Search tips
Search criteria 


Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. 1996 July; 62(7): 2264–2272.
PMCID: PMC168007

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


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 (362K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Bowden WB. Comparison of two direct-count techniques for enumerating aquatic bacteria. Appl Environ Microbiol. 1977 May;33(5):1229–1232. [PMC free article] [PubMed]
  • Buttner MP, Stetzenbach LD. Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae. Appl Environ Microbiol. 1991 Apr;57(4):1268–1270. [PMC free article] [PubMed]
  • Byrd JJ, Xu HS, Colwell RR. Viable but nonculturable bacteria in drinking water. Appl Environ Microbiol. 1991 Mar;57(3):875–878. [PMC free article] [PubMed]
  • Chang CW, Grinshpun SA, Willeke K, Macher JM, 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. [PubMed]
  • Chang CW, Hwang YH, Grinshpun SA, Macher JM, Willeke K. Evaluation of counting error due to colony masking in bioaerosol sampling. Appl Environ Microbiol. 1994 Oct;60(10):3732–3738. [PMC free article] [PubMed]
  • Hobbie JE, Daley RJ, Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. [PMC free article] [PubMed]
  • Hugenholtz P, Fuerst JA. Heterotrophic bacteria in an air-handling system. Appl Environ Microbiol. 1992 Dec;58(12):3914–3920. [PMC free article] [PubMed]
  • Jensen PA, Todd WF, Davis GN, Scarpino PV. Evaluation of eight bioaerosol samplers challenged with aerosols of free bacteria. Am Ind Hyg Assoc J. 1992 Oct;53(10):660–667. [PubMed]
  • Jones JG, Simon BM. 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. [PubMed]
  • Juozaitis A, Willeke K, Grinshpun SA, 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. [PMC free article] [PubMed]
  • Karol MH. Allergic reactions to indoor air pollutants. Environ Health Perspect. 1991 Nov;95:45–51. [PMC free article] [PubMed]
  • Kjelleberg S, Humphrey BA, Marshall KC. Effect of interfaces on small, starved marine bacteria. Appl Environ Microbiol. 1982 May;43(5):1166–1172. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PubMed]
  • Korgaonkar KS, Ranade SS. Evaluation of acridine orange fluorescence test in viability studies on Escherichia coli. Can J Microbiol. 1966 Feb;12(1):185–190. [PubMed]
  • Lauer BA, Reller LB, 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. [PMC free article] [PubMed]
  • Madelin TM, Johnson HE. Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer. J Appl Bacteriol. 1992 May;72(5):400–409. [PubMed]
  • Maki JS, Remsen CC. Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl Environ Microbiol. 1981 May;41(5):1132–1138. [PMC free article] [PubMed]
  • Marcellino SN, Benson DR. Scanning electron and light microscopic study of microbial succession on bethlehem st. Nectaire cheese. Appl Environ Microbiol. 1992 Nov;58(11):3448–3454. [PMC free article] [PubMed]
  • McCarthy LR, Senne JE. Evaluation of acridine orange stain for detection of microorganisms in blood cultures. J Clin Microbiol. 1980 Mar;11(3):281–285. [PMC free article] [PubMed]
  • Paul JH. Use of hoechst dyes 33258 and 33342 for enumeration of attached and planktonic bacteria. Appl Environ Microbiol. 1982 Apr;43(4):939–944. [PMC free article] [PubMed]
  • Paul JH, Myers B. Fluorometric determination of DNA in aquatic microorganisms by use of hoechst 33258. Appl Environ Microbiol. 1982 Jun;43(6):1393–1399. [PMC free article] [PubMed]
  • Pedersen JC, Jacobsen CS. 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. [PMC free article] [PubMed]
  • Ray B, Speck ML. Repair of injury induced by freezing Escherichia coli as influenced by recovery medium. Appl Microbiol. 1972 Aug;24(2):258–263. [PMC free article] [PubMed]
  • Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol. 1992 Jun;58(6):1801–1808. [PMC free article] [PubMed]
  • Roszak DB, Colwell RR. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987 Sep;51(3):365–379. [PMC free article] [PubMed]
  • Roszak DB, Grimes DJ, Colwell RR. Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems. Can J Microbiol. 1984 Mar;30(3):334–338. [PubMed]
  • Schaule G, Flemming HC, Ridgway HF. 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. [PMC free article] [PubMed]
  • Singh A, Yu FP, McFeters GA. 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. [PMC free article] [PubMed]
  • Stewart SL, Grinshpun SA, 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. [PMC free article] [PubMed]
  • Walter MV, Marthi B, Fieland VP, Ganio LM. Effect of aerosolization on subsequent bacterial survival. Appl Environ Microbiol. 1990 Nov;56(11):3468–3472. [PMC free article] [PubMed]
  • Watson SW, Novitsky TJ, Quinby HL, Valois FW. Determination of bacterial number and biomass in the marine environment. Appl Environ Microbiol. 1977 Apr;33(4):940–946. [PMC free article] [PubMed]
  • Welch LS. Severity of health effects associated with building-related illness. Environ Health Perspect. 1991 Nov;95:67–69. [PMC free article] [PubMed]
  • Winding A, Binnerup SJ, Sørensen J. Viability of indigenous soil bacteria assayed by respiratory activity and growth. Appl Environ Microbiol. 1994 Aug;60(8):2869–2875. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Yu W, Dodds WK, Banks MK, Skalsky J, Strauss EA. 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]

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