Modification of delayed-incubation procedure for detection of fecal coliforms in water.

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Elimination of overgrowth in delayed-incubation membrane filter test for total coliforms by m-ST holding medium

Two holding media were compared for their effects on total coliform recovery by the delayed-incubation membrane filter procedure. LES-MF holding medium contains tryptone, m-Endo broth, dipotassium hydrogen phosphate, sodium benzoate, sulfanilamide, para-aminobenzoic acid, and cycloheximide (pH 7.0). m-ST holding medium contains ethanol, sodium monophosphate, dipotassium hydrogen phosphate, sulfanilamide, and Tris (pH 8.6). In tests with natural water and wastewater samples from various sources, recovery with LES-MF and m-ST were similar after a 1-day holding period. With LES-MF, however, after a 2- or 3-day holding period, coliform bacteria frequently were partially or totally overgrown by noncoliforms, causing significant reductions in coliform counts. No significant overgrowth was observed with m-ST. We propose that m-ST be used for all holding periods longer than 1 day.     

Elimination of overgrowth in delayed-incubation membrane filter test for total coliforms by m-ST holding medium.

Two holding media were compared for their effects on total coliform recovery by the delayed-incubation membrane filter procedure. LES-MF holding medium contains tryptone, m-Endo broth, dipotassium hydrogen phosphate, sodium benzoate, sulfanilamide, para-aminobenzoic acid, and cycloheximide (pH 7.0). m-ST holding medium contains ethanol, sodium monophosphate, dipotassium hydrogen phosphate, sulfanilamide, and Tris (pH 8.6). In tests with natural water and wastewater samples from various sources, recovery with LES-MF and m-ST were similar after a 1-day holding period. With LES-MF, however, after a 2- or 3-day holding period, coliform bacteria frequently were partially or totally overgrown by noncoliforms, causing significant reductions in coliform counts. No significant overgrowth was observed with m-ST. We propose that m-ST be used for all holding periods longer than 1 day.     

Delayed-Incubation Membrane-Filter Test for Fecal Coliforms

A delayed-incubation membrane-filter technique for fecal coliforms was developed and compared with the immediate fecal coliform test described in Standard Methods for the Examination of Water and Wastewater (13th ed., 1971). Laboratory and field evaluations demonstrated that the delayed-incubation test, with the use of the proposed vitamin-free Casitone holding medium, produces fecal coliform counts which very closely approximate those from the immediate test, regardless of the source or type of fresh-water sample. Limited testing indicated that the method is not as effective when used with saline waters. The delayed-incubation membrane-filter test will be especially useful in survey monitoring or emergency situations when the standard immediate fecal coliform test cannot be performed at or near the sample site or when time and temperature limitations for water sample storage cannot be met. The procedure can also be used for analyzing the bacterial quality of water or waste discharges by a standardized procedure in a central examining laboratory remote from the sample source.     

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.     

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.     

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.     

Rapid Determination of the Presence of Enteric Bacteria in Water

A rapid and sensitive method is described for the detection of bacteria in water and various other natural substrates by the isolation of specific bacteriophage. By the addition of large numbers of the organism in question to the sample, the presence of virulent bacteriophage can be demonstrated in as little as 6 to 8 h. Fecal coliform, total coliform, and total coliphage counts were determined for over 150 water samples from several geographical areas over a period of 2 years. Computer analysis of the data shows a high degree of correlation between fecal coliforms and the coliphage present in the samples. With a high correlation coefficient between fecal coliform and coliphage counts, predictions of the fecal coliforms may be made by enumeration of the phage.     

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.     

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.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

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.     

Gas Chromatographic Presumptive Test for Coliform Bacteria in Water

A gas chromatographic procedure which shows promise as a presumptive test for coliform bacteria in water is described. Total coliform bacteria concentrations were determined from the incubation times at 37 C required for ethanol to be produced. Fecal coliform densities were determined in a similar manner at 44.5 C. The culture medium was filter sterilized M-9 salts supplemented with 1% lactose, 0.1% Casamino Acids, and 0.1% yeast extract. Best results were obtained when the initial total coliform concentrations were 5 per ml or higher and when fecal coliform concentrations were 50 per ml or higher. Minimum detection times at these concentrations were 9 and 12 h, respectively.     

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.     

Optimum Membrane Structures for Growth of Coliform and Fecal Coliform Organisms

The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 μm and a retention pore size of 0.7 μm.     

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.     

Modification of M-FC medium by eliminating rosolic acid.

Eliminating rosolic acid from M-FC medium improves the MFC procedure by allowing higher fecal coliform colony recoveries with greater ease in counting. Samples of unchlorinated and chlorinated domestic sewage, creek, lake, and river water were analyzed for fecal coliforms by standard procedures. Results of 200 comparisons of fecal coliform counts on M-FC medium without and with rosolic acid showed that higher counts were obtained 71% of the time when rosolic acid was excluded without an overgrowth of background colonies. Results from analyzing chlorinated sewage showed that eliminating rosolic acid improved the recovery of fecal coliform bacteria by 49%. A total of 1,675 blue colonies and 766 nonblue colonies were verified. Of the 1,675 blue colonies, 1,566 were confirmed as fecal coliform bacteria, for a verification of 93.5%. The percent verification of nonblue colonies as noncoliform bacteria was 84.2% (644/766).     

Evaluation of porcelain cup soil water samplers for bacteriological sampling

The validity of obtaining soil water for fecal coliform analyses by porcelain cup soil water samplers was examined. Numbers from samples of manure slurry drawn through porcelain cups were reduced 100- to 10,000,000-fold compared to numbers obtained from the external manure slurry, and 65% of the cups yielded coliform-free samples. Fecal coliforms adsorbed to cups apparently were released, thus influencing the counts of subsequent samples. Fecal coliforms persisted in soil water samplers buried in soil and thus could significantly influence the coliform counts of water samples obtained a month later. These studies indicate that porcelain cup soil water samplers do not yield valid water samples for fecal coliform analyses.