An instrument for the immediate quantification of bacteria in potable waters

A new semiautomated instrument is described which quantifies the number of bacteria in potable waters within 3 min, providing a permanent colorimetric record of the results. The bacteria detection device can measure as few as 100 CFU/ml in potable waters. In brief, a 100- to 1,000-ml sample of tap water is drawn through a large surface, customized filter housed in the device, and bacteria, rust, and humic acid in the water are concentrated thereon. A reducing agent is used to remove the rust and humic acid from the filter. The filter is inverted and backflushed to elute the bacteria which are collected and reconcentrated onto a 7-mm-diameter filter surface. The reconcentrated bacteria are stained, and the filter fibers are preferentially decolorized without removing the dye from the bacteria. The color intensity of the filter surface is compared with a color guide to determine the amount of bacteria in the test water.     

Concentration of enteroviruses from large volumes of water

An improved method for concentrating viruses from large volumes of clean waters is described. It was found that, by acidification, viruses in large volumes of water could be efficiently adsorbed to epoxy-fiber-glass and nitrocellulose filters in the absence of exogenously added salts. Based upon this finding, a modified version of our previously described virus concentration system was developed for virus monitoring of clean waters. In this procedure the water being tested is acidified by injection of N HCl prior to passage through a virus adsorber consisting of a fiber-glass cartridge depth filter and an epoxy-fiber-glass membrane filter in series. The adsorbed viruses are then eluted with a 1-liter volume of pH 11.5 eluent and reconcentrated by adsorption to and elution from a small epoxy-fiber-glass filter series. With this method small quantities of poliovirus in 100-gallon (378.5-liter) volumes of tapwater were concentrated nearly 40,000-fold with an average virus recovery efficiency of 77%.     

Failure of the most-probable-number technique to detect coliforms in drinking water and raw water supplies.

A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.     

Membrane Filter Technique for Enumeration of Pseudomonas aeruginosa

A membrane filter procedure for the quantitation of Pseudomonas aeruginosa (mPA procedure) has been developed. Through the use of inhibitors and an elevated incubation temperature, the level of background microbial flora was decreased approximately 10,000-fold. Using P. aeruginosa cells suspended in sea water and held for 24 hr, between 70 and 100% of the „viable” cells could be recovered by the mPA procedure. Assay variability was found to be insignificant. The recoveries of P. aeruginosa from surface (fresh and salt) waters, potable waters, and sewage by the mPA procedure exceeded those obtainable by current methods. Subsequent to its development and evaluation, the mPA procedure was used at three other laboratories for the enumeration of P. aeruginosa in potable and recreational waters and in sewage samples. It was found amenable to routine use, and confirmation of typical colonies approached 100%.     

Higher prevalence of Alternaria spp. in marine and river waters than in potable samples

Alternaria species have been recognized as important cause of allergic diseases and are considered opportunistic pathogens for immunocompromised individuals. In order to assess the contribution of waters to the spread of Alternaria, we examined 390 water samples of various origin for the presence of these microorganisms in parallel with the standard pollution indicator bacteria, i.e. total coliforms, faecal coliforms and enterococci. Alternaria spp. were isolated from 42 out of 196 (21.4%) marine water samples, from 13 out of 68 (19.1%) river samples, whereas out of 126 potable waters only two (1.6%) were found positive. The incidence of Alternaria was significantly higher (p < 0.001) in marine and river samples than in potable waters. The mean values of the colony-forming units of the pollution indicator bacteria did not significantly differ between Alternaria-positive and Alternaria-negative samples.     

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.     

Preformed magnesium hydroxide precipitate for second-step concentration of enteroviruses from drinking and surface waters

A method is described for the second-step concentration of viruses from large volumes of drinking and surface waters. Seeded viruses present in the first eluate, performed with 50 mM glycine buffer, pH 11.5, were adsorbed on a preformed magnesium hydroxide precipitate. After low-speed centrifugation they were desorbed and adjusted to pH 7 with McIlvaine citrate-phosphate buffer. In these experimental conditions 90% of the viruses present in the 300-mL first eluate were reconcentrated in a final volume of 40 mL. The recovery efficiency was independent of either virus concentration or water quality.     

Application of laser scanning for the rapid and automated detection of bacteria in water samples

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Nonphotosynthetic pigmented bacteria in a potable water treatment and distribution system

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to distribution water, including dead-end locations, was compared at sample sites in a large municipal water system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35 degrees C. The predominant pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from 2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all correlations between yellow and orange pigmented bacteria were positive. The study results indicate that pigmented bacteria could readily be detected by using plate counting media developed for heterotroph enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an additional marker for monitoring changes in water quality. High numbers of heterotrophs, including pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when the water temperature was greater than 16 degrees C. The association of some pigmented bacteria with nosocomial and other infections raises concern that the organisms may have originated from the potable water supply. High levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals. Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of high concentrations of heterotrophic plate count bacteria, including the pigmented forms.     

Nonphotosynthetic pigmented bacteria in a potable water treatment and distribution system.

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to distribution water, including dead-end locations, was compared at sample sites in a large municipal water system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35 degrees C. The predominant pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from 2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all correlations between yellow and orange pigmented bacteria were positive. The study results indicate that pigmented bacteria could readily be detected by using plate counting media developed for heterotroph enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an additional marker for monitoring changes in water quality. High numbers of heterotrophs, including pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when the water temperature was greater than 16 degrees C. The association of some pigmented bacteria with nosocomial and other infections raises concern that the organisms may have originated from the potable water supply. High levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals. Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of high concentrations of heterotrophic plate count bacteria, including the pigmented forms.     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Electrostatic mechanism of survival of virulent Aeromonas salmonicida strains in river water.

The ecological mechanism of survival of Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, in river water was investigated by laboratory-based experiments with two virulent strains (which were autoagglutinating) and two virulent strains (which were nonagglutinating). A difference in net electrical charge of A. salmonicida cells was detected by electrophoresis; cells of the virulent strains were negative, whereas cells of the avirulent strains were positive. Despite the loss of viable cells within a week in distilled water and physiological saline (0.85% sodium chloride), the cells of the virulent strains survived for more than 15 weeks in the presence of diluted humic acid (10 micrograms/ml), tryptone (10 micrograms/ml), and cleaned river sand (100 g/100 ml of medium), but loss of viable cells occurred within 5 weeks in the absence of sand. The cells of the avirulent strains lost viability within 2 weeks with no relation to the presence of sand. Using ion-exchange columns, humic acid and the amino acids of tryptone were found to be anionic and cationic in water (pH 7.0), respectively. Sand particles had a high capacity to adsorb humic acid alone and amino acid-humic acid complexes. Thirty to fifty times the environmental concentration of amino acids (10 micrograms/ml) were accumulated on the surface of sand particles, thereby permitting only bacterial cells carrying net negative electrical charges (virulent cells) to survive for a long period on the surface of the sand particles. These electrostatic interrelationships among river sand, humic acid, and bacterial cells are closely implicated in the mechanism of long-term survival of virulent A. salmonicida in river sediments.     

Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-beta-D-galactoside

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.     

Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-beta-D-galactoside.

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration.

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

TRANSFORMATION AND ALLELOPATHY OF NATURAL DISSOLVED ORGANIC CARBON AND TANNIC ACID ARE AFFECTED BY SOLAR RADIATION AND BACTERIA1

The aim of this study was to test whether abiotic and biotic factors may affect allelopathic properties. Therefore, we investigated how solar radiation and bacteria influence allelopathic effects of the plant‐derived, polyphenolic tannic acid (TA) on microalgae. Using a block design, lake water samples with and without TA were exposed to solar radiation or kept in darkness with or without bacteria for 3 weeks. Dissolved organic carbon (DOC), specific size fractions of DOC analyzed by chromatography–organic carbon detection (LC‐OCD), and concentrations of total phenolic compounds (TPC) were measured to follow the fate of TA in lake water with natural DOC exposed to photolytic and microbial degradation. DOC and TPC decreased in dark‐incubated lake water with TA and bacteria indicating microbial degradation. In contrast, exposure to solar radiation of lake water with TA and bacteria did not decrease DOC. Chromatographic analyses documented an accumulation of DOC mean size fraction designated as humic substances (HS) in sunlit water samples with TA. The recalcitrance of the humic fraction indicates that photolytic degradation may contribute to a DOC less available for bacterial degradation. Subsequent growth tests with Desmodesmus armatus (Chodat) E. Hegewald showed low but reproducible difference in algal growth with lower algal growth rate cultured in photolytically and microbially degraded TA in lake water than cultured in respective dark treatments. This finding highlights the importance of photolytic processes and microbial degradation influencing allelopathic effects and may explain the high potential of allelochemicals for structuring the phytoplankton community composition in naturally illuminated surface waters.