Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.     

Membrane filter method for recovery of fecal coliforms in chlorinated sewage effluents.

The standard one-step M-FC broth-membrane-filter procedure for recovery of fecal coliforms from chlorinated sewage effluents is much less effective than the multiple-tube (most-probable-number) technique. A two-step membrane-filter method, using a pre-enrichment technique with phenol red lactose broth and incubation at 35 degrees C for 4 h, followether 18+/-2 h, enhanced fecal coliform recovery from chlorinated effluents. The results of 126 comparisons using chlorinated effluents from five wastewater plants showed that fecal coliform recovery by using the two-step membrane-filter method is comparable to that using the multiple-tube procedure.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

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.     

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.     

Membrane filter technique for the quantification of stressed fecal coliforms in the aquatic environment.

A two-layer membrane filtration (MF) medium (injury-mitigating MF [IM-MF]) and a procedure for the enumeration of injured fecal coliforms are described. These procedures included the addition of glycerol and acetate plus reducing agents to both layers of a two-layer medium and rinsing of the filter with a rich resuscitation medium. Some changes in incubation time and temperatures were used. This method was compared with the multiple-tube fermentation most-probable-number procedure and the one-step M-FC agar-membrane filter method (direct M-FC) in terms of fecal coliform recovery from various aquatic environments that cause bacterial injury. With chlorinated sewage effluents, results of the IM-MF technique were equal to or greater than the most probable number in 9 of 18 trials and were 1.3 to 19 times greater than the M-FC method. When sewage samples were chlorinated in the laboratory, fecal coliform counts with IM-MF equaled or exceeded the most probable number in 7 of 15 trials and always exceeded the M-FC. M-FC was exceeded by IM-MF in 30 of 33 trials with clean mountain stream water. Fecal coliform bacteria that were exposed to low levels of an iodophore in the laboratory produced IM-MF counts 3 to 10 times greater than those with M-FC. A biochemical rationale for the formation of the IM-MF medium is discussed.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

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.     

Isolation of fecal coliforms from pristine sites in a tropical rain forest

Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.     

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.     

Isolation of fecal coliforms from pristine sites in a tropical rain forest.

Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.     

Evaluation of millipore HA and HC membrane filters for the enumeration of indicator bacteria.

Millipore type HA and HC membrane filters were compared for recoveries of total coliforms, fecal coliforms and fecal streptococci from natural waters and wastewater effluents. Bacterial and fecal coliform recovery was better on HC than on HA filters.     

Comparison of bacterial indicators and sampling programs for drinking water systems

A total of 48 French villages were selected for a survey of water quality from February 1983 to June 1984 as part of an epidemiological study conducted in the rural area of the Rh?ne-Alpes region. Water samples were collected and analyzed on a weekly basis in each village. Bacteriological analysis of each water sample included enumeration of standard plate count bacteria, total and thermotolerant coliforms, and fecal streptococci. The water quality regulations are examined as to the analysis frequency, the volume of samples, and the relationship between the various bacterial indicators. Analyzing 300-ml, instead of 100-ml, samples tends to generate better information on single water samples. However, if many samples are analyzed over time from the same community, the value of using large volumes of water samples is diminished. The comparisons between bacterial indicators showed that the information obtained from the various indicators was very similar. However, fecal streptococci had a better predictive value of a negative test than coliforms with respect to the French standards.     

Comparison of bacterial indicators and sampling programs for drinking water systems.

A total of 48 French villages were selected for a survey of water quality from February 1983 to June 1984 as part of an epidemiological study conducted in the rural area of the Rhône-Alpes region. Water samples were collected and analyzed on a weekly basis in each village. Bacteriological analysis of each water sample included enumeration of standard plate count bacteria, total and thermotolerant coliforms, and fecal streptococci. The water quality regulations are examined as to the analysis frequency, the volume of samples, and the relationship between the various bacterial indicators. Analyzing 300-ml, instead of 100-ml, samples tends to generate better information on single water samples. However, if many samples are analyzed over time from the same community, the value of using large volumes of water samples is diminished. The comparisons between bacterial indicators showed that the information obtained from the various indicators was very similar. However, fecal streptococci had a better predictive value of a negative test than coliforms with respect to the French standards.     

Anaerobic incubation of membrane filter cultures for improved detection of fecal coliforms from recreational waters.

Anaerobic incubation of membrane filter cultures significantly enhanced detection of fecal coliforms in surface-water samples from recreational beaches. In contrast to standard aerobic incubation, anaerobic incubation suppressed overgrowth of masking, noncoliform bacteria but did not increase the frequency of fecal coliform recovery.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.