Comparison of Verification Procedures for the Membrane Filter Total Coliform Technique

Verification of membrane filter total coliform colonies from drinking water was increased 87% by testing for the presence of β-galactosidase and cytochrome oxidase, compared with verification by determination of gas production in lauryl tryptose broth. Over 90% of the coliforms verified by testing for β-galactosidase and cytochrome oxidase were representative of the typical coliform genera.     

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.     

Evaluation of factors affecting the membrane filter technique for testing drinking water.

The following studies were done in response to questions regarding the adoption and use of the membrane filter (MF) technique for testing drinking water for the total coliform indicator group. A comparison with the most-probable-number technique showed that MF procedures with m-Endo agar LES were somewhat superior to the most-probable-number methods in terms of numbers of coliform organims recovered. Medium preparation and storage studies indicated that rehydration of m-Endo agar LES should be done with boiling water for less than 15 min, that m-Endo agar LES should not be exposed to light for more than 4 to 6 h, and that m-Endo agar LES plates may be used for up to 4 weeks and broth verification media for up to 3 weeks under given storage conditions. MF culture colonies were commonly found which did not produce sheen as expected for coliforms and yet were verified as coliforms. The occurrence and morphology of these atypical colonies were studied. Parallel inoculation of both lauryl tryptose (LT) and brilliant green bile (BGB) broth was found to be a better colony verification approach than recommended LT preenrichment before transfer to BGB. Comparison of parallel verification results indicated very little justification for the use of LT medium in MF verification procedures. In the case of overgrown or confluent cultures, the best coliform recoveries resulted from swabbing the MF plate and directly inoculating BGB medium with the swab. The occurrence of overgrowth was defined and evidence was collected suggesting that overgrowth is a function of sample holding time. Evaluation of routine test data and bacterial population reductions as a function of time indicated that nonquantitative recovery of coliforms may not be significantly affected for at least a 72-h sample holding time.     

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique.

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Pollution indicator bacteria associated with municipal raw and drinking water supplies.

A total of 3819 bacterial cultures isolated from municipal water samples were identified using a combination of Enterotubules and confirmatory media. Frequency distributions for the different genera or groups of bacteria were similar for raw water and drinking water isolations, except for Escherichia organisms which doubled their frequency in raw water. Differences between the membrane filter (MF) and presence-absence (P-A) test with regard to types of organisms isolated were limited to Klebsiella organisms which were preferentially cultured from MF plates. Members of the genus Enterobacter were isolated more than twice as frequently as any of the other coliform genera dealt with in this study. Aeromonas organisms were detected almost as often as such individual genera as Escherichia, Citrobacter, or Klebsiella. Although non-lactose fermenting colonies (false-negatives) of the coliform genera would not be detected by the MF technique, their lack of detection would likely be offset by the Aeromonas colonies (false-positives). At least 25% of the coliform isolates were either anaerogenic or non-lactose fermenters and would therefore go undetected by the most probably number (MPN) technique.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

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.     

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.     

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.     

Comparison of media for recovery of total coliform bacteria from chemically treated water.

Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.     

Comparison of media for recovery of total coliform bacteria from chemically treated water

Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.     

Comparison of Five Media for the Isolation of Coliform Organisms from Dehydrated and Deep Frozen Foods

S ummary . A significantly higher number of coliform and faecal coliform organisms were isolated using lactose-glutamic acid medium than by using lauryl sulphate-tryptone, lactose, brilliant green-bile or EE broths. With brilliant green-bile and lauryl sulphate-tryptone broth, coliforms/Enterobacteriaceae were isolated less often than with the others. For the detection of faecal coliforms, EE broth proved less good than all other media. All 5 media showed more coliforms/Enterobacteriaceae after 48 h than after 24 h at 30°. Few false positive results were obtained using any of these media.     

Effect of turbidity on chlorination efficiency and bacterial persistence in drinking water.

To define interrelationships between elevated turbidities and the efficiency of chlorination in drinking water, experiments were performed to measure bacterial survival, chlorine demand, and interference with microbiological determinations. Experiments were conducted on the surface water supplies for communities which practice chlorination as the only treatment. Therefore, the conclusions of this study apply only to such systems. Results indicated that disinfection efficiency (log10 of the decrease in coliform numbers) was negatively correlated with turbidity and was influenced by season, chlorine demand of the samples, and the initial coliform level. Total organic carbon was found to be associated with turbidity and was shown to interfere with maintenance of a free chlorine residual by creating a chlorine demand. Interference with coliform detection in turbid waters could be demonstrated by the recovery of typical coliforms from apparently negative filters. The incidence of coliform masking in the membrane filter technique was found to increase as the turbidity of the chlorinated samples increased. the magnitude of coliform masking in the membrane filter technique increased from less than 1 coliform per 100 ml in water samples of less than 5 nephelometric turbidity units to greater than 1 coliform per 100 ml in water samples of greater than 5 nephelometric turbidity units. Statistical models were developed to predict the impact of turbidity on drinking water quality. The results justify maximum contaminant levels for turbidity in water entering a distribution system as stated in the National Primary Drinking Water Regulations of the Safe Drinking Water Act.     

Removal of fluoride, arsenic and coliform bacteria by modified homemade filter media from drinking water

An attempt was made to investigate the removal of fluoride, arsenic and coliform bacteria from drinking water using modified homemade filter media. Batch mode experimental study was conducted to test the efficiency of modified homemade filter for reduction of impurities under the operating condition of treatment time. The physico-chemical and biological analysis of water samples had been done before and after the treatment with filter media, using standard methods. Optimum operating treatment time was determined for maximum removal of these impurities by running the experiment for 2, 4, 6, 8, 10 and 12h, respectively. The maximum reduction of fluoride, arsenic and coliform bacteria in percentage was 85.60%, 93.07% and 100% and their residual values were 0.72 mg/l, 0.009 mg/l and 0 coliform cells/100ml, respectively after a treatment time of 10h. These residual values were under the permissible limits prescribed by WHO. Hence this could be a cheap, easy and an efficient technique for removal of fluoride, arsenic and coliform bacteria from drinking water.     

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.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.     

Differential susceptibility of aeromonads and coliforms to cefsulodin.

Cefsulodin was evaluated as a potential selective agent for aeromonads. Resistance of Aeromonas and coliform isolates was determined by using a standard disk diffusion technique. A total of 119 Aeromonas and 78 coliform strains were isolated. For 102 of 130 [corrected] Aeromonas isolates (environmental and reference strains), the MIC of cefsulodin was < 8 micrograms/ml. Results of MIC tests by the agar dilution method showed that a concentration of cefsulodin of 10 micrograms/ml or less inhibited the growth of 96% of isolates. In comparison, for 81 of 94 coliform isolates (environmental and reference strains), the MIC of cefsulodin was > 32 micrograms/ml. Because cefsulodin suppresses growth of Aeromonas and other oxidase-positive organisms, total coliform (TC) and Escherichia coli counts on Chromocult Coliform agar (CC agar) without cefsulodin and on CC agar with 10 mg of cefsulodin per liter (CC-CFS) were compared. Variance analysis of data from 14 sewage-polluted irrigation water specimens did not demonstrate any statistically significant difference in the enumeration of E. coli with CC and CC-CFS media. On average, the CC agar recovered 2.46 times as many TCs as CC-CFS. However, Aeromonas colonies made up an average of 58.6% of the TC counts on CC agar. Because no Aeromonas spp. were recovered on CC-CFS, background interference was eliminated and the counts that were obtained reflected more accurately the number of TCs. Results of this study suggest that cefsulodin may be a useful selective agent against Aeromonas spp. which should be included in coliform chromogenic media when high levels of accompanying flora are expected.