Evaluation of the 7-h membrane filter test for quantitation of fecal coliforms in water.

The 7-h fecal coliform (FC) test for detection of FC organisms in water was evaluated to establish its validity and usefulness for emergency and disaster situations. The waters tested consisted of routine samples collected for public health surveillance and enforcement purposes. A total of 984 water samples from throughout California were assayed. These included samples from coastal salt waters, rivers, canals, and reservoirs, in addition to potable and miscellaneous freshwater sources. A portion of each sample was tested concurrently by both the 7-h FC test and the most-probable-number FC five-tube test. The 7-h FC test samples were incubated for 7 to 7.25 h at 41.5 degrees C. Overall, greater than 90% agreement was obtained between the methods in determining whether the water quality was acceptable or unacceptable. Statistical analysis of the 984 samples confirmed that the 7-h FC method was a suitable alternative to the most-probable-number FC method for evaluation of freshwater samples. During emergencies or disasters, the 7-h FC test could provide a means for detection of fecal contamination of water with results available in less than 1 day.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters.

Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.     

Enchanced accuracy of coliform testing in seawater by a modification of the most-probable-number method.

A 1-year study of marine water sample from six beach locations showed that the most-probable-number method failed to recover significant numbers of coli-forms. Modifying this method by transferring, after 48 h, presumptive negatives (growth and no gas production) to confirmed and fecal coliform media significantly improved recovery. Tests which were presumptive negative but confirmed as fecal coliform positive were designated as false negatives. Most-probable-number method false negatives occurred throughout the year, with 143 of 270 samples collected producing false negatives. More than 50% of fecal coliform false-negative isolates were Escherichia coli. Inclusion of false-negative tubes into the coliform most-probable-number method data resulted in increased violation of the California ocean water contact sports standard at all sites. More than 20% of the samples collected were in violation of this standard. These data indicate that modification of the most-probable-number method increases detection of coliform numbers in the marine environment.     

Enchanced accuracy of coliform testing in seawater by a modification of the most-probable-number method.

A 1-year study of marine water sample from six beach locations showed that the most-probable-number method failed to recover significant numbers of coli-forms. Modifying this method by transferring, after 48 h, presumptive negatives (growth and no gas production) to confirmed and fecal coliform media significantly improved recovery. Tests which were presumptive negative but confirmed as fecal coliform positive were designated as false negatives. Most-probable-number method false negatives occurred throughout the year, with 143 of 270 samples collected producing false negatives. More than 50% of fecal coliform false-negative isolates were Escherichia coli. Inclusion of false-negative tubes into the coliform most-probable-number method data resulted in increased violation of the California ocean water contact sports standard at all sites. More than 20% of the samples collected were in violation of this standard. These data indicate that modification of the most-probable-number method increases detection of coliform numbers in the marine environment.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water.

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Comparison of methods of enumerating coliforms after UV disinfection.

In view of the differences that have been found between the most-probable-number and membrane filtration methods for the recovery of coliforms from chlorinated samples, the survival of total and fecal coliforms in UV-irradiated effluent samples, as tested by the most-probable-number and standard single-step membrane filtration methods, was compared. There were no significant differences in the survival of total and fecal coliforms, as tested by the two methods. In a separate set of experiments comparing total and fecal coliform survival, as tested by the most-probable-number method, only a very small but statistically significant difference of 0.1 log survival units was found. For UV-disinfected wastewater effluents, standard one-step membrane filtration procedures are comparable to standard most-probable-number procedures.     

Detection of fecal coliforms in water by using [14C]mannitol

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Detection of fecal coliforms in water by using [14C]mannitol.

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Improved Membrane Filter Method for Fecal Coliform Analysis

A two-layer agar method has been developed which consistently yields higher recovery of fecal coliforms on membrane filters when compared to the existing membrane fecal coliform procedure. This method has been evaluated by three laboratories using samples of raw and chlorinated waste water, and reservoir, river, and marine waters. Verification of 1,013 fecal coliform colonies isolated from 61 water samples averaged 92% on this proposed procedure. Comparison with the Standard Methods membrane fecal coliform procedure revealed the two-layer agar method had an overall increased sensitivity to fecal coliform detection in these waters. It is therefore proposed that this procedure be evaluated as an alternative to the Standard Methods fecal coliform membrane Filter test in the examination of chlorinated secondary effluents, marine waters, and any natural waters that may contain pollutants with heavy metal ions.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Evaluation of a rapid method for the quantitative estimation of coliforms in meat by impedimetric procedures.

A 24-h instrumental procedure is described for the quantitative estimation of coliforms in ground meat. The method is simple and rapid, and it requires but a single sample dilution and four replicates. The data are recorded automatically and can be used to estimate coliforms in the range of 100 to 10,000 organisms per g. The procedure is an impedance detection time (IDT) method using a new medium, tested against 131 stock cultures, that markedly enhances the impedance response of gram-negative organisms, and it is selective for coliforms. Seventy samples of ground beef were analyzed for coliforms by the IDT method and the conventional three-dilution, two-step most-probable-number test tube procedure. Seventy-nine percent of the impedimetric estimates fell within the 95% confidence limits of the most-probable-number values. This corresponds to the criteria used to evaluate other coliform tests, with the added advantage of a single dilution and more rapid results.     

Effect of sunlight on enumeration of indicator bacteria under field conditions.

The effect of sunlight on the enumeration of fecal coliform (FC) and fecal streptococcal (FS) bacteria when water samples are collected in containers and brought back to the laboratory for analysis or when the water samples are filtered through membranes on site was determined. FC and FS in raw sewage stored in clear glass or translucent polyethylene containers were resistant to the effects of sunlight. However, under the same conditions of storage and exposure to sunlight, 90% of FC and FS in sewage diluted 1:100 in seawater were inactivated within 13 to 32 min. When sewage was similarly diluted in stream water and exposed to sunlight, 90% of FC were inactivated after 28 to 38 min, whereas 90% of FS were not inactivated even after a 2-h exposure to sunlight. Other experiments showed that 90 to 99% of FC and FS retained on membranes were inactivated when these membranes were exposed to sunlight for 10 to 15 min. FS were inherently more resistant to sunlight inactivation than were FC. Finally, evidence was obtained to show that sunlight initially stresses the bacteria but eventually causes cell death.     

Evaluation of a Most-Probable-Number Technique for the Enumeration of Pseudomonas aeruginosa

A most-probable-number (MPN) technique was evaluated for detecting and enumerating Pseudomonas aeruginosa in water and wastewater. Both the presumptive and confirmatory media, as described in the 13th edition of Standard Methods for the Examination of Water and Wastewater, as well as modifications of these media were included in evaluations. Various samples of water were tested, namely chlorinated tap water, creek water, and influent to a wastewater treatment plant. Modified media repeatedly gave higher estimated MPNs of P. aeruginosa than media listed in Standard Methods. P. aeruginosa was detected and recovered from all creek water and wastewater samples, but not from tap water samples tested. This organism was determined to be present in as large numbers as the fecal coliforms and in even greater quantities than the fecal streptococci in all samples, whenever MPN estimations were determined from those positive tubes containing the modified confirmatory medium.     

Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.