Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and coliform bacteria in traditional brass and earthernware water storage vessels

The detection and enumeration of indicator bacteria such as Escherichia coli is used to assess the extent of faecal contamination of drinking water. On the basis of this approach, the effectiveness of storing water contaminated with faecal indicator bacteria in brass or earthern vessels (mutkas) of the type used in rural India have been investigated. Suspensions of bacteria in sterile distilled water were maintained for up to 48 h in each vessel and enumerated by surface plate counts on nutrient agar (non-selective) and several selective coliform media at 37 °C either under standard aerobic conditions, or under conditions designed to neutralise reactive oxygen species (ROS), e.g. using an anaerobic cabinet to prepare plates of pre-reduced growth medium or by inclusion of sodium pyruvate in the growth medium, with incubation of aerobically-prepared plates in an anaerobic jar. The counts obtained for E. coli decreased on short-term storage in a brass mutka; counts for selective media were lower than for equivalent counts for non-selective medium, with ROS-neutralised conditions giving consistently higher counts than aerobic incubation. However, after 48 h, no bacteria were cultivable under any conditions. Similar results were obtained using water from environmental sources in the Panjab, and from rural households where brass and earthern mutkas are used for storage of drinking water, with enumeration on selective coliform media (presumptive total coliforms). In all cases results indicated that, while storage of water in a brass mutka can inactivate E. coli and coliforms over a 48 h period, standard aerobic plate counting using selective media may not be fully effective in enumerating sub-lethally damaged bacteria.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Repair of Heat-Injured Clostridium perfringes During Outgrowth

Clostridium perfringens strain NCTC 8798 spores were injured by ultrahigh temperature treatment and were unable to outgrow in the presence of antibiotics used in selective enumeration media. Injured spores underwent repair in a nonselective laboratory medium and in foods.     

Model studies on a membrane filtration method for the enumeration of coagulase-positive staphylococci in swimming-pool water using rabbit plasma-bovine fibrinogen agar

The recovery of Staphylococcus aureus from swimming-pool water by membrane filtration was studied in model experiments. On the nonselective medium tryptone soya agar (TSA) there was no difference in counts of noninjured S. aureus with all membrane filters tested and with pour plates. Chlorine-injured S. aureus was enumerated most efficiently on TSA by Gelman Tuffryn HT-450 and Sartorius SM 13806 filters. Tuffryn filters were also most productive when used in combination with the selective medium rabbit plasma - bovine fibrinogen agar (RPFA). Other filters, particularly Gelman GN-6 and Millipore HAWP, when used on RPFA were shown to have a synergistic inhibitory effect on both noninjured and chlorine-injured S. aureus. This effect was not found on Baird-Parker agar. Using Tuffryn filters, counts on RPFA were equal to those on TSA for noninjured S. aureus and 0.1-2.0 log units less for chlorine-injured S. aureus. Despite this, the possibility for reading the in situ coagulase reaction for individual colonies on RPFA is considered such an advantage of this medium that its general use for enumeration of S. aureus in swimming pools is recommended. Further studies should be carried out to allow better resuscitation.     

Comparison and recovery of Escherichia coli and thermotolerant coliforms in water with a chromogenic medium incubated at 41 and 44.5 degrees C.

This study compared the performance of a commercial chromogenic medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate (CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA) for enumeration of Escherichia coli and non-E. coli thermotolerant coliforms (KEC). To establish that we could recover the maximum KEC and E. coli population, we compared two incubation temperature regimens, 41 and 44.5 degrees C. Statistical analysis by the Fisher test of data did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli for the different media (CECC and CECCP) and incubation temperatures. Variance analysis of data performed on KEC counts showed significant differences (P = 0.01) between KEC counts at 41 and 44.5 degrees C on both CECC and CECCP. Analysis of variance demonstrated statistically significant differences (P = 0.05) in the enumeration of total thermotolerant coliforms (TTCs) on CECC and CECCP compared with mLSA. Target colonies were confirmed to be E. coli at a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4% when using CECCP incubated at 41 degrees C. The results of this study showed that CECCP agar incubated at 41 degrees C is efficient for the simultaneous enumeration of E. coli and KEC from river and marine waters.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation.

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Improved detection of acid mine water stressed coliform bacteria on media containing catalase and sodium pyruvate

Pure culture suspensions of two strains of exponential and stationary phase Escherichia coli exhibited significant reductions in catalase activity following exposure to acid mine water (AMW). The exogenous addition of catalase (500-2000 U) or sodium pyruvate (0.05-5%) to a nonselective recovery medium resulted in enhanced detection (12- to 465-fold) of AMW-stressed E. coli as compared with recovery on the medium lacking these supplements, whereas addition of 3,3'-thiodipropionic acid failed to improve recovery. Additional in vitro experiments utilizing selective M-FC, mT7, and M-Endo media containing 1000 U catalase or 1.0% pyruvate similarly resulted in improved detection of AMW-stressed cells, with the exception of M-Endo containing pyruvate. Appropriately modified media were then used to analyze an AMW-impacted stream by the membrane filtration technique. Addition of catalase, pyruvate, or a combination of both significantly improved recovery of fecal and total coliforms without promoting growth of noncoliforms. Supplementation of plate count agar with pyruvate and (or) catalase enhanced detection of total heterotrophs. These findings suggest that addition of catalase or pyruvate to standard recovery media may improve detection of coliform and total heterotrophic bacteria in AMW-impacted waters.     

Solar inactivation of faecal bacteria in water : the critical role of oxygen

Suspensions of the faecal indicator bacteria Escherichia coli and Enterococcus faecalis were incubated in full sunlight in plastic bottles containing either (i) air-equilibrated (oxygenated) water or (ii) anaerobic (deoxygenated) water. A rapid decrease in cfu ml⁻¹ was observed for actively growing and stationary phase cells of both types of faecal bacteria when illuminated under aerobic conditions, with Ent. faecalis showing the greater enhancement in the rate of inactivation in air-equilibrated water. The demonstration of an oxygen requirement for the inactivation of faecal bacteria in sunlight indicates that solar-based water disinfection systems are likely to require fully aerobic conditions in order to function effectively.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Efficiency of Various Growth Media in Recovering Oral Bacterial Flora from Human Dental Plaque

MM10 sucrose blood agar (MM10 SB agar), N(2)C agar, Schaedler agar (SH agar), and mitis salivarius agar (MS agar) were tested for their ability to recover human dental plaque flora by a continuous anaerobic procedure and by a conventional anaerobic method. MM10 SB agar yielded higher recovery of bacteria from plaque samples as determined by the enumeration of colony-forming units (CFU). The CFU on N(2)C agar, SH agar, and MS agar were lower than MM10 SB agar when the continuous anaerobic procedure was used. The superior performance of MM10 SB agar was much more apparent when used for the cultivation of dental plaque by the conventional anaerobic method. Under these conditions the counts were consistently higher on MM10 SB agar as compared to the other media tested. However, the differential counts of Streptococcus sanguis and S. mutans from carious plaque samples were in general comparable on all culture media. Deletion of blood from MM10 SB agar did not lower counts. The elimination of dithiothreitol from this medium resulted in a significantly lower recovery of bacteria from the plaque samples when cultured by the conventional anaerobic method. The storage of MM10 SB agar for varying periods of time aerobic conditions did not seem to affect its performance. These findings suggest that MM10 SB agar is an ideal culture medium for the isolation, nonselective enumeration, and differential counts of bacteria present in normal and disease-associated plaques.     

The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide

B olton , F.J. C oates D. & H utchinson , D.N. 1984. The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. Journal of Applied Bacteriology 56 , 151–157.
Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C. coli and nalidixic acid-resistant, therrnophilic campylobacter (NARTC) strains. All five campylobacter test strains showed > 5 log reduction in counts on media which had been stored in light and air. Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters. Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air. All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains. In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active. The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.     

Enumeration of Enterobacter cloacae after chloramine exposure.

Growth of Enterobacter cloacae on various media was compared after disinfection. This was done to examine the effects of monochloramine and chlorine on the enumeration of coliforms. The media used were TLY (nonselective; 5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, and 1.5% Bacto-Agar), m-T7 (selective; developed to recover injured coliforms), m-Endo (selective; contains sodium sulfite), TLYS (TLY with sodium sulfite), and m-T7S (m-T7 with sodium sulfite). Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae. However, sodium sulfite in TLYS and m-T7S did not significantly improve the detection of chlorine-treated E. cloacae, and m-Endo was the least effective medium for recovering chlorinated bacteria. Differences in recovery of chlorine- and chloramine-treated E. cloacae are consistent with mechanistic differences between the disinfectants.     

Enumeration of Enterobacter cloacae after chloramine exposure

Growth of Enterobacter cloacae on various media was compared after disinfection. This was done to examine the effects of monochloramine and chlorine on the enumeration of coliforms. The media used were TLY (nonselective; 5.5% tryptic soy broth, 0.3% yeast extract, 1.0% lactose, and 1.5% Bacto-Agar), m-T7 (selective; developed to recover injured coliforms), m-Endo (selective; contains sodium sulfite), TLYS (TLY with sodium sulfite), and m-T7S (m-T7 with sodium sulfite). Sodium sulfite in any medium improved the recovery of chloramine-treated E. cloacae. However, sodium sulfite in TLYS and m-T7S did not significantly improve the detection of chlorine-treated E. cloacae, and m-Endo was the least effective medium for recovering chlorinated bacteria. Differences in recovery of chlorine- and chloramine-treated E. cloacae are consistent with mechanistic differences between the disinfectants.     

Inhibitory activity of 2-nitropropanol against select food-borne pathogens in vitro

AIMS: To test the inhibitory activity of 2-nitro-1-propanol (2NPOH) against Salmonella Typhimurium, Escherichia coli O157:H7 and Enterococcus faecalis. METHODS AND RESULTS: Specific growth rates (h(-1)) of S. Typhimurium, E. coli O157:H7 and Ent. faecalis were determined during culture in tryptic soya broth (TSB) supplemented with 0-10 mm 2NPOH. Growth rates were inhibited by 2NPOH, with nearly complete inhibition observed with 10 mm. Studies with S. Typhimurium revealed that its survivability during culture in TSB containing 5 or 10 mm 2NPOH was lower (P < 0.05) under aerobic than anaerobic conditions. The survivability of Salmonella during anaerobic culture in TSB containing 2.5 mm 2NPOH was less at pH 5.6 than at pH 7.0 and 8.0. No Salmonella survived anaerobic incubation in TSB supplemented with 10 mm 2NPOH regardless of pH. When incubated in suspensions of freshly collected populations of ruminal and faecal bacteria, Salmonella concentrations were lower (P < 0.05) in suspensions containing 10 mm 2NPOH than in suspensions containing no 2NPOH. CONCLUSIONS: 2NPOH inhibited S. Typhimurium, E. coli O157:H7 and Ent. faecalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that 2NPOH may be a useful antimicrobial supplement to reduce carriage of certain food-borne pathogens in food animals.     

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with³²P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli , faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli /faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the mictrotiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli, faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli/faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the microtiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.