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.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.     

Evidence for the role of copper in the injury process of coliform bacteria in drinking water.

Low levels of copper in chlorine-free distribution water caused injury of coliform populations. Monitoring of 44 drinking water samples indicated that 64% of the coliform population was injured. Physical and chemical parameters were measured, including three heavy metals (Cu, Cd, and Pb). Copper concentrations were important, ranging from 0.007 to 0.54 mg/liter. Statistical analyses of these factors were used to develop a model to predict coliform injury. The model predicted almost 90% injury with a copper concentration near the mean observed value (0.158 mg/liter) in distribution waters. Laboratory studies with copper concentrations of 0.025 and 0.050 mg/liter in an inorganic carbon buffer under controlled conditions of temperature and pH caused over 90% injury within 6 and 2 days, respectively. Studies of the metabolism of injured Escherichia coli cells indicated that the respiratory chain is at least one site of damage in injured cells.     

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.     

Evidence for the role of copper in the injury process of coliform bacteria in drinking water

Low levels of copper in chlorine-free distribution water caused injury of coliform populations. Monitoring of 44 drinking water samples indicated that 64% of the coliform population was injured. Physical and chemical parameters were measured, including three heavy metals (Cu, Cd, and Pb). Copper concentrations were important, ranging from 0.007 to 0.54 mg/liter. Statistical analyses of these factors were used to develop a model to predict coliform injury. The model predicted almost 90% injury with a copper concentration near the mean observed value (0.158 mg/liter) in distribution waters. Laboratory studies with copper concentrations of 0.025 and 0.050 mg/liter in an inorganic carbon buffer under controlled conditions of temperature and pH caused over 90% injury within 6 and 2 days, respectively. Studies of the metabolism of injured Escherichia coli cells indicated that the respiratory chain is at least one site of damage in injured cells.     

Effects of medium composition on the recovery of bacteria from sea water

Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample.Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.     

Antibiotic-resistant bacteria in drinking water.

We analyzed drinking water from seven communities for multiply antibiotic-resistant (MAR) bacteria (bacteria resistant to two or more antibiotics) and screened the MAR bacterial isolates obtained against five antibiotics by replica plating. Overall, 33.9% of 2,653 standard plate count bacteria from treated drinking waters were MAR. Two different raw water supplies for two communities carried MAR standard plate count bacteria at frequencies of 20.4 and 18.6%, whereas 36.7 and 67.8% of the standard plate count populations from sites within the respective distribution systems were MAR. Isolate identification revealed that MAR gram-positive cocci (Staphylococcus) and MAR gram-negative, nonfermentative rods (Pseudomonas, Alcaligenes, Moraxella-like group M, and Acinetobacter) were more common in drinking waters than in untreated source waters. Site-to-site variations in generic types and differences in the incidences of MAR organisms indicated that shedding of MAR bacteria living in pipelines may have contributed to the MAR populations in tap water. We conclude that the treatment of raw water and its subsequent distribution select for standard plate count bacteria exhibiting the MAR phenotype.     

Characterization of dysgonic, heterotrophic bacteria from drinking water.

Only a small percentage of the heterotrophic bacteria encountered in water distribution systems are identifiable, because of these organisms fail to grow on the conventional media used for biochemical characterization. Organisms that would not subculture from the same standard plate count agar used for initial isolation were successfully subcultured on a low-nutrient medium, R3A. These cultures were then inoculated to a modified O/F base medium containing specific substrates. This, combined with a lower incubation temperature (30 degrees C), increased the enzymatic activity of many of the organisms. These reactions established a groundwork for tentative taxonomy.     

Full-scale studies of factors related to coliform regrowth in drinking water.

An 18-month survey of 31 water systems in North America was conducted to determine the factors that contribute to the occurrence of coliform bacteria in drinking water. The survey included analysis of assimilable organic carbon (AOC), coliforms, disinfectant residuals, and operational parameters. Coliform bacteria were detected in 27.8% of the 2-week sampling periods and were associated with the following factors: filtration, temperature, disinfectant type and disinfectant level, AOC level, corrosion control, and operational characteristics. Four systems in the study that used unfiltered surface water accounted for 26.6% of the total number of bacterial samples collected but 64.3% (1,013 of 1,576) of the positive coliform samples. The occurrence of coliform bacteria was significantly higher when water temperatures were > 15 degrees C. For filtered systems that used free chlorine, 0.97% of 33,196 samples contained coliform bacteria, while 0.51% of 35,159 samples from chloraminated systems contained coliform bacteria. The average density of coliform bacteria was 35 times higher in free-chlorinated systems than in chloraminated water (0.60 CFU/100 ml for free-chlorinated water compared with 0.017 CFU/100 ml for chloraminated water). Systems that maintained dead-end free chlorine levels of < 0.2 mg/liter or monochloramine levels of < 0.5 mg/liter had substantially more coliform occurrences than systems that maintained higher disinfectant residuals. Free-chlorinated systems with AOC levels greater than 100 micrograms/liter had 82% more coliform-positive samples and 19 times higher coliform levels than free-chlorinated systems with average AOC levels less than 99 micrograms/liter. Systems that maintained a phosphate-based corrosion inhibitor and limited the amount of unlined cast iron pipe had fewer coliform bacteria. Several operational characteristics of the treatment process or the distribution system were also associated with increased rates of coliform occurrence. The study concludes that the occurrence of coliform bacteria within a distribution system is dependent upon a complex interaction of chemical, physical, operational, and engineering parameters. No one factor could account for all of the coliform occurrences, and one must consider all of the parameters described above in devising a solution to the regrowth problem.     

Holding effects on coliform enumeration in drinking water samples

Standard procedures for analyzing drinking water stress the need to adhere to the time and temperature conditions recommended for holding samples collected for microbiological testing. The National Drinking Water Laboratory Certification Program requires compliance with these holding limits, but some investigators have reported difficulties in meeting them. Research was conducted by standard analytical methods to determine if changes occurred when samples were held at 5 and 22 degrees C and analyzed at 0, 24, 30, and 48 h. Samples were analyzed for coliforms by the membrane filter and fermentation-tube procedures and for heterotrophs by the pour plate method. A total of 17 treated water samples were collected from a large municipal distribution system from August to December 1981, and 12 samples were collected from January to May 1983. The samples were dosed with coliforms previously isolated from the water system, Enterobacter cloacae in 1981 and Citrobacter freundii in 1983. The coliform counts declined linearly over time, and the rates of decline were significant at both 5 and 22 degrees C. Within 24 h at 22 degrees C, levels of E. cloacae and C. freundii decreased by 47 and 26%, respectively, and at 5 degrees C, E. cloacae numbers declined by 23%. Results from these representative laboratory-grown coliforms reinforced those previously obtained with naturally occurring coliforms under the same experimental conditions. Significantly, some samples with initially unacceptable counts (greater than 4/100 ml) met the safe drinking water limits after storage at 24 h at 5 and 22 degrees C and would have been classified as satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

Chlorine resistance patterns of bacteria from two drinking water distribution systems.

The relative chlorine sensitivities of bacteria isolated from chlorinated and unchlorinated drinking water distribution systems were compared by two independent methods. One method measured the toxic effect of free chlorine on bacteria, whereas the other measured the effect of combined chlorine. Bacteria from the chlorinated system were more resistant to both the combined and free forms of chlorine than those from the unchlorinated system, suggesting that there may be selection for more chlorine-tolerant microorganisms in chlorinated waters. Bacteria retained on the surfaces of 2.0-microns Nuclepore membrane filters were significantly more resistant to free chlorine compared to the total microbial population recovered on 0.2-micron membrane filters, presumably because aggregated cells or bacteria attached to suspended particulate matter exhibit more resistance than unassociated microorganisms. In accordance with this hypothesis, scanning electron microscopy of suspended particulate matter from the water samples revealed the presence of attached bacteria. The most resistant microorganisms were able to survive a 2-min exposure to 10 mg of free chlorine per liter. These included gram-positive spore-forming bacilli, actinomycetes, and some micrococci. The most sensitive bacteria were readily killed by chlorine concentrations of 1.0 mg liter-1 or less, and included most gram-positive micrococci, Corynebacterium/Arthrobacter, Klebsiella, Pseudomonas/Alcaligenes, Flavobacterium/Moraxella, and Acinetobacter.     

Characterization of dysgonic, heterotrophic bacteria from drinking water

Only a small percentage of the heterotrophic bacteria encountered in water distribution systems are identifiable, because of these organisms fail to grow on the conventional media used for biochemical characterization. Organisms that would not subculture from the same standard plate count agar used for initial isolation were successfully subcultured on a low-nutrient medium, R3A. These cultures were then inoculated to a modified O/F base medium containing specific substrates. This, combined with a lower incubation temperature (30 degrees C), increased the enzymatic activity of many of the organisms. These reactions established a groundwork for tentative taxonomy.     

Viable but nonculturable bacteria in drinking water.

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.