the logarithmic phase of growth. The plates were incubated for at least 48 h, and the assay was performed when sufficient growth was obtained. Antisera were diluted in phosphate-buffered saline, and 2 drops were added to each well. The plates were then allowed to incubate for 30 min at room temperature. Antisera were removed by simply turning the plate, taking care not to mix the different antisera. Immediately thereafter, the wells were thoroughly washed two times with buffer and left covered with buffer for 10 min. This washing procedure was repeated twice. After the wells were washed, the plates were inverted on a clean blotting paper until no fluid remained in the wells. Fluorescein-conjugated goat anti-rabbit immunoglobulin G (2 drops) was then added. The plates were incubated for another 30 min and again washed as before. The plates were read with a Dialux microscope (E. Leitz Inc.) equipped with Ploem epi-illumination, with a 50-W mercury light source and an 12 filter block; total magnification was x 100 with a 10x objective lens (numerical aperture: 0.25; mechanical tube length: 170 mm). The wells were first screened with visible transmitted light, and after visualization of U. urealyticum colonies the light was switched to fluorescent
Corresponding author. 191
NOTES
192
J. CLIN. MICROBIOL.
6.
7. 8.
9. FIG. 1. 24-Well tissue culture plate filled with A7
agar.
light. With this method, however, background fluorescence appeared to be relatively high. An initial wash with phosphate-buffered saline before performance of the assay or an overnight incubation of the stained plates covered with buffer did not reduce the background fluorescence significantly. A reduction was obtained by counterstaining with Evans blue. A dilution of Evans blue (1/1,500) was added to each well, incubated for 10 min, and washed as before. Since this counterstaining inserted an additional step in the serotyping procedure and thus additional work, we tried to omit this step by diluting the conjugate in Evans blue. The optimal dilution was found to be 1/1,500. This modified method was used to serotype about 200 clinical isolates. Ten antisera corresponding to U. urealyticum serotypes 1 to 10 were used. Our method proved to be very handy, especially for performance of large series. The time needed for the washing was greatly reduced, and the reading of the plates was easy. Mixed serotypes were easily detected, since many colonies were screened on a relatively large surface. In a single tissue culture plate, two different isolates can be serotyped against 12 different anti-
10.
11.
12.
13.
14.
15. 16. 17.
sera.
1. 2.
3.
4.
5.
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