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.     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

The occurrence of fungi in hospital and community potable waters

The prevalence of fungi was investigated in 126 potable water samples (84 hospital and 42 community samples), in parallel with the standard pollution indicator micro-organisms. Filamentous fungi were isolated from 104 of 126 (82.5%) samples and yeasts from 14 (11.1%), whereas their mean counts were 36.6 and 4.4, respectively. Fungi were isolated from 95.2% of community and 76.2% of hospital water samples, with the difference being statistically significant (P < 0.05), while yeasts were isolated from 9.5 and 11.9%, respectively. Prevailing genera were Penicillium spp., isolated from 64, Aspergillus spp., from 53, and Candida, from nine of the examined samples. Colony-forming units of yeasts were significantly correlated with those of total and faecal coliforms, whereas the counts of filamentous fungi were significantly correlated with total heterotrophic bacteria counts. These results suggest that tap water is a potential transmission route for fungi both in hospitals and the community in the examined region and may pose a health hazard mainly for the immunocompromised host.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water.

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

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.     

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.     

Iron bacteria of the genus Siderocapsa in mineral waters.

The occurrence of iron bacteria in mineral waters has been under study. It could be shown that Siderocapsa caronata Redlinger 1931 and S. treubii Molisch 1909 are synonymous, as well as S. eusphaera Skuja 1948 and S. major Molisch 1909, and S. botryoides Berger 1949 and S. monoica 1922. Two new species, S. hexagonata and S. quadrata, have been described. A simplified key for determining the species of the genus Sideracapsa is presented.     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

An intracellular metabolite quantification technique applicable to polysaccharide-producing bacteria

An experimental procedure for the quantification of intracellular concentrations of metabolites in exoploysacharide-producing bacteria has been developed. This simple technique is based on the simultaneous quenching and extraction of metabolites using cold ethanol. Extracellular polysaccharide is precipitated with the cell matter generating clean samples which can be further concentrated using evaporation techniques or solid state extraction columns. Intracellular pools, coherent with the operation of the Entner–Doudoroff pathway were observed in Xanthomonas campestris.     

Visual quantification of DNA double-strand breaks in bacteria.

In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.     

A simple and cost-effective method for the quantification of total coliforms and Escherichia coli in potable water

In this study, a new simple and cost-effective method for the study of total coliforms and Escherichia coli in potable water, combining the use of lactose TTC agar and TBX agar, was developed and compared with methods using Chromocult agar and coli ID. The statistical analysis showed no significant difference and a good correlation (R(2)) between the three methods.     

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference ( P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus , (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.     

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.     

Rapid quantification of bacterial cells in potable water using a simplified microfluidic device

A simplified microfluidic device for quantification of bacteria in potable water was fabricated and examined. Comparisons of counts of Escherichia coli by the microfluidic system and by epifluorescence microscopy closely correlated (r2=0.99). Bacteria in natural mineral water and in purified household tap water were accurately enumerated by using this system within 15 min after fluorescent staining.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

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.     

Bioinformatics and molecular biology for the quantification of closely related bacteria

Molecular biological methods for mixed culture analysis outshine conventional culture-based techniques in terms of better sensitivity and reliability. The majority of these methods exploit the 16S rRNA sequences of the community DNA, which often fall short for the analysis of closely related microorganisms. This research details the development and validation of a comprehensive methodology to differentiate and quantitatively characterize two Pseudomonas species in a mixed culture. A bioinformatics tool based on whole-genome polymorphism comparison was used to identify marker sequences to differentiate the two bacteria using quantitative real-time PCR. The quantification of the two species was achieved through a correlation of the genomic DNA versus cell number (genomic DNA purification) and threshold cycle number versus genomic DNA (real-time PCR). Several factors including the limitation of genomic DNA purification, effects of substrate concentrations and growth phase on cellular DNA, and choice of simplex or duplex reaction for real-time PCR were considered and evaluated. The developed method was experimentally validated against synthetically constructed consortia.     

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower.

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).