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

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.

[1]  A. Singh,et al.  Rapid enumeration of viable bacteria by image analysis. , 1989, Journal of microbiological methods.

[2]  G A McFeters,et al.  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. , 1993, Water science and technology : a journal of the International Association on Water Pollution Research.

[3]  H. Jannasch,et al.  Bacterial Populations in Sea Water as Determined by Different Methods of Enumeration1 , 1959 .

[4]  M. W. Jennison The Relations Between Plate Counts and Direct Microscopic Counts of Escherichia coli During the Logarithmic Growth Period , 1937, Journal of bacteriology.

[5]  G. McFeters,et al.  Physiological responses of bacteria in biofilms to disinfection , 1994, Applied and environmental microbiology.

[6]  R. Colwell,et al.  Application of a direct microscopic method for enumeration of substrate-responsive marine bacteria , 1981 .

[7]  J. Hobbie,et al.  Direct counts of aquatic bacteria by a modified epifluorescence technique1 , 1975 .

[8]  J. Schillinger,et al.  New methods to assess bacterial injury in water , 1980, Applied and environmental microbiology.

[9]  D B Kell,et al.  Dormancy in non-sporulating bacteria. , 1993, FEMS microbiology reviews.

[10]  D. Danielsson A MEMBRANE FILTER METHOD FOR THE DEMONSTRATION OF BACTERIA BY THE FLUORESCENT ANTIBODY TECHNIQUE. 1. A METHODOLOGICAL STUDY. , 1965, Acta pathologica et microbiologica Scandinavica.

[11]  D. B. Smith,et al.  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 , 1988, Applied and environmental microbiology.

[12]  M. H. McCrady,et al.  The Numerical Interpretation of Fermentation-Tube Results , 1915 .

[13]  R. Colwell,et al.  An improved filter method for direct viable count of Salmonella in seawater , 1992 .

[14]  R. Mah,et al.  Acridine orange-epifluorescence technique for counting bacteria in natural waters. , 1973, Transactions of the American Microscopical Society.

[15]  Gordon A. McFeters,et al.  Enumeration, Occurrence, and Significance of Injured Indicator Bacteria in Drinking Water , 1990 .

[16]  G. Laurell,et al.  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. , 2009, Acta pathologica et microbiologica Scandinavica.

[17]  G. McFeters,et al.  Physiological aspects of disinfection resistance in Pseudomonas cepacia. , 1994, Journal of Applied Bacteriology.

[18]  G. McFeters,et al.  Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes. , 1994, Journal of microbiological methods.

[19]  P. Shaw,et al.  A comparison of tetrazolium reduction and FDA hydrolysis with other measures of microbial activity , 1990 .

[20]  M. Tortorello,et al.  Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef , 1994, Applied and environmental microbiology.

[21]  A. Matin,et al.  Effect of Starvation on Bacterial Resistance to Disinfectants , 1990 .

[22]  G. Laurell,et al.  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. , 2009, Acta pathologica et microbiologica Scandinavica.

[23]  A. Rompré,et al.  The optimization and application of two direct viable count methods for bacteria in distributed drinking water. , 1994, Canadian journal of microbiology.

[24]  H. Ridgway,et al.  Use of a fluorescent redox probe for direct visualization of actively respiring bacteria , 1992, Applied and environmental microbiology.

[25]  A. Mills,et al.  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 , 1982, Applied and environmental microbiology.

[26]  K. Schleifer,et al.  Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. , 1991, Journal of General Microbiology.

[27]  B. Mackey,et al.  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. , 1994, The Journal of applied bacteriology.

[28]  K Kogure,et al.  A tentative direct microscopic method for counting living marine bacteria. , 1979, Canadian journal of microbiology.

[29]  A. Winding,et al.  Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth , 1994, Applied and environmental microbiology.

[30]  P. Callis,et al.  Acridine orange staining reaction as an index of physiological activity in Escherichia coli. , 1991, Journal of microbiological methods.

[31]  J. Pratt,et al.  Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. , 1994, Microbiological reviews.

[32]  E. L. Schmidt,et al.  The Immunofluorescence Approach in Microbial Ecology , 1980 .

[33]  H. O. Halvorson,et al.  Application of Statistics to Problems in Bacteriology , 1935, Journal of bacteriology.

[34]  G. Pettipher Review: The direct epifluorescent filter technique , 1986 .

[35]  J. Clark The detection of various bacteria indicative of water pollution by a presence-absence (P-A) procedure. , 1969, Canadian journal of microbiology.

[36]  A. Matin,et al.  Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium , 1984, Journal of bacteriology.

[37]  D. B. Smith,et al.  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 , 1988, Applied and environmental microbiology.

[38]  M. Adams,et al.  Production and viability of coccoid forms of Campylobacter jejuni. , 1994, The Journal of applied bacteriology.

[39]  R. Atlas,et al.  Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. , 1990, Molecular and cellular probes.

[40]  D. Kell,et al.  Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation , 1993, Applied and environmental microbiology.

[41]  P. Stewart,et al.  Physiological assessment of bacteria using fluorochromes. , 1995, Journal of microbiological methods.

[42]  R. Colwell,et al.  Indicator organisms for estuarine and marine waters , 1985, FEMS Microbiology Letters.

[43]  R. Herbert,et al.  1 Methods for Enumerating Microorganisms and Determining Biomass in Natural Environments , 1990 .

[44]  James J. Smith,et al.  Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment , 1994, Applied and environmental microbiology.

[45]  R. G. Kroll,et al.  Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique. , 1990, The Journal of applied bacteriology.

[46]  H. Flemming,et al.  Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water , 1993, Applied and environmental microbiology.

[47]  A. Singh,et al.  Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis , 1990, Applied and environmental microbiology.

[48]  A. M. Mckay,et al.  Viable but non‐culturable forms of potentially pathogenic bacteria in water , 1992 .

[49]  O. Nybroe,et al.  Effects of starvation and osmotic stress on viability and heat resistance of Pseudomonas fluorescens AH9 , 1994 .