Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction.

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).     

Conjugative Plasmid in Corynebacterium flaccumfaciens subsp. oortii That Confers Resistance to Arsenite, Arsenate, and Antimony(III)

The membrane filter (MF) method for detection and enumeration of coliform bacteria in drinking water requires that the coliforms both grow and produce a green metallic sheen when the filter is incubated on modified Endo medium at 35 degrees C for 22 h. Large numbers of noncoliform bacteria, which are enumerated by the standard plate count (SPC) technique, can interfere with the detection of coliforms on MF. This paper presents quantitative evidence from laboratory experiments on the interference of specific SPC bacteria on coliform colony sheen production on MF. Pseudomonas aeruginosa and Aeromonas hydrophila caused significant reductions in Escherichia coli sheen colony counts when present at 3,000 and 220 per filter, respectively. The Flavobacterium sp. and Bacillus sp. selected for this study from SPC did not interfere with coliform colony sheen production. Excessive crowding of E. coli and Enterobacter cloacae colonies on MF also caused a reduction in the number of colonies that produced sheen. Even when there was no crowding (14 colonies per filter), only a fraction of the E. cloacae colonies produced sheen colonies on modified Endo medium.     

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).     

Rapid enumeration of Fecal Coliforms in water by a colorimetric beta-galactosidase assay.

The colorimetric beta-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-beta-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45 micrometer membrane filter. This filter was then incubated at 37 degrees C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5 degrees C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. A total of 302 isolates randomly selected from positive ONPG-EC media, which were derived from 11 different habitats, were identified as E. coli (96.69 percent), Enterobacter cloacae (2.32 percent), Klebsiella pneumoniae (0.66 percent), and Citrobacter freundii (0.33 percent).     

Effect of ambient temperature storage on potable water coliform population estimations

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

Effect of ambient temperature storage on potable water coliform population estimations.

The effect of the length of time between sampling potable water and performing coliform analyses has been a long-standing controversial issue in environmental microbiology. The issue is of practical importance since reducing the sample-to-analysis time may substantially increase costs for water analysis programs. Randomly selected samples (from those routinely collected throughout the State of Wisconsin) were analyzed for total coliforms after being held at room temperature (20 +/- 2 degrees C) for 24 and 48 h. Differences in results for the two holding times were compared with differences predicted by probability calculations. The study showed that storage of the potable water for up to 48 h had little effect on the public health significance of most samples containing more than two coliforms per 100 ml.     

Coliform species recovered from untreated surface water and drinking water by the membrane filter, standard, and modified most-probable-number techniques.

The species of total coliform bacteria isolated from drinking water and untreated surface water by the membrane filter (MF), the standard most-probable-number (S-MPN), and modified most-probable-number (M-MPN) techniques were compared. Each coliform detection technique selected for a different profile of coliform species from both types of water samples. The MF technique indicated that Citrobacter freundii was the most common coliform species in water samples. However, the fermentation tube techniques displayed selectivity towards the isolation of Escherichia coli and Klebsiella. The M-MPN technique selected for more C. freundii and Enterobacter spp. from untreated surface water samples and for more Enterobacter and Klebsiella spp. from drinking water samples than did the S-MPN technique. The lack of agreement between the number of coliforms detected in a water sample by the S-MPN, M-MPN, and MF techniques was a result of the selection for different coliform species by the various techniques.     

Antimicrobial resistance in Gram-negative bacilli isolated from infant formulas

A total of 90 samples of infant formula (IF) were collected from the lactary of a teaching hospital, during a 4-month period from July to August 1999. The sanitary conditions of the formulas were analyzed, and a physiological characterization of Gram-negative bacillus isolates and antimicrobial susceptibility testing were performed. Colony counts were considered to be unacceptable for the majority of the IF samples and the contamination rates were related to inadequate handling. Coliforms (35 degrees C and 45 degrees C growth) were detected in most of the IF tested. Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae, Klebsiella planticola and Enterobacter cloacae were the isolates most commonly identified. Antimicrobial susceptibility testing showed significant resistance rates, particularly to amoxicillin/clavulanic acid, cefoxitin, cephalotin or ampicillin. One extended-spectrum beta-lactamase-producing K. pneumoniae strain was also recovered.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Bacteriological examinations of drinking water in the district of Melut (Upper Nile Province) South Sudan

In October and November 1983 bacteriological examinations of drinking water were done in the district of Melut/Upper Nile Province/South Sudan. The samples were taken from the middle of the White Nile, from handpumps along the river-bank, from the bank of the river and from drinking water bowls in the village of Melut and other surrounding villages within about 100 kilometers. All together 213 water samples were bacteriologically examined. The examinations of the samples taken from the river-bank and from the drinking water bowls showed very high plate counts (10(4)-10(7) colonies/ml) and a very high identification rate of Escherichia coli (11.1-31.5%) and of coliforms (82.1-92.8%). The water from the middle of the White Nile and from the handpumps, which are pumping water from a distance of 25 meters from the river-bank, had a better bacteriological quality. The plate counts were about 10(3)-10(4) colonies/ml. Escherichia coli could only be identified in one sample from the handpumps (4.4%) and coliforms were found in 40% of the samples of the middle of the Nile and in 48.8% of the samples from the handpumps. The trial to get qualitative better water with handpumps from regions far away from the river-bank, can be viewed as successful.     

Bacterial flora in bottled uncarbonated mineral drinking water

A quantitative study of bacterial populations in mineral water was carried out. Samples were stored at 6 and 20 degrees C, and the colony counts were determined on tryptone agar plates incubated at 22 and 37 degrees C. Samples were collected from the spring source in sterile glass flasks and from the bottling factory in conventional plastic and glass containers. In both cases, the initial population (10(1)-10(2) cfu/mL water) increased to 10(5)-10(6) cfu/mL after 3 days storage as determined from plate counts incubated at 22 degrees C. The levels reached by this population were similar to those of samples of mineral water obtained at the market stage. Results from plate counts incubated at 37 degrees C showed that populations in samples collected at the bottling factory reached 10(2)-10(3) cfu/mL. No growth was observed in water collected from spring source. Bacterial multiplication was not stopped even when water was stored at 6 degrees C. Caulobacter was the genus found most frequently in both types of samples, followed by Sphaerotilus-Leptothrix. Acinetobacter calcoaceticus and Pseudomonas fluorescens were frequently found in only two springs, and Pseudomonas putida, Arthrobacter, Aeromonas hydrophilia, and Corynebacterium were isolated less frequently. Janthinobacterium was recovered only once from a single spring. A giant bacterium closely resembling Hyphomicrobium and a budding one similar to Pasteuria were recovered from all samples of a single spring and from some of the commercial samples.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants

Seven drinking water treatment plants were sampled twice a month for 12 months to evaluate the removal of indicator bacteria and cytopathogenic enteric viruses. Samples were obtained at each level of treatment: raw water, postchlorination, postsedimentation, postfiltration, postozonation, and finished (tap) water. Raw water quality was usually poor, with total coliform counts exceeding 105 to 106 CFU/liter and the average virus count in raw water of 3.3 most probable number of cytopathogenic units (MPNCU)/liter; several samples contained more than 100 MPNCU/liter. All plants distributed finished water that was essentially free of indicator bacteria as judged by analysis of 1 liter for total coliforms, fecal coliforms, fecal streptococci, coagulase-positive staphylococci, and Pseudomonas aeruginosa. The total plate counts at 20 and 35 degrees C were also evaluated as a measure of the total microbial population and were usually very low. Viruses were detected in 7% (11 of 155) of the finished water samples (1,000 liters) at an average density of 0.0006 MPNCU/liter the highest virus density measured being 0.2 MPNCU/liter. The average cumulative virus reduction was 95.15% after sedimentation and 99.97% after filtration and did not significantly decrease after ozonation or final chlorination. The viruses isolated from treated waters were all enteroviruses: poliovirus types 1, 2, and 3, coxsackievirus types B3, B4, and B5, echovirus type 7, and untyped picornaviruses.     

Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants.

Seven drinking water treatment plants were sampled twice a month for 12 months to evaluate the removal of indicator bacteria and cytopathogenic enteric viruses. Samples were obtained at each level of treatment: raw water, postchlorination, postsedimentation, postfiltration, postozonation, and finished (tap) water. Raw water quality was usually poor, with total coliform counts exceeding 105 to 106 CFU/liter and the average virus count in raw water of 3.3 most probable number of cytopathogenic units (MPNCU)/liter; several samples contained more than 100 MPNCU/liter. All plants distributed finished water that was essentially free of indicator bacteria as judged by analysis of 1 liter for total coliforms, fecal coliforms, fecal streptococci, coagulase-positive staphylococci, and Pseudomonas aeruginosa. The total plate counts at 20 and 35 degrees C were also evaluated as a measure of the total microbial population and were usually very low. Viruses were detected in 7% (11 of 155) of the finished water samples (1,000 liters) at an average density of 0.0006 MPNCU/liter the highest virus density measured being 0.2 MPNCU/liter. The average cumulative virus reduction was 95.15% after sedimentation and 99.97% after filtration and did not significantly decrease after ozonation or final chlorination. The viruses isolated from treated waters were all enteroviruses: poliovirus types 1, 2, and 3, coxsackievirus types B3, B4, and B5, echovirus type 7, and untyped picornaviruses.     

National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escherichia coli from drinking water: comparison with presence-absence techniques

A defined substrate method was applied to drinking water to simultaneously enumerate total coliforms and total Escherichia coli directly from samples. After incubation at 35 degrees C for 24 h, the development of yellow in an initially colorless solution was specific for total coliforms; fluorescence at 366 nm in the same tube(s) or vessel demonstrated the presence of E. coli. No confirmatory or completed steps were necessary. Known as autoanalysis colilert (AC), this method was constituted as a presence-absence test and compared with the methods described in Standard Methods (SM) in the P-A format. Seven water utilities representing a wide geological and hydrological spectrum participated in the evaluation. A total of 702 split drinking water samples were analyzed. Of these, 358 were negative in both tests (SM- and AC-); 302 were positive (SM+ and AC+); and 42 were mixed (SM+ and AC-, 20; AC+ and SM-, 22). The overall agreement rate was 94%. Comparison of the SM and AC results by nonparametric statistics demonstrated no differences. Heterotrophic plate count bacteria exerted no discernible effect on the AC test. By subculture, each time the AC test was yellow, a total coliform was present; when the test was fluorescent, E. coli was isolated.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

National field evaluation of a defined substrate method for the simultaneous detection of total coliforms and Escherichia coli from drinking water: comparison with presence-absence techniques.

A defined substrate method was applied to drinking water to simultaneously enumerate total coliforms and total Escherichia coli directly from samples. After incubation at 35 degrees C for 24 h, the development of yellow in an initially colorless solution was specific for total coliforms; fluorescence at 366 nm in the same tube(s) or vessel demonstrated the presence of E. coli. No confirmatory or completed steps were necessary. Known as autoanalysis colilert (AC), this method was constituted as a presence-absence test and compared with the methods described in Standard Methods (SM) in the P-A format. Seven water utilities representing a wide geological and hydrological spectrum participated in the evaluation. A total of 702 split drinking water samples were analyzed. Of these, 358 were negative in both tests (SM- and AC-); 302 were positive (SM+ and AC+); and 42 were mixed (SM+ and AC-, 20; AC+ and SM-, 22). The overall agreement rate was 94%. Comparison of the SM and AC results by nonparametric statistics demonstrated no differences. Heterotrophic plate count bacteria exerted no discernible effect on the AC test. By subculture, each time the AC test was yellow, a total coliform was present; when the test was fluorescent, E. coli was isolated.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Effect of transport at ambient temperature on detection and isolation of Vibrio cholerae from environmental samples

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31 degrees C to 35 degrees C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.