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).     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Comparison of fecal coliform agar and violet red bile lactose agar for fecal coliform enumeration in foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 +/- 1 degrees C and transfer to 44 +/- 1 degrees C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.     

Fecal coliform elevated-temperature test: a physiological basis.

The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.5 degrees C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5 degrees C. A temperature increase from 35 to 44.5 degrees C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform beta-galactosidase activity coupled with thermal inactivation of this enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature.     

Fecal coliform elevated-temperature test: a physiological basis.

The physiological basis of the Eijkman elevated-temperature test for differentiating fecal from nonfecal coliforms was investigated. Manometric studies indicated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.5 degrees C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experiments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5 degrees C. A temperature increase from 35 to 44.5 degrees C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform beta-galactosidase activity coupled with thermal inactivation of this enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature.     

Membrane filter method for recovery of fecal coliforms in chlorinated sewage effluents.

The standard one-step M-FC broth-membrane-filter procedure for recovery of fecal coliforms from chlorinated sewage effluents is much less effective than the multiple-tube (most-probable-number) technique. A two-step membrane-filter method, using a pre-enrichment technique with phenol red lactose broth and incubation at 35 degrees C for 4 h, followether 18+/-2 h, enhanced fecal coliform recovery from chlorinated effluents. The results of 126 comparisons using chlorinated effluents from five wastewater plants showed that fecal coliform recovery by using the two-step membrane-filter method is comparable to that using the multiple-tube procedure.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water.

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

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).     

Anaerobic incubation of membrane filter cultures for improved detection of fecal coliforms from recreational waters.

Anaerobic incubation of membrane filter cultures significantly enhanced detection of fecal coliforms in surface-water samples from recreational beaches. In contrast to standard aerobic incubation, anaerobic incubation suppressed overgrowth of masking, noncoliform bacteria but did not increase the frequency of fecal coliform recovery.     

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.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Evaluation of commercial presence-absence test kits for detection of total coliforms, Escherichia coli, and other indicator bacteria.

Evaluations of several commercial presence-absence (P-A) test kits were performed over a 6-month period in 1990 by using the Ontario Ministry of the Environment (MOE) P-A test for comparison. The general principles of the multiple-tube fermentation technique formed the basis for conducting the product evaluations. Each week, a surface water sample was diluted and inoculated into 25 99-ml dilution blanks for each of three dilutions. The inoculated dilution blanks from each dilution series were randomly sorted into sets of five. Three of these sets were inoculated into the P-A test kits or vice versa, as required. The other two sets were passed through membrane filters, and one set of five membrane filters was placed onto m-Endo agar LES to give replicate total coliform counts and the other set was placed onto m-TEC agar to give replicate fecal coliform results. A statistical analysis of the results was performed by a modified logistic transform method, which provided an improved way to compare binary data obtained from the different test kits. The comparative test results showed that three of the four commercial products tested gave very good levels of recovery and that the fourth commercial product gave only fair levels of recovery when the data were compared with the data from MOE P-A tests and membrane filter tests. P-A bottles showing positive results after 18 h of incubation that were subcultured immediately in ECMUG tubes frequently could be confirmed as containing total coliforms, fecal coliforms, or Escherichia coli after 6 h of incubation; thus, the total incubation time was only 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)