Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Jul;61(7):2614–2619. doi: 10.1128/aem.61.7.2614-2619.1995

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

B H Pyle 1, S C Broadaway 1, G A McFeters 1
PMCID: PMC167532  PMID: 7618872

Abstract

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.

Full Text

The Full Text of this article is available as a PDF (255.3 KB).

Selected References

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

  1. Baker K. H., Mills A. L. Determination of the Number of Respiring Thiobacillus ferrooxidans Cells in Water Samples by Using Combined Fluorescent Antibody-2-(p-Iodophenyl)-3-(p-Nitrophenyl)-5-Phenyltetrazolium Chloride Staining. Appl Environ Microbiol. 1982 Feb;43(2):338–344. doi: 10.1128/aem.43.2.338-344.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bej A. K., Mahbubani M. H., Miller R., DiCesare J. L., Haff L., Atlas R. M. Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. Mol Cell Probes. 1990 Oct;4(5):353–365. doi: 10.1016/0890-8508(90)90026-v. [DOI] [PubMed] [Google Scholar]
  3. Boucher S. N., Slater E. R., Chamberlain A. H., Adams M. R. Production and viability of coccoid forms of Campylobacter jejuni. J Appl Bacteriol. 1994 Sep;77(3):303–307. doi: 10.1111/j.1365-2672.1994.tb03078.x. [DOI] [PubMed] [Google Scholar]
  4. Bovill R. A., Shallcross J. A., Mackey B. M. Comparison of the fluorescent redox dye 5-cyano-2,3-ditolyltetrazolium chloride with p-iodonitrotetrazolium violet to detect metabolic activity in heat-stressed Listeria monocytogenes cells. J Appl Bacteriol. 1994 Oct;77(4):353–358. doi: 10.1111/j.1365-2672.1994.tb03434.x. [DOI] [PubMed] [Google Scholar]
  5. Clark J. A. The detection of various bacteria indicative of water pollution by a presence-absence (P-A) procedure. Can J Microbiol. 1969 Jul;15(7):771–780. doi: 10.1139/m69-135. [DOI] [PubMed] [Google Scholar]
  6. Coallier J., Prévost M., Rompré A., Duchesne D. The optimization and application of two direct viable count methods for bacteria in distributed drinking water. Can J Microbiol. 1994 Oct;40(10):830–836. doi: 10.1139/m94-132. [DOI] [PubMed] [Google Scholar]
  7. Danielsson D., Laurell G. A membrane filter method for the demonstration of bacteria by the fluorescent antibody technique. 4. Experimental studies of the demonstration of Shigellae in water from various sources. Acta Pathol Microbiol Scand. 1968;72(2):251–262. [PubMed] [Google Scholar]
  8. Danielsson D., Laurell G., Nordbring F., Sandler O. A membrane filter method for the demonstration of bacteria by the fluorescent antibody technique. 3. The application of the method for the demonstration of enteropathogenic Escherichia coli in drinking water. Acta Pathol Microbiol Scand. 1968;72(1):118–124. doi: 10.1111/j.1699-0463.1968.tb00439.x. [DOI] [PubMed] [Google Scholar]
  9. Edberg S. C., Allen M. J., Smith D. B. National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: comparison with the standard multiple tube fermentation method. Appl Environ Microbiol. 1988 Jun;54(6):1595–1601. doi: 10.1128/aem.54.6.1595-1601.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Francisco D. E., Mah R. A., Rabin A. C. Acridine orange-epifluorescence technique for counting bacteria in natural waters. Trans Am Microsc Soc. 1973 Jul;92(3):416–421. [PubMed] [Google Scholar]
  11. Jennison M. W. The Relations Between Plate Counts and Direct Microscopic Counts of Escherichia coli During the Logarithmic Growth Period. J Bacteriol. 1937 May;33(5):461–477. doi: 10.1128/jb.33.5.461-477.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaprelyants A. S., Gottschal J. C., Kell D. B. Dormancy in non-sporulating bacteria. FEMS Microbiol Rev. 1993 Apr;10(3-4):271–285. doi: 10.1111/j.1574-6968.1993.tb05871.x. [DOI] [PubMed] [Google Scholar]
  13. Kaprelyants A. S., Kell D. B. Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation. Appl Environ Microbiol. 1993 Oct;59(10):3187–3196. doi: 10.1128/aem.59.10.3187-3196.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kepner R. L., Jr, Pratt J. R. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol Rev. 1994 Dec;58(4):603–615. doi: 10.1128/mr.58.4.603-615.1994. [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. McFeters G. A., Pyle B. H., Gillis S. J., Acomb C. J., Ferrazza D. Chlorine injury and the comparative performance of Colisure (TM), ColiLert (TM) and ColiQuik (TM) for the enumeration of coliform bacteria and E.coli in drinking water. Water Sci Technol. 1993;27(3-4):261–265. [PubMed] [Google Scholar]
  17. McFeters G. A., Singh A., Byun S., Callis P. R., Williams S. Acridine orange staining reaction as an index of physiological activity in Escherichia coli. J Microbiol Methods. 1991;13:87–97. doi: 10.1016/0167-7012(91)90009-f. [DOI] [PubMed] [Google Scholar]
  18. McFeters G. A., Yu F. P., Pyle B. H., Stewart P. S. Physiological assessment of bacteria using fluorochromes. J Microbiol Methods. 1995 Jan;21(1):1–13. doi: 10.1016/0167-7012(94)00027-5. [DOI] [PubMed] [Google Scholar]
  19. Pyle B. H., Watters S. K., McFeters G. A. Physiological aspects of disinfection resistance in Pseudomonas cepacia. J Appl Bacteriol. 1994 Feb;76(2):142–148. doi: 10.1111/j.1365-2672.1994.tb01609.x. [DOI] [PubMed] [Google Scholar]
  20. Reeve C. A., Bockman A. T., Matin A. Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium. J Bacteriol. 1984 Mar;157(3):758–763. doi: 10.1128/jb.157.3.758-763.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rodrigues U. M., Kroll R. G. Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique. J Appl Bacteriol. 1990 Mar;68(3):213–223. doi: 10.1111/j.1365-2672.1990.tb02567.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Singh A., Pyle B. H., McFeters G. A. Rapid enumeration of viable bacteria by image analysis. J Microbiol Methods. 1989;10:91–101. doi: 10.1016/0167-7012(89)90005-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Smith J. J., Howington J. P., McFeters G. A. Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment. Appl Environ Microbiol. 1994 Aug;60(8):2977–2984. doi: 10.1128/aem.60.8.2977-2984.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stellmach J. Fluorescent redox dyes. 1. Production of fluorescent formazan by unstimulated and phorbol ester- or digitonin-stimulated Ehrlich ascites tumor cells. Histochemistry. 1984;80(2):137–143. doi: 10.1007/BF00679987. [DOI] [PubMed] [Google Scholar]
  29. Tortorello M. L., Stewart D. S. Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef. Appl Environ Microbiol. 1994 Oct;60(10):3553–3559. doi: 10.1128/aem.60.10.3553-3559.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Yu F. P., McFeters G. A. Physiological responses of bacteria in biofilms to disinfection. Appl Environ Microbiol. 1994 Jul;60(7):2462–2466. doi: 10.1128/aem.60.7.2462-2466.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yu F. P., McFeters G. A. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes. J Microbiol Methods. 1994;20:1–10. doi: 10.1016/0167-7012(94)90058-2. [DOI] [PubMed] [Google Scholar]
  33. Zarda B., Amann R., Wallner G., Schleifer K. H. Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. J Gen Microbiol. 1991 Dec;137(12):2823–2830. doi: 10.1099/00221287-137-12-2823. [DOI] [PubMed] [Google Scholar]
  34. Zaske S. K., Dockins W. S., Schillinger J. E., McFeters G. A. New methods to assess bacterial injury in water. Appl Environ Microbiol. 1980 Mar;39(3):656–658. doi: 10.1128/aem.39.3.656-658.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ziegler N. R., Halvorson H. O. Application of Statistics to Problems in Bacteriology: IV. Experimental Comparison of the Dilution Method, the Plate Count, and the Direct Count for the Determination of Bacterial Populations. J Bacteriol. 1935 Jun;29(6):609–634. doi: 10.1128/jb.29.6.609-634.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES