Effect of freezing of water samples on viable counts of environmental mycobacteria

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at —75°C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0·9–10·4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.     

Factors Stimulating Propagation of Legionellae in Cooling Tower Water

Our survey of cooling tower water demonstrated that the highest density of legionellae, ≥10⁴ CFU/100 ml, appeared in water containing protozoa, ≥10² MPN/100 ml, and heterotrophic bacteria, ≥10⁶ CFU/100 ml, at water temperatures between 25 and 35°C. Viable counts of legionellae were detected even in the winter samples, and propagation, up to 10⁵ CFU/100 ml, occurs in summer. The counts of legionellae correlated positively with increases in water temperature, pH, and protozoan counts, but not with heterotrophic bacterial counts. The water temperature of cooling towers may promote increases in the viable counts of legionellae, and certain microbes, e.g., protozoa or some heterotrophic bacteria, may be a factor stimulating the propagation of legionellae.     

A Note on the Total Viable Counts and Selective Enumeration of Anaerobic Bacteria in the Caecal Content, Soft and Hard Faeces of Rabbit

Total viable counts, proteolytic, ureolytic, cellulolytic and NH⁺₄ utilizing anaerobic bacteria in caecal contents, soft and hard faeces of rabbits were studied. No significant differences between the bacterial counts from the caecum and soft faeces were observed ( P < 0·05) but there were significant differences between the bacterial counts of the soft and hard faeces ( P < 0·01).     

Quantification of Staphylococcus aureus and Escherichia coli in the liquid medium by fluorimetry and its use in phagocytosis assay

A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence ( r = 0·99 for both Staph. aureus and E. coli ). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline ( r = 0·99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Environmental factors affecting the occurrence of mycobacteria in brook sediments

The occurrence of mycobacteria was studied in aerobic brook sediments from 39 drainage areas in Finland. The culturable counts of mycobacteria were related to climatic conditions, characteristics of the drainage area, chemical characteristics of the sediment and water, culturable counts of other heterotrophic bacteria, and microbial respiration rate in the sediment. The counts of mycobacteria varied from 1·1 × 10² to 1·5 × 10⁴ cfu g⁻¹ dry weight of sediment. They correlated positively with the proportion of the drainage area consisting of peatland, total content of C, content of Pb, microbial respiration rate in the sediment, and chemical oxygen demand of the water. The correlations of the mycobacterial counts with pH of sediment and alkalinity of water were negative. The results of the present sediment study and of the forest soil study published earlier strongly suggest that an increase in acidity increases the counts of mycobacteria and decreases the counts and activity of other heterotrophic bacteria. Mycobacterial counts were more than 100 times higher (per dry weight) in forest soils with pH 3·5–4·3 than in sediments with pH 4·5–6·3.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity on loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity no loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

Environmental Factors Affecting the Occurrence of Mycobacteria in Brook Waters

To evaluate the impact of environmental factors on the occurrence of environmental mycobacteria, viable counts of mycobacteria were measured in samples of brook water collected from 53 drainage areas located in a linear belt crossing Finland at 63° north latitude. The numbers of mycobacteria were correlated with characteristics of the drainage area, climatic parameters, chemical and physical characteristics of the water, and counts of other heterotrophic bacteria in the water. The numbers of mycobacteria in the water ranged from 10 to 2,200 CFU/liter. The counts correlated positively (P < 0.001) with the presence of peatlands, precipitation data, chemical oxygen demand, water color, and concentrations of Fe, Al, Cu, Co, and Cr. The mycobacterial counts correlated negatively (P < 0.001) with water pH, whereas other heterotrophic bacterial counts lacked any correlation with pH. A linear regression model with four independent variables (i.e., peatlands in the drainage area, chemical oxygen demand, concentration of potassium, and pH) explained 83% of the variation in mycobacterial counts in brook waters. Our results suggest that acidification may enhance the growth of environmental mycobacteria.     

Bacterial colonization of domestic reverse-osmosis water filtration units

We have analyzed the bacterial content of water from the reservoirs of 300 reverse-osmosis units installed in households. The heterotrophic plate counts on R2A medium (20 and 35 degrees C) ranged from 0 to 10(7) colony forming units per millilitre (cfu/mL). Most reservoirs contained water with bacterial counts between 10(4) and 10(5) cfu/mL. The bacteria identified were Pseudomonas (not aeruginosa), Alcaligenes or Moraxella, Acinetobacter, Flavobacterium, and Chromobacterium. This report emphasizes the importance of bacterial colonization by heterotrophic bacteria in water reservoirs from domestic reverse-osmosis units.     

Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents

Suspensions of Pseudomonas aeruginosa and Staphylococcus epidermidis , and biofilms established (16 h) on submerged glass and stainless steel (216 2B) coupons, were exposed to sodium hypochlorite (0·02% or 0·015% w/v), Dodigen (0·0015% w/v or 0·0006% w/v), sodium dodecylsulphate (6% w/v or 0·1% w/v) and Tween-80 (6% w/v) for 5 min at 20 °C. Survival was assessed by viable counts and blot succession. Biofilm bacteria were significantly less susceptible to these biocides than were planktonic cells, but their attachment to the surfaces was loosened by such treatments. Treatment with the non-ionic surfactant, Tween-80, however, strengthened the attachment of Staph. epidermidis to stainless steel. Such effects on attachment strength, which are species and surface dependent, have profound implications on post-treatment cleansing and possible re-contamination of product in clean-in-place (CIP) systems.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0·45 μm membrane filters which were then placed upon appropriate media and incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37°C in 2·5% CO₂ for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia , and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

Evaluation of the antimicrobial activity of citral

Citral showed appreciable antimicrobial activity against Gram-positive and Gram-negative bacteria as well as fungi. Media composition and inoculum size had no observable effect on activity but alkaline pH increased citral activity. The growth rates of Escherichia coli cultures were reduced at concentrations of citral ≥0·01% v/v while concentrations ≥0·03% v/v produced rapid reduction in viable cells followed by limited regrowth. In a non-growth medium, 0·08% and 0·1% v/v showed rapid bactericidal effects. Citral may therefore be of preservative use in addition to its other uses in the food, soap and cosmetic industries.     

Bacterial Activity in a Reservoir Determined by Autoradiography and its Relationships to Phyto- and Zooplankton

In the drinking water reservoir Římov (Southern Bohemia) bacterioplankton was studied during 1983. Special attention was given to the relationships between parameters of bacterial abundance, total and individual activity. Bacterial counts and biomass was assessed and autoradiographic determinations of the proportion of active bacteria incorporating thymidine (Th) and mixture of amino acids (AA) and total uptake rate of AA were made over a year in the surface layer and during summer stratification from the thermocline and 15 m depth. Specific activity of metabolically active bacteria (SAMAB) and specific activity per unit of biomass (SAUB) were negatively correlated with counts of metabolizing cells and with bacterial biomass, respectively. Total and individual heterotrophic activity and counts of bacteria coincided with the changes of phytoplankton biomass, whereas bacteria incorporating Th were more tightly correlated with primary production. The most significant relation of metabolically active bacteria was found to cladoceran biomass. Thus, this part of heterotrophic bacterial activity seems to be stimulated by leakage of dissolved organic matter from phytoplankton being disrupted and incompletely digested by cladocerans rather than from healthy photosynthetizing cells.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

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.     

River bacteria time series analysis: a field and laboratory study which demonstrates aquatic ecosystem health

Time series analysis of heterotrophic baeterial viable counts on agar plates incubated at 20° and 4°C from weekly river water samples for 1 year has revealed a periodicity of 6 to 7 weeks in the total numbers. A cycle of increase and decrease in the counts which could not be attributed to season, river water temperature, pH, and flow rate has been observed in the heterotrophic bacteria which were isolated from river water samples. Spectral analysis has demonstrated a hidden periodicity in population increase and decrease which could not be detected by examination of the raw count data. Fluctuations with periodicity could imply that this is the expected behaviour in a well-balanced ecosystem. Pseudomonas fluorescens, the predominant species isolated from a river water sample which was at 0°C at the time of sampling, utilized two different pathways at 5° and 30°C. In addition, 2-ketogluconate, a product of the utilization of glucose, was utilized by the same organism. Fluctuations in enzymic responses could contribute to the overall periodicity in bacterial numbers. The flexibility in bacterial metabolism would serve to maintain P. fluorescens in the predominant flora. A feature which sustains health in this ecosystem, P. fluorescens, is one of the most successful of all biodegraders in the aquatic system.     

Oligotrophic bacteria in ultra-pure water systems: Media selection and process component evaluations

Summary Presently, tryptic soy agar (TSA) medium is used in the semiconductor industry to determine the concentration of viable oligotrophic bacteria in ultra-pure water systems. Deionized water from an ultra-pure water pilot plant was evaluated for bacterial growth at specific locations, using a non-selective medium (R2A) designed to detect injured heterotrophic as well as oligotrophic bacteria. Results were compared to those obtained using Tryptic Soy Agar. Statistically greater numbers of bacteria were observed when R2A was used as the growth medium. Total viable bacterial numbers were compared both before and after each treatment step of the recirculating loop to determine their effectiveness in removing bacteria. The reduction in bacterial numbers for the reverse osmosis unit, the ion exchange bed, and the ultraviolet sterilizer were 97.4%, 31.3%, and 72.8%, respectively, using TSA medium, and 98.4%, 78.4%, and 35.8% using R2A medium. The number of viable bacteria increased by 60.7% based on TSA medium and 15.7% based on R2A medium after passage of the water through an in-line 0.2-m pore size nylon filter, probably because of the growth of bacteria on the filter. Our results suggest that R2A medium may give a better representation of the microbial water quality in ultra-pure water systems and therefore a better idea of the effectiveness of the various treatment processes in the control of bacteria.     

An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein

During growth of Escherichia coli in broth at pH 5·0, an extracellular protein termed an extracellular induction component (EIC) appears in the medium, this component being essential for acid tolerance induction. The present study establishes that the EIC arises from an extracellular precursor which is formed during growth at pH 7·0, and that conversion of the precursor to the EIC occurs at pH 5·0 (and other mildly acidic pH values) in the absence of organisms. On the basis that this precursor is produced by non-stressed cells as well as by stressed ones, and that it is converted to the EIC (which in turn induces the tolerance response) by the stress, the precursor can be considered to be a stress sensor, the first extracellular stimulus sensor to be reported. The EIC formed at pH 5·0 was inactivated at pH 9·0. This inactivation probably involved conversion back to the precursor as EIC was reformed if the alkali-inactivated component was incubated at pH 5·0. Both mild heat treatments (exposure to 40–55 °C) and u.v. irradiation also activated the precursor; the active induction component formed by the mild heat treatments was reversibly inactivated at pH 9·0 and so it seems likely that the component formed by heat treatment is similar or identical to the EIC produced at acidic pH. In contrast, the EIC produced by u.v. irradiation was not inactivated at pH 9·0, suggesting that it is different in some way to the EICs produced from the precursor by acidity or by heat treatment. It is likely that many responses affecting stress tolerance involve the functioning of such extracellular sensors, as similar components were shown to be involved in the acid tolerance responses induced at pH 7·0 by glucose, l -aspartate and l -glutamate. Extracellular stimulus sensors may also be needed for other inducible responses.     

Enumeration of Bacteriophages and Host Bacteria in Sewage and the Activated-Sludge Treatment Process

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 × 10⁷, 9.5 × 10⁷, and 8.4 × 10⁷/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 × 10⁷ and 1.3 × 10⁹/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.