1887

Abstract

A procedure was devised to produce preparations free of adsorbed components of the growth medium, which contains high concentrations of serum. The ureaplasmas were cultivated in a medium containing PPLO-serum fraction as a replacement for horse serum. High titres of ureaplasmas (> 10 c.f.u. ml-) were obtained. Harvested cells were then purified by Urografin density gradient centrifugation. By use of H-labelled ureaplasma cells and I-labelled medium components, a distinct band of viable cells devoid of serum constituents was demonstrated. The absence of medium components was verified by immunoblotting cells from this band with antiserum to medium components. Medium components that had been present before the purification procedure were undetectable in the purified cell fraction obtained. The viability of the purified ureaplasma cells represented an 85% recovery rate and their antigenicity, examined with anti-serotype specific antiserum, remained intact. This easy and reproducible procedure can be used to prepare purified ureaplasmas for investigation of ureaplasmal antigens and their expression and/or role in disease.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-137-5-1087
1991-05-01
2021-05-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/137/5/mic-137-5-1087.html?itemId=/content/journal/micro/10.1099/00221287-137-5-1087&mimeType=html&fmt=ahah

References

  1. Bradbury J. M., Jordan F. T. W. 1972; Studies on the adsorption of certain medium proteins to Mycoplasma gallisepticum and their influence on agglutination and haemagglutination reactions. Journal of Hygiene 70:267–278
    [Google Scholar]
  2. Caldwell H. D., Kromhout J., Schächter J. 1981; Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis . Infection and Immunity 31:1161–1176
    [Google Scholar]
  3. Cassell G. H., Clyde W. A., Kenny G. E., McCormack W. M., Taylor-Robinson D. 1986; Ureaplasmas of humans with emphasis upon maternal and neonatal infections. Pediatric Infectious Disease 5:S221–S354
    [Google Scholar]
  4. Cassell G. H., Davis J. K., Waites K. B., Rudd P. T., Talkinton D., Crouse D., Horowitz S. A. 1987; Pathogenesis and significance of urogenital mycoplasmal infections. Urogenital Infections93–115 Stieritz D. D., Campos J. M., Miller L. A. New York: Plenum Press;
    [Google Scholar]
  5. Grdina D. J., Milas L., Hewitt R. R., Withers H. R. 1973; Buoyant density separation of human blood cells in renografin gradients. Experimental Cell Research 81:250–254
    [Google Scholar]
  6. Hillier S. L., Martins J., Krohn M., Kiviate N., Holmes K. K., Eschenback D. A. 1988; A case-control study of chorioamnio-nitic infection and histologic chorioamnionitis in prematurity. New England Journal of Medicine 319:972–978
    [Google Scholar]
  7. Horowitz S. A., Duffy L., Garrett B., Stephens J., Davis J. K., Cassell G. H. 1986; Can group and serovar-specific proteins be detected. Ureaplasma urealyticum? Pediatric Infectious Disease 5:S325–S331
    [Google Scholar]
  8. Johnson J. K., Somerson N. L. 1980; Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae. Applied and Environmental Microbiology 40:391–399
    [Google Scholar]
  9. Kirshner N., Wallach D., Sharoni Y., Schramm M. 1973; Purification of secretory granules on a Urografin gradient. Analytical Biochemistry 52:589–594
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature London: 227680–685
    [Google Scholar]
  11. Lee G. Y., Kenny G. E. 1987; Humoral response to Ureaplasma urealyticum polypeptides. Journal of Clinical Microbiology 25:1841–1844
    [Google Scholar]
  12. Markwell M. A. K., Fox C. F. 1978; Surface-specific iodination of membrane proteins of viruses and eukaryotic cells using 1,3,4,6-tetrachloro-3,6-diphenylglycoluvil. Biochemistry 17:4807–4817
    [Google Scholar]
  13. Naessens A., Foulon W., Breynaert J., Lauwers S. 1988; Serotypes of Ureaplasma urealyticum isolated from normal pregnant women and patients with pregnancy complications. Journal of Microbiology 26:319–322
    [Google Scholar]
  14. Nicolet J., Paroz Ph., Kristensen B. 1980; Growth medium constituents contaminating mycoplasma preparations and their role in the study of membrane glycoproteins in porcine mycoplasmas. Journal of General Microbiology 119:17–26
    [Google Scholar]
  15. Razin S., Rottem S. 1976; Techniques for the manipulation of mycoplasma membrane. Biochemical Analysis of Membranes3–26 Maddy A. H. London: Chapman and Hall;
    [Google Scholar]
  16. Razin S., Yogev D. 1986; Genetic relatedness among Ureaplasma urealyticum serotypes (serovars). Pediatric Infectious Disease 5:S300–S304
    [Google Scholar]
  17. Robertson J. A., Stemke G. W. 1982; Expanded serotyping scheme for Ureaplasma urealyticum strains isolated from humans. Journal of Clinical Microbiology 15:873–878
    [Google Scholar]
  18. Robertson J. A., Honore L. H., Stemke G. W. 1986; Serotypes of Ureaplasma urealyticum in spontaneous abortion. Pediatric Infectious Disease 5:S270–S272
    [Google Scholar]
  19. Rottem S., Pfendt E. A., Hayflick L. 1971; Sterol requirements of T-strain mycoplasmas. Journal of Bacteriology 105:323–330
    [Google Scholar]
  20. Rottem S., Trotter S. L., Barille M. F. 1977; Membrane bound thioesterase activity in mycoplasmas. Journal of Bacteriology 129:707–713
    [Google Scholar]
  21. Sayed I. A., Kenny G. E. 1980; Comparison of the proteins and polypeptides of the eight serotypes of Ureaplasma urealyticum by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. International Journal of Systematic Bacteriology 30:33–41
    [Google Scholar]
  22. Serwer P. 1975; Buoyant density sedimentation of macromolecules in sodium iothalamate density gradients. Journal of Molecular Biology 92:433–448
    [Google Scholar]
  23. Shepard M. C. 1983; Culture media for ureaplasma. Methods in Mycoplasmology 1137–146 Razin S., Tully J. G. New York: Academic Press;
    [Google Scholar]
  24. Smith P. F., Lecce J. G., Lynn R. J. 1954; A lipoprotein as a growth factor for certain pleuropneumonia-like organisms. Journal of Bacteriology 68:627–633
    [Google Scholar]
  25. Stemke G. W., Robertson J. A. 1985; Problems associated with serotyping strains of Ureaplasma urealyticum. Diagnostic Microbiology and Infectious Diseases 3:311–320
    [Google Scholar]
  26. Taylor-Robinson D. 1986; Mycoplasma and mixed infections of the human male urogenital tract and their possible implications. The Mycoplasmas 429–63 Razin S., Barile M. New York: Academic Press;
    [Google Scholar]
  27. Thirkell D., Myles A. D., Russell W. C. 1909; Serotype 8-and serocluster-specific surface-expressed antigens of Ureaplasma urealyticum. Infection and Immunity 57:1697–1701
    [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76:4350–4354
    [Google Scholar]
  29. Tully J. G., Rose D. L. 1983; Sterility and quality control of mycoplasma culture media. Methods in Mycoplasmology 1121–125 Razin S., Tully J. G. New York: Academic Press;
    [Google Scholar]
  30. Washburn L. R., Hughes J. H., Somerson N. L. 1978; Mycoplasma growth factors in bovine serum fractions. Journal of Bacteriology 135:818–827
    [Google Scholar]
  31. Williams M. H. 1968; Recovery of Mycoplasma from Rheumatoid Synovial Fluid, Third Pfizer International Symposium on Rheumatic Diseases. 171–181 Duthie J. J. R., Alexander W. R. M. Edinburgh: University Press;
    [Google Scholar]
  32. Wise K. S., Minion C. F., Cheung H. C. 1982; Translocation of Thy-1 antigen and a fluorescent lipid probe during lymphoblastoid cell interaction with Mycoplasma hyorhinis. Reviews of Infectious Diseases 4:S210–S218
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-137-5-1087
Loading
/content/journal/micro/10.1099/00221287-137-5-1087
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error