New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.     

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.     

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.     

Synergistic False-Positive Coliform Reaction on M-Endo MF Medium

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.     

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.     

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.     

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55°C for 5 min), freezing (−20°C for 24 h, thawed, frozen again at −20°C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21°C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

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.     

A method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay

The mouse lymphoma assay (MLA) is the most widely used in vitro mammalian gene mutation assay. It detects various mutation events involving the thymidine kinase (Tk) gene in L5178Y/Tk+/- -3.7.2C mouse lymphoma cells. Mutants are detected using a thymidine analogue that arrests the growth of cells containing a functional Tk gene. However, there are a number of potential test chemicals that are thymidine analogues, and there is a problem when using the MLA to evaluate the mutagenicity of these chemicals. Thymidine analogues are activated by Tk before eliciting their toxicity. Therefore, any pre-existing Tk-/- mutants may avoid the toxicity of the test chemical and obtain a growth advantage over the Tk+/- cells, increasing the Tk mutant frequency (MF) in the culture via a selection mechanism. This potential mutant selection effect needs to be distinguished from de novo mutant induction in order to properly evaluate the mutagenicity of these chemicals. Here we describe a simple MLA study design that can differentiate between the selection of pre-existing mutants and de novo mutant induction. Trifluorothymidine (TFT), a thymidine analogue and the selection agent normally used in the MLA, and 4-nitroquinoline-1-oxide (4-NQO), a potent mutagen, were used to treat cells from two different Tk+/- mouse lymphoma cell cultures with different background MFs (approximately 112 and 305x10(-6)). Both agents significantly increased the Tk MFs in both the normal and high background cultures (p<0.01). In 4-NQO-treated cultures, the induced MFs (MF of treated culture-MF of control) for the cultures with different background MFs were about the same (p>0.1), while in TFT-treated cultures, they were significantly different (p<0.01). In TFT-treated cultures, the fold-increases of MF (MF of treated culture/MF of control) for the cultures with different background MFs were about the same (p>0.1), while in 4-NQO-treated cultures, they were significantly different (p<0.01). This study confirms that, when de novo mutations are induced, the induced MF is the same for cultures with normal and artificially high background MFs. In situations where the increase in MF is due solely to selection of pre-existing mutants, the "induced" MF will be a multiple of the background MF and the magnitude of the increase of the induced MF will depend upon the magnitude of the background MF. Our results demonstrate that it is possible, using this experimental design, to distinguish between chemicals acting primarily via the selection of pre-existing Tk mutants and those inducing de novo mutants in the MLA.     

Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental Protection Agency-approved membrane filter method.

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

Performance of media for recovering stressed cells of Enterobacter sakazakii as determined using spiral plating and ecometric techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55 degrees C for 5 min), freezing (-20 degrees C for 24 h, thawed, frozen again at -20 degrees C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21 degrees C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

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 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.     

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.     

Microbiological quality of bottled water sold in Canada

The microbiological quality of bottled water sold in Canada was evaluated. A total of 114 lots of bottled water, both domestic and imported, were analyzed for aerobic colony count, coliforms, fecal coliforms, and Escherichia coli. No fecal coliforms or E. coli were found. Nineteen (46%) of the 41 lots of domestic purified water were found to exceed aerobic colony count standards and another lot exceeded coliform standards. One lot each of domestic and imported mineral water exceeded coliform standards. If mineral water were governed by the aerobic colony count standards for bottled water, then five lots each of both domestic and imported mineral water would have been found to be unsatisfactory. More surveillance of the bottled water industry in Canada is recommended.     

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.