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. 1997 Apr;63(4):1557–1563. doi: 10.1128/aem.63.4.1557-1563.1997

Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

J L Lange 1, P S Thorne 1, N Lynch 1
PMCID: PMC168448  PMID: 9097451

Abstract

Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.

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Selected References

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  1. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990 Jun;56(6):1919–1925. doi: 10.1128/aem.56.6.1919-1925.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulanger C. A., Edelstein P. H. Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation. Appl Environ Microbiol. 1995 May;61(5):1805–1809. doi: 10.1128/aem.61.5.1805-1809.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braun-Howland E. B., Vescio P. A., Nierzwicki-Bauer S. A. Use of a simplified cell blot technique and 16S rRNA-directed probes for identification of common environmental isolates. Appl Environ Microbiol. 1993 Oct;59(10):3219–3224. doi: 10.1128/aem.59.10.3219-3224.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  6. Donham K. J. Hazardous agents in agricultural dusts and methods of evaluation. Am J Ind Med. 1986;10(3):205–220. doi: 10.1002/ajim.4700100305. [DOI] [PubMed] [Google Scholar]
  7. Fouchet P., Jayat C., Héchard Y., Ratinaud M. H., Frelat G. Recent advances of flow cytometry in fundamental and applied microbiology. Biol Cell. 1993;78(1-2):95–109. doi: 10.1016/0248-4900(93)90120-4. [DOI] [PubMed] [Google Scholar]
  8. Kirchman D., Sigda J., Kapuscinski R., Mitchell R. Statistical analysis of the direct count method for enumerating bacteria. Appl Environ Microbiol. 1982 Aug;44(2):376–382. doi: 10.1128/aem.44.2.376-382.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. MAY K. R., HARPER G. J. The efficiency of various liquid impinger samplers in bacterial aerosols. Br J Ind Med. 1957 Oct;14(4):287–297. doi: 10.1136/oem.14.4.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Macher J. M., Huang F. Y., Flores M. A two-year study of microbiological indoor air quality in a new apartment. Arch Environ Health. 1991 Jan-Feb;46(1):25–29. doi: 10.1080/00039896.1991.9937425. [DOI] [PubMed] [Google Scholar]
  11. Macnaughton S. J., O'Donnell A. G., Embley T. M. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes. Microbiology. 1994 Oct;140(Pt 10):2859–2865. doi: 10.1099/00221287-140-10-2859. [DOI] [PubMed] [Google Scholar]
  12. Monfort P., Baleux B. Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems. Cytometry. 1992;13(2):188–192. doi: 10.1002/cyto.990130213. [DOI] [PubMed] [Google Scholar]
  13. Morey P. R. Practical aspects of sampling for organic dusts and microorganisms. Am J Ind Med. 1990;18(3):273–278. doi: 10.1002/ajim.4700180306. [DOI] [PubMed] [Google Scholar]
  14. Palmgren U., Ström G., Blomquist G., Malmberg P. Collection of airborne micro-organisms on Nuclepore filters, estimation and analysis--CAMNEA method. J Appl Bacteriol. 1986 Nov;61(5):401–406. doi: 10.1111/j.1365-2672.1986.tb04303.x. [DOI] [PubMed] [Google Scholar]
  15. Robertson B. R., Button D. K. Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry. Cytometry. 1989 Jan;10(1):70–76. doi: 10.1002/cyto.990100112. [DOI] [PubMed] [Google Scholar]
  16. Salvaggio J. E. Inhaled particles and respiratory disease. J Allergy Clin Immunol. 1994 Aug;94(2 Pt 2):304–309. [PubMed] [Google Scholar]
  17. Stewart C. C., Steinkamp J. A. Quantitation of cell concentration using the flow cytometer. Cytometry. 1982 Jan;2(4):238–243. doi: 10.1002/cyto.990020407. [DOI] [PubMed] [Google Scholar]
  18. Thorne P. S., Kiekhaefer M. S., Whitten P., Donham K. J. Comparison of bioaerosol sampling methods in barns housing swine. Appl Environ Microbiol. 1992 Aug;58(8):2543–2551. doi: 10.1128/aem.58.8.2543-2551.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Thorne P. S., Lange J. L., Bloebaum P., Kullman G. J. Bioaerosol sampling in field studies: can samples be express mailed? Am Ind Hyg Assoc J. 1994 Nov;55(11):1072–1079. doi: 10.1080/15428119491018367. [DOI] [PubMed] [Google Scholar]
  20. Wagner M., Assmus B., Hartmann A., Hutzler P., Amann R. In situ analysis of microbial consortia in activated sludge using fluorescently labelled, rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy. J Microsc. 1994 Dec;176(Pt 3):181–187. doi: 10.1111/j.1365-2818.1994.tb03513.x. [DOI] [PubMed] [Google Scholar]
  21. Wallner G., Amann R., Beisker W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry. 1993;14(2):136–143. doi: 10.1002/cyto.990140205. [DOI] [PubMed] [Google Scholar]

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