Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1977 May;5(5):535–542. doi: 10.1128/jcm.5.5.535-542.1977

Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay.

A S Randhawa, G J Stanton, J A Green, S Baron
PMCID: PMC274646  PMID: 194918

Abstract

A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.

Full text

PDF
535

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARON S., PORTERFIELD J. S., ISAACS A. The influence of oxygenation of virus growth I. Effect on plaque formation by different viruses. Virology. 1961 Aug;14:444–449. doi: 10.1016/0042-6822(61)90336-1. [DOI] [PubMed] [Google Scholar]
  2. BLACK F. L., MELNICK J. L. Microepidemiology of poliomyelitis and herpes-B infections: spread of the viruses within tissue cultures. J Immunol. 1955 Mar;74(3):236–242. [PubMed] [Google Scholar]
  3. Baker D. A., Glasgow L. A. Rapid plaque assay for encephalomyocarditis virus. Appl Microbiol. 1969 Nov;18(5):932–934. doi: 10.1128/am.18.5.932-934.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baron S., Buckler C. E., Takemoto K. K. RACK permitting efficient handling of tissue cultures. Appl Microbiol. 1966 Nov;14(6):1042–1043. doi: 10.1128/am.14.6.1042-1043.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CARTER G. B. THE RAPID DETECTION, TITRATION, AND DIFFERENTIATION OF VARIOLA AND VACCINIA VIRUSES BY A FLUORESCENT ANTIBODY-COVERSLIP CELL MONOLAYER SYSTEM. Virology. 1965 Apr;25:659–662. doi: 10.1016/0042-6822(65)90094-2. [DOI] [PubMed] [Google Scholar]
  6. COOPER P. D. A method for producing plaques in agar suspensions of animal cells. Virology. 1955 Nov;1(4):397–401. doi: 10.1016/0042-6822(55)90033-7. [DOI] [PubMed] [Google Scholar]
  7. Coates H. V., Alling D. W., Chanock R. M. An antigenic analysis of respiratory syncytial virus isolates by a plaque reduction neutralization test. Am J Epidemiol. 1966 Mar;83(2):299–313. doi: 10.1093/oxfordjournals.aje.a120586. [DOI] [PubMed] [Google Scholar]
  8. DEMAEYER E., SCHONNE E. STARCH GEL AS AN OVERLAY FOR THE PLAQUE ASSAY OF ANIMAL VIRUSES. Virology. 1964 Sep;24:13–18. doi: 10.1016/0042-6822(64)90142-4. [DOI] [PubMed] [Google Scholar]
  9. Daniel M. D., Rabin H., Barahona H. H., Meléndez L. V. Herpesvirus saimiri. 3. Plaque formation under multi agar, methyl cellulose and starch overlays. Proc Soc Exp Biol Med. 1971 Apr;136(4):1192–1196. doi: 10.3181/00379727-136-35456. [DOI] [PubMed] [Google Scholar]
  10. De Madrid A. T., Porterfield J. S. A simple micro-culture method for the study of group B arboviruses. Bull World Health Organ. 1969;40(1):113–121. [PMC free article] [PubMed] [Google Scholar]
  11. Dolan T. M., Fenters J. D., Fordyce P. A., Holper J. C. Rhinovirus plaque formation in WI-38 cells with methylcellulose overlay. Appl Microbiol. 1968 Sep;16(9):1331–1336. doi: 10.1128/am.16.9.1331-1336.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green J. A., Stanton G. J., Goode J., Baron S. Vesicular stomatitis virus plaque production in monolayer cultures with liquid overlay medium: description and adaptation to a one-day, human interferon-plaque. J Clin Microbiol. 1976 Dec;4(6):479–485. doi: 10.1128/jcm.4.6.479-485.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HENDERSON J. R., TAYLOR R. M. Propagation of certain arthropod-borne viruses in avian and primate cell cultures. J Immunol. 1960 Jun;84:590–598. [PubMed] [Google Scholar]
  14. HOLLAND J. J., McLAREN L. C. Improved method for staining cell monolayers for virus plaque counts. J Bacteriol. 1959 Oct;78:596–597. doi: 10.1128/jb.78.4.596-597.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. HOTCHIN J. E. Use of methyl cellulose gel as a substitute for agar in tissue-culture overlays. Nature. 1955 Feb 19;175(4451):352–352. doi: 10.1038/175352a0. [DOI] [PubMed] [Google Scholar]
  16. Mengeling W. L. A fluorescent micro-plaque assay for hog cholera virus. Localization of infection with homologous antiserum. Arch Gesamte Virusforsch. 1968;23(1):27–39. doi: 10.1007/BF01242111. [DOI] [PubMed] [Google Scholar]
  17. NOYES W. F. A simple technic for demonstrating plaque formation with virus of vaccinia. Proc Soc Exp Biol Med. 1953 Jul;83(3):426–429. doi: 10.3181/00379727-83-20378. [DOI] [PubMed] [Google Scholar]
  18. PARSONS R., TYRRELL D. A. A plaque method for assaying some viruses isolated from common colds. Nature. 1961 Feb 25;189:640–642. doi: 10.1038/189640a0. [DOI] [PubMed] [Google Scholar]
  19. PLOWRIGHT W. Rinderpest virus. Ann N Y Acad Sci. 1962 Nov 30;101:548–563. doi: 10.1111/j.1749-6632.1962.tb18896.x. [DOI] [PubMed] [Google Scholar]
  20. POSTLETHWAITE R. A plaque technique for the titration of vaccinia virus in chick embryo cells and some features of vaccinial infection in this system. Virology. 1960 Apr;10:466–482. doi: 10.1016/0042-6822(60)90130-6. [DOI] [PubMed] [Google Scholar]
  21. RAPP F., SELIGMAN S. J., JAROSS L. B., GORDON I. Quantitative determination of infectious units of measles virus by counts of immunofluorescent foci. Proc Soc Exp Biol Med. 1959 Jun;101(2):289–294. doi: 10.3181/00379727-101-24915. [DOI] [PubMed] [Google Scholar]
  22. RUSSELL W. C. A sensitive and precise plaque assay for herpes virus. Nature. 1962 Sep 8;195:1028–1029. doi: 10.1038/1951028a0. [DOI] [PubMed] [Google Scholar]
  23. SCHULZE I. T., SCHLESINGER R. W. Plaque assay of dengue and other group B arthropod-borne viruses under methyl cellulose overlay media. Virology. 1963 Jan;19:40–48. doi: 10.1016/0042-6822(63)90022-9. [DOI] [PubMed] [Google Scholar]
  24. SCOTT T. F., McLEOD D. L., TOKUMARU T. A biologic comparison of two strains of Herpesvirus hominis. J Immunol. 1961 Jan;86:1–12. [PubMed] [Google Scholar]
  25. SOMMERVILLE R. G. A microplaque method for counting enterovirus particles. Virology. 1959 Dec;9:701–702. doi: 10.1016/0042-6822(59)90159-x. [DOI] [PubMed] [Google Scholar]
  26. TAKEMOTO K. K., LIEBHABER H. Virus-polysaccharide interactions. I. An agar polysaccharide determining plaque morphology of EMC virus. Virology. 1961 Aug;14:456–462. doi: 10.1016/0042-6822(61)90338-5. [DOI] [PubMed] [Google Scholar]
  27. TYTELL A. A., NEUMAN R. E. A medium free of agar, serum and peptone for plaque assay of herpes simplex virus. Proc Soc Exp Biol Med. 1963 Jun;113:343–346. doi: 10.3181/00379727-113-28362. [DOI] [PubMed] [Google Scholar]
  28. WHEELER C. E. Further studies on the effect of neutralizing antibody upon the course of herpes simplex infections in tissue culture. J Immunol. 1960 Apr;84:394–403. [PubMed] [Google Scholar]
  29. WHEELOCK E. F., TAMM I. Enumeration of cell-infecting particles of Newcastle disease virus by the fluorescent antibody technique. J Exp Med. 1961 Feb 1;113:301–316. doi: 10.1084/jem.113.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES