Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Comparison of four membrane filter methods for fecal coliform enumeration.

Four membrane filter methods fecal coliform enumeration were evaluated and compared in six laboratories based on determination of accuracy, specificity, upper counting limit, and recovery comparability. Recovery accuracy with pure cultures ranged from 89 to 100% for m-FC, mTEC (a procedure developed for thermotolerant Escherichia coli), and m-FC2 methods (the latter incorporating a 2-h, 35 degrees C resuscitation period), but was less than 60% for the MacConkey membrane broth method. These figures dropped by approximately 40 to 55% when the cultures were subjected to temperature (10 degrees C) stress. Close to 800 colonies were verified to determine specificity. False-positive colonies occurred most frequently with the m-FC2 method (18%), whereas false-negative colonies were most common on MacConkey membrane broth (26%). In counting range experiments using a variety of samples, the highest upper counting limit was 130 colonies per filter with the mTEC procedure. Recovery comparisons were based on over 130 samples including raw surface waters, raw sewage, and chlorinated and unchlorinated sewage effluents. In general, recoveries were significantly higher with the m-FC2 and mTEC methods; however, on m-FC2, growth of nontarget background organisms was also higher in most cases. Highest recoveries from chlorinated sewage effluents were obtained by the mTEC method, and the addition of a similar resuscitation period to the m-FC procedure (m-FC2) improved fecal coliform recovery from such samples. The best overall performance for fecal coliform enumeration was obtained with the mTEC method with high recovery and low levels of background colonies, good specificity and accuracy, and a high upper counting limit. This procedure also offers the advantage of enumerating E. coli within 24 h.     

Comparison of nine brands of membrane filter and the most-probable-number methods for total coliform enumeration in sewage-contaminated drinking water.

Nine different brands of membrane filter were compared in the membrane filtration (MF) method, and those with the highest yields were compared against the most-probable-number (MPN) multiple-tube method for total coliform enumeration in simulated sewage-contaminated tap water. The water was chlorinated for 30 min to subject the organisms to stresses similar to those encountered during treatment and distribution of drinking water. Significant differences were observed among membranes in four of the six experiments, with two- to four-times-higher recoveries between the membranes at each extreme of recovery. When results from the membranes with the highest total coliform recovery rate were compared with the MPN results, the MF results were found significantly higher in one experiment and equivalent to the MPN results in the other five experiments. A comparison was made of the species enumerated by these methods; in general the two methods enumerated a similar spectrum of organisms, with some indication that the MF method was subject to greater interference by Aeromonas.     

Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters.

Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.     

Comparison of Clark's presence-absence test and the membrane filter method for coliform detection in potable water samples.

A total of 2,601 water samples from six different water systems were tested for coliform bacteria by Clark's presence-absence (P-A) test and by the membrane filter (MF) method. There was no significant difference in the fraction of samples positive for coliform bacteria for any of the systems tested. It was concluded that the two tests are equivalent for monitoring purposes. However, 152 samples were positive for coliform bacteria by the MF method but negative by the P-A test, and 132 samples were positive by the P-A test but negative by the MF method. Many of these differences for individual samples can be explained by random dispersion of bacteria in subsamples when the coliform density is low. However, 15 samples had MF counts greater than 3 and gave negative P-A results. The only apparent explanation for most of these results is that coliform bacteria were present in the P-A test bottles but did not produce acid and gas. Two other studies have reported more samples positive by Clark's P-A test than by the MF method.     

Comparison of verification procedures for the membrane filter total coliform technique

[This corrects the article on p. 1127 in vol. 45.].     

Comparison of the presence-absence and membrane filter techniques for coliform detection in small, nonchlorinated water distribution systems.

The traditional membrane filter (American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th ed., American Public Health Association, Washington, D.C., 1985) and presence-absence (P-A) (J. A. Clark, Can. J. Microbiol. 14:13-18, 1968) techniques for the detection of coliform bacteria were compared in a small nonchlorinated drinking water distribution system by using total positive samples and frequency-of-occurrence analyses. No significant differences (P less than 0.05) were found in detection of the presence of coliform bacteria or in changes in the frequency of occurrence with time. A reduction in P-A sample volume (to 50 ml) was not found to statistically affect the comparative results of traditional membrane filter and P-A tests.     

Comparison of Clark's presence-absence test and the membrane filter method for coliform detection in potable water samples

A total of 2,601 water samples from six different water systems were tested for coliform bacteria by Clark's presence-absence (P-A) test and by the membrane filter (MF) method. There was no significant difference in the fraction of samples positive for coliform bacteria for any of the systems tested. It was concluded that the two tests are equivalent for monitoring purposes. However, 152 samples were positive for coliform bacteria by the MF method but negative by the P-A test, and 132 samples were positive by the P-A test but negative by the MF method. Many of these differences for individual samples can be explained by random dispersion of bacteria in subsamples when the coliform density is low. However, 15 samples had MF counts greater than 3 and gave negative P-A results. The only apparent explanation for most of these results is that coliform bacteria were present in the P-A test bottles but did not produce acid and gas. Two other studies have reported more samples positive by Clark's P-A test than by the MF method.     

Comparison of the presence-absence and membrane filter techniques for coliform detection in small, nonchlorinated water distribution systems

The traditional membrane filter (American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th ed., American Public Health Association, Washington, D.C., 1985) and presence-absence (P-A) (J. A. Clark, Can. J. Microbiol. 14:13-18, 1968) techniques for the detection of coliform bacteria were compared in a small nonchlorinated drinking water distribution system by using total positive samples and frequency-of-occurrence analyses. No significant differences (P less than 0.05) were found in detection of the presence of coliform bacteria or in changes in the frequency of occurrence with time. A reduction in P-A sample volume (to 50 ml) was not found to statistically affect the comparative results of traditional membrane filter and P-A tests.     

Modification of the standard most-probable-number procedure for fecal coliform bacteria in seawater and shellfish.

To determine whether or not presumptive tubes which take 48 h to become positive need to be transferred to EC medium during the fecal coliform multitube most-probable-number procedure, 572 seawater and shellfish samples were considered. When the 24-hour positive tubes alone were transferred to EC, it appeared that incorrect dilution data would be rare.     

False-positive coliform readings using membrane filter techniques for seawater

Seawater samples that had been analysed by membrane filter techniques produced positive coliform readings that were found to be false. Apparently, the culture media and the incubation temperatures used in these methods permit the growth of vibrios which are abundant in the seawater. The vibrio colonies isolated by membrane filter were oxidase-positive Gram-negative short rods that grew on TCBS and required NaCl.     

Comparison of four-hour and twenty-four-hour refrigerated storage of nonpotable water for fecal coliform analysis.

The problem of extending the storage time of water samples for fecal coliform analysis was addressed. Included in this report is a literature review of the storage problem. Twenty-eight samples were analyzed in replicate to determine the effect of 24-h storage of water samples at 4 degrees C. A new statistical approach to data analysis, coupled with the concept of practical acceptability, is presented. According to our results, many samples can successfully be stored at 4 degrees C for 24 h.     

Comparison of four-hour and twenty-four-hour refrigerated storage of nonpotable water for fecal coliform analysis.

The problem of extending the storage time of water samples for fecal coliform analysis was addressed. Included in this report is a literature review of the storage problem. Twenty-eight samples were analyzed in replicate to determine the effect of 24-h storage of water samples at 4 degrees C. A new statistical approach to data analysis, coupled with the concept of practical acceptability, is presented. According to our results, many samples can successfully be stored at 4 degrees C for 24 h.     

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 methods for determining the numbers and species distribution of coliform bacteria in well water samples

AIMS: Enumeration of coliform bacteria and Escherichia coli is the most widely used method in the estimation of hygienic quality of drinking water. The yield of target bacteria and the species composition of different populations of coliform bacteria may depend on the method.Three methods were compared. METHODS AND RESULTS: Three membrane filtration methods were used for the enumeration of coliform bacteria in shallow well waters. The yield of confirmed coliform bacteria was highest on Differential Coliform agar, followed by LES Endo agar. Differential Coliform agar had the highest proportion of typical colonies, of which 74% were confirmed as belonging to the Enterobacteriaceae. Of the typical colonies on Lactose Tergitol 7 TTC agar, 75% were confirmed as Enterobacteriaceae, whereas 92% of typical colonies on LES Endo agar belonged to the Enterobacteriaceae. LES Endo agar yielded many Serratia strains, Lactose Tergitol 7 TTC agar yielded numerous strains of Rahnella aquatilis and Enterobacter, whereas Differential Coliform agar yielded the widest range of species. CONCLUSION: The yield of coliform bacteria varied between methods. Each method compared had a characteristic species distribution of target bacteria and a typical level of interference of non-target bacteria. Identification with routine physiological tests to distinct species was hampered by the slight differences between species. High yield and sufficient selectivity are difficult to achieve simultaneously, especially if the target group is diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed that several aspects of method performance should be considered, and that the target group must be distinctly defined to enable method comparisons.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.