Enumeration of Escherichia coli and coliforms in surface water by multiple tube fermentation and membrane filter methods

The current investigation was carried out in order to compare directly the multiple tube fermentation method (MTF), using standard procedures (lactose broth, LB) and the Colilert reagent, with the membrane filter method (MF) using Les Endo agar (LEA), m-faecal coliform agar (mFCA) and chromogenic coliform agar (CCA), for recovery of coliforms and Escherichia coli in 80 surface water samples. Total coliforms were isolated from 100% of samples by all methodologies. Faecal coliforms/E. coli were detected in 100% of samples by MTF methods, but only in 75.5% by MF-mFCA and in 86.2% by MF-CCA. Even if MTF-LB counts were consistently higher, the Colilert reagent accurately determined total coliforms and E. coli levels within 24 h with no additional confirmatory tests. Therefore, it could be a powerful tool for rapidly assessing possible faecal contamination and a suitable alternative to the traditional MTF and MF techniques utilized for coliform detection.     

Evaluation of a rapid, defined substrate technology method for enumeration of total coliforms and Escherichia coli in chlorinated drinking water

A rapid, defined substrate technology method, commercially available as Colilert, simultaneously enumerates total coliforms and Escherichia coli in drinking water samples in 24 h without the need for confirmatory tests. The ability of this method to enumerate both total coliforms and E. coli in simulated chlorine-treated drinking water samples was compared with the standard UK method (minerals-modified glutamate most probable number) which requires up to 96 h to complete including confirmation. Statistical analysis by a nonparametric matched-pair test showed the Colilert method to be less efficient at detecting down to one E. coli in these samples compared to the standard UK method. No statistically significant difference between the two methods of enumeration for total coliforms was detected.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Comparison of culturable fecal coliforms and Escherichia coli enumeration in freshwaters

Fecal coliforms (FC) counts were compared with Escherichia coli counts in differently contaminated freshwater samples (n = 166). FC were enumerated by plate count on triphenyl 2,3,5-tetrazolium chloride Tergitol medium. Escherichia coli were enumerated by the most probable number microplate method based on the detection of glucuronidase activity. FC and E. coli counts were highly correlated; an average E. coli/FC ratio equal to 0.77 was found, meaning that on average, 77% of FC were E. coli. Knowing the E. coli/FC ratio allows us to convert the historical microbiological quality data expressed in FC counts into E. coli abundance and thus to compare with present and future monitoring data that are (or will be) based on E. coli enumeration.     

Evaluation of the MicroFoss system for enumeration of total viable count, Escherichia coli and coliforms in ground beef

This study was conducted to evaluate the performance of the MicroFoss system (Biosys, Ann Arbor, MI) for enumeration of total viable organisms, Escherichia coli and coliforms in ground beef. The system performance was compared to that of the USDA Bacteriological Analytical Method (BAM) reference culture methods. The correlation coefficients for the regression lines comparing the MicroFoss system detection times to the results of plate count methods for the total viable counts, coliform counts and the most probable number (MPN) method for E. coli were -0.95, -0.96 and -0.97, respectively. Tests comparing the reproducibility of data generated independently by two technicians on the same batch of samples showed no significant differences (P>0.05) in the MicroFoss detection times and culture results. The plate count methods for the total viable counts and coliform counts, and the MPN method for E. coli required 10, 11 and 22 times, respectively, the amount of time to complete tests compared to the length of time required to perform these tests using the MicroFoss system. The MicroFoss system produced reproducible data and provided a rapid and cost-efficient alternative method for enumeration of TVC, coliforms and E. coli in ground beef.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Quantitative determination of Escherichia coli from coliforms and faecal coliforms in sea water.

Escherichia coli concentration in sea water was determined by the MUG test after primary growth on membrane filters used to determine total coliforms or faecal coliforms. A good correlation (r = 0.86) was found between E. coli obtained from coliforms versus those from faecal coliforms. Verification procedures showed that all the MUG-positive colonies obtained on both media were E. coli. Evaluation of this data and the literature indicated that this technique for estimation of E. coli in sea water is a useful addition to laboratory procedures without generally increasing the time and the expense of the analysis of recreational water.     

Evaluation of coliforms as indicators of water quality in India.

The total and thermotolerant coliform counts in rural drinking water derived from ground water, piped supplies and surface water are reported for a number of areas in India. To evaluate these counts as indicators of recent faecal contamination the total coliforms and thermotolerant coliforms isolated have been identified. Thermotolerant Escherichia coli formed 11.7% of the total coliforms and 75.1% of the thermotolerant coliforms. Citrobacter sp. (20.4%) and Klebsiella sp. (50.9%) were the other common total coliforms isolated and, among the thermotolerant coliforms, Klebsiella sp. (16.4%) was the only other organism frequently encountered. The total coliform counts were significantly correlated with water temperature. The applicability in tropical areas of standards developed for temperate climates is discussed.     

Evaluation of colilert-marine water for detection of total coliforms and Escherichia coli in the marine environment.

A test that allows for early detection of fecally contaminated coastal water would enhance public health protection. Colilert-Marine Water (Colilert-MW; Environetics, Branford, Conn.) is a rapid 24-h test that has recently been developed to detect total coliforms and Escherichia coli in coastal water. We performed a premarketing evaluation of the Colilert-MW product, testing it in parallel with the multiple tube fermentation (MTF) method for 86 coastal water samples in southern California. Statistical analysis was performed by using paired t tests and linear regression. Bacterial isolates were evaluated by biochemical and genetic analysis. The results of this study showed a strong correlation between the traditional MTF and the Colilert-MW method for detection of total coliforms (r = 0.95) and E. coli (r = 0.89) in ocean water samples. Paired t-test results indicated that the Colilert-MW and MTF were equivalent in detecting E. coli and that the Colilert-MW may be more sensitive in the detection of total coliforms. We conclude that Colilert-MW would be a useful tool with which to monitor coastal beach water.     

EU Drinking Water Directive reference methods for enumeration of total coliforms and Escherichia coli compared with alternative methods

AIMS: The reference methods for enumeration of total coliforms and Escherichia coli as stated in the European Drinking Water Directive were compared with alternative methods. METHODS AND RESULTS: Laboratories used the reference method on Lactose TTC agar (LTTC), the Colilert/18 system, Laurysulphate Agar (LSA), Chromocult Coliform Agar and the E. coli Direct Plating (DP) method. They enumerated more total coliforms on LTTC than on LSA. CONCLUSIONS: LTTC is suitable for analysis of very clean water samples only, due to heavy background growth. Colilert/18 is a good alternative but it enumerates a broader group of total coliforms, resulting in higher counts. The DP method appeared to be the best choice for enumeration of E. coli because Colilert/18 produces lower counts and false-negative results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the limitations of the EU reference method on LTTC due to lack of selectivity and suggests alternative methods for the enumeration of total coliforms and E. coli.     

Evaluation of coliforms as indicators of water quality in India

P.W. RAMTEKE, J.W. BHATTACHARJEE, S.P. PATHAK AND N. KALRA. 1992. The total and thermotolerant coliform counts in rural drinking water derived from ground water, piped supplies and surface water are reported for a number of areas in India. To evaluate these counts as indicators of recent faecal contamination the total coliforms and thermotolerant coliforms isolated have been identified. Thermotolerant Escherichia coli formed 11.7% of the total coliforms and 75.1% of the thermotolerant coliforms. Citrobacter sp. (20.4%) and Klebsiella sp. (50.9%) were the other common total coliforms isolated and, among the thermotolerant coliforms, Klebsiella sp. (16.4%) was the only other organism frequently encountered. The total coliform counts were significantly correlated with water temperature. The applicability in tropical areas of standards developed for temperate climates is discussed.     

Microbiological Evaluation of Cold-Water Shrimp (Pandalus borealis)

A bacteriological survey of the Maine shrimp industry was conducted to investigate the conditions associated with the production of frozen, raw, peeled shrimp. In-plant samples and finished product units were collected from seven plants. The most probable number of Escherichia coli, coliforms, and coagulase-positive staphylococci, as well as aerobic plate counts (APC), were determined. Freshly harvested shrimp collected from fishing vessels had an APC geometric mean of 510/g, and E. coli, coliforms, and coagulase-positive staphylococci were absent. Subsequent storage and insanitary practices during processing increased the APC and introduced coliforms. However, the low air temperatures (18 to 45 F) in the plants and the large volumes of cold water (34 F) used during processing inhibited significant bacterial buildup in the finished product.     

Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms.

Recovery of total coliforms and Escherichia coli on a new membrane filtration (MF) medium was evaluated with 25 water samples from seven states. Testing of the new medium, m-ColiBlue24 broth, was conducted according to a U.S. Environmental Protection Agency protocol. For comparison, this same protocol was used to measure recovery of total coliforms and E. coli with two standard MF media, m-Endo broth and mTEC broth. E. coli recovery on the new medium was also compared to recovery on nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Comparison of specificity, sensitivity, false positive error, undetected target error, and overall agreement indicated E. coli recovery on m-ColiBlue24 was superior to recovery on mTEC for all five parameters. Recovery of total coliforms on the new medium was comparable to recovery on m-Endo.     

Enumeration of heterotrophs, fecal coliforms and Escherichia coli in water: comparison of 3M Petrifilm plates with standard plating procedures

A total of 177 naturally contaminated water samples were analyzed by membrane filtration according to the Standard Methods for the Examination of Water and Wastewater published by the American Public Health Association. Filters were incubated in parallel on mHPC-agar and 3M™ Petrifilm™ Aerobic Count Plates (Petrifilm™ AC plates) for heterotrophic counts. Fecal coliforms and Escherichia coli were enumerated on mFC-agar and 3M™ Petrifilm™ E. coli/Coliform Count Plates (Petrifilm™ EC plates). Typical colonies on each media type were confirmed following standard procedures. Heterotrophic counts were between 10³ and 10⁴ CFU/mL and the average log₁₀ counts obtained on Petrifilm™ AC plates were about two-fold lower than on mHPC-agar. Counts for fecal coliforms and E. coli were between 10² and 10³ CFU/mL. Average log₁₀ counts for confirmed fecal coliforms obtained on Petrifilm™ EC plates were slightly lower than on mFC agar with a correlation coefficient of 0.949. The average log₁₀ counts for confirmed E. coli on Petrifilm™ EC plates and on mFC agar were statistically not different (P=0.126) with a correlation coefficient of 0.879. Specificity of Petrifilm™ EC plates and mFC agar was evaluated by comparing typical colony counts with confirmed counts. On mFC agar, counts for typical colonies were by 2 log₁₀ CFU higher than the actual confirmed counts. In contrast, on Petrifilm™ EC plates typical colony counts were almost identical to confirmed colony counts for both fecal coliforms and E. coli. This comparison illustrates the high specificity of Petrifilm™ EC plates for enumeration of both fecal coliforms and E. coli in water.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental Protection Agency-approved membrane filter method.

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.     

Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction.

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10³/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa ) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5–27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

Holding effects on coliform enumeration in drinking water samples

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