Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Jul;62(7):2264–2272. doi: 10.1128/aem.62.7.2264-2272.1996

Comparison of methods for detection and enumeration of airborne microorganisms collected by liquid impingement.

S Terzieva 1, J Donnelly 1, V Ulevicius 1, S A Grinshpun 1, K Willeke 1, G N Stelma 1, K P Brenner 1
PMCID: PMC168007  PMID: 8779564

Abstract

Bacterial agents and cell components can be spread as bioaerosols, producing infections and asthmatic problems. This study compares four methods for the detection and enumeration of aerosolized bacteria collected in an AGI-30 impinger. Changes in the total and viable concentrations of Pseudomonas fluorescens in the collection fluid with respect to time of impingement were determined. Two direct microscopic methods (acridine orange and BacLight) and aerodynamic aerosol-size spectrometry (Aerosizer) were employed to measure the total bacterial cell concentrations in the impinger collection fluid and the air, respectively. These data were compared with plate counts on selective (MacConkey agar) and nonselective (Trypticase soy agar) media, and the percentages of culturable cells in the collection fluid and the bacterial injury response to the impingement process were determined'. The bacterial collection rate was found to be relatively unchanged during 60 min of impingement. The aerosol measurements indicated an increased amount of cell fragments upstream of the impinger due to continuous bacterial nebulization. Some of the bacterial clusters, present in the air upstream of the impinger, deagglomerated during impingement, thus increasing the total bacterial count by both direct microscopic methods. The BacLight staining technique was also used to determine the changes in viable bacterial concentration during the impingement process. The percentage of viable bacteria, determined as a ratio of BacLight live to total counts was only 20% after 60 min of sampling. High counts on Trypticase soy agar indicated that most of the injured cells could recover. On the other hand, the counts from the MacConkey agar were very low, indicating that most of the cells were structurally damaged in the impinger. The comparison of data on the percentage of injured bacteria obtained by the traditional plate count with the data on percentage of nonviable bacteria obtained by the BacLight method showed good agreement.

Full Text

The Full Text of this article is available as a PDF (361.7 KB).

Selected References

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

  1. Bergström I., Heinänen A., Salonen K. Comparison of acridine orange, acriflavine, and bisbenzimide stains for enumeration of bacteria in clear and humic waters. Appl Environ Microbiol. 1986 Mar;51(3):664–667. doi: 10.1128/aem.51.3.664-667.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bitton G., Koopman B. Tetrazolium reduction-malachite green method for assessing the viability of filamentous bacteria in activated sludge. Appl Environ Microbiol. 1982 Apr;43(4):964–966. doi: 10.1128/aem.43.4.964-966.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowden W. B. Comparison of two direct-count techniques for enumerating aquatic bacteria. Appl Environ Microbiol. 1977 May;33(5):1229–1232. doi: 10.1128/aem.33.5.1229-1232.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buttner M. P., Stetzenbach L. D. Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae. Appl Environ Microbiol. 1991 Apr;57(4):1268–1270. doi: 10.1128/aem.57.4.1268-1270.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byrd J. J., Xu H. S., Colwell R. R. Viable but nonculturable bacteria in drinking water. Appl Environ Microbiol. 1991 Mar;57(3):875–878. doi: 10.1128/aem.57.3.875-878.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang C. W., Grinshpun S. A., Willeke K., Macher J. M., Donnelly J., Clark S., Juozaitis A. Factors affecting microbiological colony count accuracy for bioaerosol sampling and analysis. Am Ind Hyg Assoc J. 1995 Oct;56(10):979–986. doi: 10.1080/15428119591016403. [DOI] [PubMed] [Google Scholar]
  7. Chang C. W., Hwang Y. H., Grinshpun S. A., Macher J. M., Willeke K. Evaluation of counting error due to colony masking in bioaerosol sampling. Appl Environ Microbiol. 1994 Oct;60(10):3732–3738. doi: 10.1128/aem.60.10.3732-3738.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hugenholtz P., Fuerst J. A. Heterotrophic bacteria in an air-handling system. Appl Environ Microbiol. 1992 Dec;58(12):3914–3920. doi: 10.1128/aem.58.12.3914-3920.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jensen P. A., Todd W. F., Davis G. N., Scarpino P. V. Evaluation of eight bioaerosol samplers challenged with aerosols of free bacteria. Am Ind Hyg Assoc J. 1992 Oct;53(10):660–667. doi: 10.1080/15298669291360319. [DOI] [PubMed] [Google Scholar]
  11. Jones J. G., Simon B. M. An investigation of errors in direct counts of aquatic bacteria by epifluorescence microscopy, with reference to a new method for dyeing membrane filters. J Appl Bacteriol. 1975 Dec;39(3):317–329. doi: 10.1111/j.1365-2672.1975.tb00578.x. [DOI] [PubMed] [Google Scholar]
  12. Juozaitis A., Willeke K., Grinshpun S. A., Donnelly J. Impaction onto a Glass Slide or Agar versus Impingement into a Liquid for the Collection and Recovery of Airborne Microorganisms. Appl Environ Microbiol. 1994 Mar;60(3):861–870. doi: 10.1128/aem.60.3.861-870.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karol M. H. Allergic reactions to indoor air pollutants. Environ Health Perspect. 1991 Nov;95:45–51. doi: 10.1289/ehp.919545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kjelleberg S., Humphrey B. A., Marshall K. C. Effect of interfaces on small, starved marine bacteria. Appl Environ Microbiol. 1982 May;43(5):1166–1172. doi: 10.1128/aem.43.5.1166-1172.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kogure K., Simidu U., Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol. 1979 Mar;25(3):415–420. doi: 10.1139/m79-063. [DOI] [PubMed] [Google Scholar]
  16. Kogure K., Simidu U., Taga N. Distribution of viable marine bacteria in neritic seawater around Japan. Can J Microbiol. 1980 Mar;26(3):318–323. doi: 10.1139/m80-052. [DOI] [PubMed] [Google Scholar]
  17. Korgaonkar K. S., Ranade S. S. Evaluation of acridine orange fluorescence test in viability studies on Escherichia coli. Can J Microbiol. 1966 Feb;12(1):185–190. doi: 10.1139/m66-024. [DOI] [PubMed] [Google Scholar]
  18. Lauer B. A., Reller L. B., Mirrett S. Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens. J Clin Microbiol. 1981 Aug;14(2):201–205. doi: 10.1128/jcm.14.2.201-205.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Madelin T. M., Johnson H. E. Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer. J Appl Bacteriol. 1992 May;72(5):400–409. doi: 10.1111/j.1365-2672.1992.tb01853.x. [DOI] [PubMed] [Google Scholar]
  20. Maki J. S., Remsen C. C. Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl Environ Microbiol. 1981 May;41(5):1132–1138. doi: 10.1128/aem.41.5.1132-1138.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marcellino S. N., Benson D. R. Scanning electron and light microscopic study of microbial succession on bethlehem st. Nectaire cheese. Appl Environ Microbiol. 1992 Nov;58(11):3448–3454. doi: 10.1128/aem.58.11.3448-3454.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McCarthy L. R., Senne J. E. Evaluation of acridine orange stain for detection of microorganisms in blood cultures. J Clin Microbiol. 1980 Mar;11(3):281–285. doi: 10.1128/jcm.11.3.281-285.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paul J. H., Myers B. Fluorometric determination of DNA in aquatic microorganisms by use of hoechst 33258. Appl Environ Microbiol. 1982 Jun;43(6):1393–1399. doi: 10.1128/aem.43.6.1393-1399.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paul J. H. Use of hoechst dyes 33258 and 33342 for enumeration of attached and planktonic bacteria. Appl Environ Microbiol. 1982 Apr;43(4):939–944. doi: 10.1128/aem.43.4.939-944.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pedersen J. C., Jacobsen C. S. Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability. Appl Environ Microbiol. 1993 May;59(5):1560–1564. doi: 10.1128/aem.59.5.1560-1564.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ray B., Speck M. L. Repair of injury induced by freezing Escherichia coli as influenced by recovery medium. Appl Microbiol. 1972 Aug;24(2):258–263. doi: 10.1128/am.24.2.258-263.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rodriguez G. G., Phipps D., Ishiguro K., Ridgway H. F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol. 1992 Jun;58(6):1801–1808. doi: 10.1128/aem.58.6.1801-1808.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roszak D. B., Colwell R. R. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987 Sep;51(3):365–379. doi: 10.1128/mr.51.3.365-379.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roszak D. B., Grimes D. J., Colwell R. R. Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems. Can J Microbiol. 1984 Mar;30(3):334–338. doi: 10.1139/m84-049. [DOI] [PubMed] [Google Scholar]
  30. Schaule G., Flemming H. C., Ridgway H. F. Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl Environ Microbiol. 1993 Nov;59(11):3850–3857. doi: 10.1128/aem.59.11.3850-3857.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Singh A., Yu F. P., McFeters G. A. Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis. Appl Environ Microbiol. 1990 Feb;56(2):389–394. doi: 10.1128/aem.56.2.389-394.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stewart S. L., Grinshpun S. A., Willeke K., Terzieva S., Ulevicius V., Donnelly J. Effect of impact stress on microbial recovery on an agar surface. Appl Environ Microbiol. 1995 Apr;61(4):1232–1239. doi: 10.1128/aem.61.4.1232-1239.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Walter M. V., Marthi B., Fieland V. P., Ganio L. M. Effect of aerosolization on subsequent bacterial survival. Appl Environ Microbiol. 1990 Nov;56(11):3468–3472. doi: 10.1128/aem.56.11.3468-3472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watson S. W., Novitsky T. J., Quinby H. L., Valois F. W. Determination of bacterial number and biomass in the marine environment. Appl Environ Microbiol. 1977 Apr;33(4):940–946. doi: 10.1128/aem.33.4.940-946.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Welch L. S. Severity of health effects associated with building-related illness. Environ Health Perspect. 1991 Nov;95:67–69. doi: 10.1289/ehp.919567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Willeke K., Qian Y., Donnelly J., Grinshpun S., Ulevicius V. Penetration of airborne microorganisms through a surgical mask and a dust/mist respirator. Am Ind Hyg Assoc J. 1996 Apr;57(4):348–355. doi: 10.1080/15428119691014882. [DOI] [PubMed] [Google Scholar]
  37. Winding A., Binnerup S. J., Sørensen J. Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl Environ Microbiol. 1994 Aug;60(8):2869–2875. doi: 10.1128/aem.60.8.2869-2875.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yu W., Dodds W. K., Banks M. K., Skalsky J., Strauss E. A. Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes. Appl Environ Microbiol. 1995 Sep;61(9):3367–3372. doi: 10.1128/aem.61.9.3367-3372.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zimmermann R., Iturriaga R., Becker-Birck J. Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl Environ Microbiol. 1978 Dec;36(6):926–935. doi: 10.1128/aem.36.6.926-935.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES