Technique for Testing Drinking Water

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1982, p /82/ $02.00/0 Vol. 44, No. 2 Evaluation of Factors Affecting the Membrane Filter Technique for Testing Drinking Water S. C. HSU AND T. J. WILLIAMS* Bureau of Disease Control and Laboratory Services, Michigan Department of Public Health, Lansing, Michigan Received 18 November 1981/Accepted 26 April 1982 The following studies were done in response to questions regarding the adoption and use of the membrane filter (MF) technique for testing drinking water for the total coliform indicator group. A comparison with the most-probablenumber technique showed that MF procedures with m-endo agar LES were somewhat superior to the most-probable-number methods in terms of numbers of coliform organisms recovered. Medium preparation and storage studies indicated that rehydration of m-endo agar LES should be done with boiling water for less than 15 min, that m-endo agar LES should not be exposed to light for more than 4 to 6 h, and that m-endo agar LES plates may be used for up to 4 weeks and broth verification media for up to 3 weeks under given storage conditions. MF culture colonies were commonly found which did not produce sheen as expected for coliforms and yet were verified as coliforms. The occurrence and morphology of these atypical colonies were studied. Parallel inoculation of both lauryl tryptose (LT) and brilliant green bile (BGB) broth was found to be a better colony verification approach than recommended LT preenrichment before transfer to BGB. Comparison of parallel verification results indicated very little justification for the use of LT medium in MF verification procedures. In the case of overgrown or confluent cultures, the best coliform recoveries resulted from swabbing the MF plate and directly inoculating BGB medium with the swab. The occurrence of overgrowth was defined and evidence was collected suggesting that overgrowth is a function of sample holding time. Evaluation of routine test data and bacterial population reductions as a function of time indicated that nonquantitative recovery of coliforms may not be significantly affected for at least a 72-h sample holding time. The membrane filter (MF) technique, which is now familiar to all environmental and sanitary microbiologists, has been fully accepted as a procedure for testing drinking water for the total coliform indicator group and is an approved method of analysis under the federal Safe Drinking Water Act regulations (1, 7). However, widespread use of the MF technique is relatively new compared with the long history of most-probable-number (MPN) methods for indicating drinking water safety. The Michigan Department of Public Health adopted the MF technique for all drinking water analysis during Because of continuing concern over the validity of the technique, various aspects of the procedure have been studied and evaluated. Before adopting the MF technique as a routine test procedure, extensive comparisons of MF and MPN procedures were done. Because the MF technique occasionally gave overgrown cultures which potentially interfere with coliform growth, new standard techniques for dealing with overgrown cultures were needed. Verification studies of many types of MF colony growth revealed that a substantial portion of nonsheen colonies gave positive brilliant green bile (BGB) broth fermentation results. Since this defines the MPN-confirmed coliform indicator group but contradicts usual MF colony interpretation, a change in verification procedures was indicated. Preliminary experience with parallel verification results with both lauryl tryptose (LT) and BGB media also suggested further study of currently recommended verification procedures (1, 7). Concerns over MF Endo medium stability are implied in federal laboratory certification guidelines (1). For this reason, aspects of preparation and storage of media were systematically studied. Finally, the undefined effects on test results of sample holding times before testing continue to be a significant problem for centralized water supply-monitoring programs, and additional data were collected in this regard. 453 MATERIALS AND METHODS Routine MF and MPN culturing. MF culturing was done as described previously (1). All procedures used

2 454 HSU AND WILLIAMS for preparing and incubating cultures were certified by federal inspection. An agar medium, m-endo agar LES, was selected for routine use based on findings by McCarthy and Delaney (3) and McCarthy et al. (4). Comparison of fecal coliform culturing with m-fc broth (1) with and without agar also suggested that an agar-based medium yields more rapid growth and increased recovery. For routine test procedures, 100- ml portions of sample were measured and filtered with cylindrical funnel assemblies graduated at 50 and 100 ml. Filters were then rinsed with sterile phosphatebuffered dilution water. Cultures were incubated on screens over water in a closed container for 18 to 24 h at 35 C. MPN testing was also done as described previously (1) by the five-tube (10-ml sample) procedure and with LT broth as the initial enrichment medium. Routine confirmation of LT cultures giving gas at 24 or 48 h was done by transfer to BGB broth fermentation tubes. Gas production in BGB within 48 h at 35 C was considered a positive result, with the MPN index determined as shown previously (1). Verification of MF colonies. Portions of MF colonies were transferred with sterile hardwood applicator sticks to LT or BGB fermentation tubes. For routine testing, transfer was made from each colony verified to both LT medium and BGB medium at the end of MF incubation. Organisms producing gas in LT medium within 48 h at 35 C, but not in BGB medium, were again transferred from the positive LT culture to fresh BGB medium. Only organisms producing gas in BGB cultures within 48 h of incubation for the first or second transfer were reported as coliforms. MF colony counts were adjusted according to the percentage of selected MF colonies which gave positive BGB results. MF colonies were selected for verification by the following criteria. All colonies producing sheen were candidates for verification. During analyst training, examples of all types of nonsheen colonies were given verification testing. Experience gained during this training provided the judgement needed to recognize "atypical noncoliform" colonies and "atypical" colonies which are likely to give positive verification. Atypical colonies may be described as nucleated, having a milky appearance, or producing a marked darkening of the medium below the colony; however, the training in recognition needs to be based on actual testing experience. A maximum of five sheen or atypical colonies was verified for routine test cultures. Sheen colonies were selected if present. If not, any colonies judged to be "atypical" were verified. Overgrowth verification. MF cultures were considered as overgrown when isolated colonies appeared to cover better than half of the filter area or when confluent growth occurred. Semiquantitative results were obtained with individual streaks of material from across the overgrown culture. These were treated as described for verification of MF colonies, with MF colony count reported as greater than the number of positive streak transfers. More commonly, the entire area of the culture was lightly brushed with a sterile cotton-tipped swab which was used to inoculate verification media. In this case, overgrowth results were stated only as positive or negative for coliforms. Inoculation and incubation of LT or BGB cultures were otherwise done as described for single MF colony verification. APPL. ENVIRON. MICROBIOL. MF and MPN comparative studies. During the routine processing of water samples, approximately 10 to 20 samples were randomly selected for comparison once or twice a week for several weeks. These samples were shaken; then a 50-ml portion was tested by MPN procedures, and 50 ml of the remaining sample was filtered for MF culturing since the total sample size of 120 ml did not permit testing of the standard 100-ml sample used for routine MF testing. Results by the two procedures were compared. Comparison work was done by three different analysts at two different laboratories. In addition, low levels (ca. 10 organisms per 100 ml) of a known Escherichia coli strain were inoculated into 100 samples of buffered dilution water, and these were tested by both MF and MPN procedures. Medium preparation and storage studies. Systematic studies of the following factors affecting medium performance were done. (i) Because of the heat sensitivity of m-endo agar LES, the effects of heating by two different methods for different lengths of time were studied. Rehydration of m-endo agar LES (Difco Laboratories, Detroit, Mich.) was done by placing the medium container on a hot plate or in a boiling water bath. After the medium reached the boiling temperature, portions were withdrawn at 5 min and at subsequent 10-min intervals up to 60 min and used to prepare MF plates. Membrane filters were placed on the solid medium and allowed to become moist. Inoculation of the filter surface was then done by lightly touching it with the tip of an inoculation needle. Four different coliform strains known to produce sheen were used for inoculation. After standard incubation the quality of sheen was observed for each preparation condition. (ii) For studying the effect of light exposure, prepared MF plates were exposed before use to ambient laboratory light for various periods of time up to 72 h. Laboratory light consisted of both fluorescent lighting and indirect sunlight. Exposed plates were inoculated and incubated as described for the heating studies. After incubation, sheen production for exposed plates was compared with that for unexposed plates from the same lot of medium. (iii) m-endo agar LES degradation with time was studied by storage of lots of medium for up to 5 weeks. The same lot of dehydrated m-endo agar LES was used for all medium preparations. Agar plates were prepared with loose-fitting petri dishes (60 by 15 mm) and stored in lots of 200 dishes in a cardboard box sealed in a plastic bag. Stored media were refrigerated at 5 C. For each period of storage, plates were inoculated and incubated as for the heating studies and compared with freshly prepared medium. (iv) When stored in the dark, the main factor affecting verification broth medium is thought to be evaporation of water from the medium. This was studied by observing weight loss as a function of the time stored. Fermentation culture tubes were prepared with metal closure caps, plastic closures, and plastic closures on tubes sealed in plastic bags. The average weight loss of 10 tubes from each type of preparation was determined as a function of the storage time. Routine testing statistics. Data resulting from routine testing activities were compiled for various periods of time. Except for additional recordkeeping, all other sample processing and testing were done according to

3 VOL. 44, 1982 TABLE 1. Comparison of routine total coliform testing results for drinking water samples by the MF and MPN techniques' No. (% of total)b of samples that indicated: Samples from MPN and MF MPN MF analyst agreement preferencec preferencec A 545 (94.6) 6 (1.0) 25 (4.4) B 333 (91.2) 5 (1.4) 27 (7.4) C 385 (95.1) 4 (1.0) 16 (3.9) Overall 1,263 (93.8) 15 (1.1) 68 (5.1) a Comparison used 50-ml samples and direct m- Endo, agar LES culturing. b The total numbers of samples for analysts A, B, and C and overall were 576, 365, 405, and 1,346, respectively. c Preferred method gave higher recovery or was positive when the other procedure was negative. standard written procedures which were not changed for the period of data compilation. Samples involved in these studies originated from the eastern half of Michigan's lower peninsula. Sample sources were categorized as municipal treatment plant taps, distribution systems of municipal supplies, private water supplies (usually single-family dwellings), and public swimming pools. Public water supplies and swimming pools were routinely sampled as part of periodic monitoring. Private water supplies were sampled on request, and the group sampled may have contained more than the average number of suspect installations. Reported studies are described as follows. (i) The occurrence of overgrown cultures were followed for summer (1 August to 31 August 1975) and winter (6 November to 10 December 1975) periods. Positive and negative overgrowth verification results were also recorded. This study involved a total of 6,215 samples from all sources. (ii) Statistics for parallel verification results were compiled for April to July Total specimens from all sources for this period were 15,654, with 2,114 verification tests done. LT and BGB broth fermentation results were recorded for the initial parallel inoculations. False-negative results, as would be interpreted for direct BGB inoculation only, were recorded for initial LT(+) and BGB(-) results, where subsequent LT to BGB transfer gave a second BGB(+) result. False negatives, as for direct LT inoculation only, were recorded for initial LT(-) and BGB(+) results. (iii) Statistics for municipal distribution, private supply, and swimming pool samples were kept for calendar year For each sample in these categories a record was retained of the verified coliform result, whether or not overgrowth occurred, and whether the sample holding time was 0, 1, 2, or >2 days from date of collection to date of testing. This study included about 26,000 samples with a minimum of 359 samples in each holding time category. Population changes for coliform strains. Various strains of organisms giving positive BGB coliform verification results were inoculated into large volumes of phosphate-buffered dilution water used for MF rinsing. These were then apportioned to 120-mI sterile containers stored together at room temperature, which MF TECHNIQUE FOR TESTING DRINKING WATER 455 were individually tested by routine MF procedures at different times. Populations immediately after inoculation ranged from several hundred to about 2,000 organisms per 100 ml. The timing of population changes was begun for all strains when periodic counts were first found to be less than 100/100 ml. Ten naturally occurring strains were tested in this manner. These included four E. coli and two Enterobacter aerogenes strains as indicated by the Standard Methods for the Examination of Water and Wastewater (1) indole, methyl red, Voges-Proskauer, citrate testing. These six strains produced normal sheen upon MF culturing. Four atypical strains which did not produce MF sheen but gave positive BGB verification tests were also tested. RESULTS Comparisons of MF and MPN testing results are shown in Table 1. The methods were assumed to be in agreement when the MF count fell within the span covered by the MPN index (i.e., from the midpoints between the next highest and next lowest MPN index from the measured index). Only a few samples gave MF counts within the quantitation range covered by the standard five-tube index, 2.2 to 16/100 ml. Samples were considered to be out of agreement whenever one method gave a positive result and the other gave a negative result, regardless of the counts. Where samples did not agree, a preferred method was selected as the one which gave the best coliform recovery. The comparison of methods for low levels of E. coli in buffered dilution water is shown in Table 2. This comparison shows consistently higher values by the MPN method, although exactly the same number of positives was obtained by either method. This difference may reflect the difference in the nature of the values (i.e., direct count versus MPN statistical estimate) determined by the two methods. MPN procedures have a statistical bias to the high side and show considerably less precision than direct plate counts (5, 6). Results of studies of medium preparation and storage are shown in Table 3 and Fig. 1. The effect on sheen production of different heating TABLE 2. Comparison of routine MF and MPN testing techniques for cultures inoculated with E. coli MF count % of MF MPN % of MPN per 100 ml samplesa index per samplesm < a A total of 200 samples was tested for each technique.

4 456 HSU AND WILLIAMS TABLE 3. Times required for change in MF culture sheen formation for two methods of heating during rehydration of m-endo agar LES medium Time (min) to first Time (min) to sheen changea Iossb Strain Direct Boiling Direct Boiling heat water heat water A B >60 C 50 >60 >60 >60 D >60 a First time that a reduction in the amount of sheen produced was noted. b First time that the complete absence of sheen formation was observed. procedures during m-endo agar LES rehydration is shown in Table 3. Light exposure studies showed an adverse effect of light on m-endo agar LES medium sheen production. A reduction in the amount of sheen produced was first observed at 4 h of exposure time. In some cases, sheen was no longer produced after a 16-h exposure of medium to ambient lighting. Storage of m-endo agar LES plates in plastic bags under refrigeration had very little effect on colony sheen formation. Some diminished sheen reduction seemed apparent after 5 weeks of storage, but no differences could be discerned for media stored for 0 to 4 weeks. Rates of weight loss or dehydration of verification broth cultures under different storage conditions are illustrated in Fig. 1. The occurrence of MF culture overgrowth is shown in Table 4. The percentages of total values show the relative proportions of cultures with and without overgrowth for each sample type. The percentages of positive values are the percentages of cultures giving verified coliform recovery within each of the 10 sample categories. Statistics for verification studies are shown in Table 5. The first two columns of numerical values result from isolated-colony verification, while the values for overgrown cultures are based on overgrowth verification procedures. Statistics regarding LT and BGB agreement are based only on the initial verification inoculation results. False-negative statistics assume verification procedures which involve initial inoculation to only one of the media (i.e., either LT or BGB). Table 6 shows the holding times for various types of samples. The calculated percentage values shown in Fig. 2 and 3 are based on the total sample groupings for each category given in Table 6. Sample holding times were determined from dates of sample collection. Data for sample holding times for synthetically prepared APPL. ENVIRON. MICROBIOL. samples are shown in Table 7. Timing of counts is in multiples of 48 ± 3 h. DISCUSSION These studies involve various aspects of the MF technique for drinking water analysis. The following discussion is divided into major areas in which the studies provided pertinent information. Some unconventional terminology is used which has specific meaning as defined in previous sections. These terms include typical, atypical and typical noncoliform MF colonies, MF overgrowth, and direct BGB verification. It will also be noted that primary emphasis has been placed on positive versus negative results rather than on actual MF colony count values, because generally agreed upon Michigan water supply protection policy calls for investigation and correction of water supply deficiencies whenever MF counts are greater than or equal to 1/100 ml. This policy also includes all state monitoring of public water supplies required by state and federal law. MF technique preference. The standard MPN test with BGB confirmation for the coliform indicator group has a long history of providing increased public health protection. For this reason, introduction of the MF technique was usually done by demonstrating equivalence between MF techniques and standard MPN methods. The MF technique is more precise as a directcount method and more sensitive in being able to analyze larger sample volumes and gives results more rapidly. However, it does not necessarily define the same total group of organisms includ- Storage Time (Days) FIG. 1. Average percent weight loss curves for LT and BGB broth fermentation culture tubes (18 by 150 mm) containing an initial average 9.35 ml of medium stored at room temperature. Curves include culture tubes with (A) metal closures only, (B) plastic closures only, and (C) plastic closures with batches of tubes sealed in plastic bags.

5 VOL. 44, 1982 TABLE 4. MF TECHNIQUE FOR TESTING DRINKING WATER 457 Relative occurrence of overgrown MF cultures Sample type Total no. of Cultures not overgrown' Overgrown cultures" samples % of total % Positive % of total % Positive' Municipal treatment (766) 3.0 (23) 5.7 (46) 15.2 (7) plants Municipal water 1, (1,005) 3.7 (37) 9.1 (100) 13.9 (14) distribution systems Private water supplies 3, (3,002) 12.0 (360) 15.5 (551) 33.9 (187) Swimming pools (608) 3.9 (24) 18.4 (137) 10.2 (14) Overall 6, (5,381) 8.3 (444) 13.4 (834) 26.6 (222) a Number of samples is given in parenthesis. b Swabs of overgrown cultures producing positive verification results when used to inoculate BGB broth fermentation tubes. ed under MPN testing, and any MPN preenrichment benefits in recovery of attenuated organisms are lost. Because of fundamental differences between direct MF counts and MPN statistical estimates, comparison of the two methods for coliform recovery is not straightforward. Comparison of test values for synthetic E. coli samples (Table 2) illustrates the problem. The MF and MPN values appear to be similar and certainly agree with respect to the number of positive and negative results. However, 92% of the MF counts are included in a fairly precise range of 2 to 10/100 ml, while the range of MPN indices is not defined, with more that 30% of the values being >16/100 ml. It is not known whether this is because the MPN procedure is more sensitive or simply because of the statistical bias and lack of precision inherent in the MPN method. The comparison of the two methods for routine sample analysis (Table 1) was done with an arbitrary definition of agreement. The limits used are probably well within the combined deviation for the two methods, but, even so, substantial agreement was observed (overall, 93.8% of the samples tested). According to given criteria regarding recovery, where the methods disagreed, the MF method was preferred in five of six such instances. These results were in excellent agreement among different analysts and with those of McCarthy et al. (4) who used m-endo agar LES and a preenrichment step. Using the same agreement and preference criteria, their work on 654 samples gave 607 (92.8%) in agreement, 4 (0.6%) MPN preferred, and 43 (6.6%) MF preferred. Because of the agreement between the two studies, we concluded that the enrichment step was not worthwhile. This conclusion has been reinforced by recent work of Evans et al. (2) who also used m-endo agar LES for initial MF culturing and found little benefit from preenrichment culturing. MF culture preparations. In preparing m-endo agar LES plates, it was found that the medium is indeed heat and light sensitive. Both heat and light cause rapid darkening of the medium; however, the immediate effect on MF colony sheen production was not substantial. Based on reported studies (Table 3 and other observations), heating with boiling water during medium rehydration appears to be an advantage, and such heating should not exceed 15 min. Also, exposure of prepared media to laboratory lighting should not exceed 4 h. Both limits can be readily met in standard preparation procedures, and any excess heat and light should be avoided. TABLE 5. Statistics for parallel routine MF verification procedures using LT and BGB broth fermentation No. of: Verification study Typical Atypical Overgrown colonies colonies cultures LT and BGB agreeing' ,996 LT(+) and BGB(-)' LT(-) and BGB(+)' False negatives by direct BGB inoculationb False negatives by initial LT inoculation onlyb a From results of initial parallel inoculation to both LT and BGB verification media. b False negatives as compared to initial parallel inoculation to both LT and BGB media with subsequent transfer for LT(+) and BGB(-) results from LT to BGB for secondary confirmation.

6 458 HSU AND WILLIAMS TABLE 6. Number of samples tested by Michigan Department of Public Health Lansing Laboratory at various sample holding times No. of samples from the following Holding time sources:' (days) Municipal Private Swimming distribution supplies pools ,511 7,385 1, ,117 4, >2 1,268 2, Unknownb 121 1, a Data include all samples for 1976 collected from municipal water supply distribution systems, private water supplies, and public swimming pools. The total numbers of samples from these sources were 6,394, 16,559, and 3,217, respectively. b Collection date not given in information submitted with sample. Some economy and certainly convenience can be achieved by preparation of large batches of culturing units. This in turn requires adequate long-term storage. Although 1-week outdating of a medium used in MF testing is recommended (7), our medium storage study observations suggest this to be overly cautious. Very little change in sheen production for natural coliform strains was noted for m-endo agar LES medium refrigerated in sealed plastic bags for up to 5 weeks. BGB and LT verification media may also be used for longer than 1 week if plastic closures are used to limit medium dehydration. Based on dehydration rates shown in Fig. 1 and an allowed 2% volume error in medium preparation, broth medium fermentation tubes with plastic A B C > > >2 Sample Holding Time (Days) FIG. 2. Percentage of test cultures showing overgrowth for different sample holding times between sample collection and testing. Zero days corresponds to same-day sample collection and testing. Different sample sources include (A) municipal water supply distribution systems, (B) private water supplies, and (C) public swimming pools. Each percentage calculation includes 330 to 7,380 total specimens APPL. ENVIRON. MICROBIOL ' '3 Sample Holding Time (Days) FIG. 3. Percentage of samples giving positive routine coliform verification tests for different sample holding times between sample collection and testing. Zero days corresponds to same-day sample collection and testing. Darkened areas represent positive MF cultures not showing overgrowth, while crosshatched areas represent additional coliform recovery from overgrown MF cultures. Different sample sources include (A) municipal water supply distribution systems, (B) private water supplies, and (C) public swimming pools. Each percentage calculation includes 330 to 7,380 total specimens. closures may be stored at room temperature for at least 2 weeks. Sealing of tubes in plastic bags extends storage time to about 3 weeks under the conservative 2% volume change criteria. MF isolated-colony evaluation. For reasons previously discussed, it is important that coliforms recovered by the MF technique include those strains previously recovered by the traditional MPN methods. These coliforms have traditionally been defined by the completed MPN test as described in Standard Methods for the Examination of Water and Wastewater (1). Isolated MF colonies giving positive BGB verification tests come closer to the completed MPN definition than do confirmed MPN results. In fact, if MF culturing provides true isolation, verification of colonies then fulfills all aspects of coliform definition except Gram staining. For this work, only when selected colonies gave positive BGB verification tests were cultures reported as positive for coliforms. This convention is also supported by average 10% falsepositive verification statistics for typical sheen MF colonies. Given this verification requirement, the judgement used in selecting colonies for verification becomes critical. As previously discussed, such judgement can come only with experience in colony verification. Records have been kept for rates of positive verification for classes of colony growth previously described. Overall average monthly positive verification rates for the period January 1977 to October 1979 were 90% for

7 VOL. 44, 1982 MF TECHNIQUE FOR TESTING DRINKING WATER 459 TABLE 7. MF counts per 100 ml of sample at various sample holding times' MF counts per 100 ml for: Time E. coli strainsb E. aerogenes Atypical strainsd (days) strains" a MF coliform counts as a function of storage time at room temperature for isolated environmental strains inoculated to buffered phosphate dilution water. b Identified as E. coli strains by indole, methyl red, Voges-Proskauer, citrate testing. c Identified as E. aerogenes strains by indole, methyl red, Voges-Proskauer, citrate testing. d Atypical strains not producing typical MF sheen cultures but giving positive BGB broth verification test results. e, Not done. typical sheen colonies and 30% for atypical nonsheen colonies. The range of monthly verification rates was 77 to 97% for sheen colonies and 19 to 54% for atypical colonies. Table 5 data indicate a proportion of 237 typical to 852 atypical cultures for 15,654 specimens. Using the lower 19% rate for atypical colonies and the upper 97% rate for typical colonies, 162 positive atypical cultures and 240 positive typical sheen cultures would be predicted. Clearly, the number of nonsheen atypical colonies conforming to the MPN definition of coliforms is substantial relative to the number of typical sheen colonies observed. If the MPN coliform definition is to be retained, MF colony interpretation should go beyond simple sheen or no sheen considerations in selecting colonies for verification. MF colony verification procedures. Routine use of parallel inoculation of suspect MF growth to both LT and BGB broth fermentation cultures for verification has indicated that changes in verification procedures are advisable. Data shown in Table 5 are typical of several years' experience. The need for an enrichment with LT medium is questionable, and these data indicate very little benefit from this step. Total agreement was found for typical sheen colonies for LT enrichment and direct BGB verification (eliminating initial LT enrichment). For atypical colonies, eight specimens (<1%) gave positive LT results which were confirmed with secondary transfer to BGB medium, while direct BGB verification was negative. On the other hand, 19 specimens gave positive direct BGB verification but would have been reported as negative with a preliminary LT enrichment step as is currently recommended in federally approved methodology. If a single medium for verification were to be selected, BGB would be preferred as recovering the greatest number of coliforms. With respect to additional cost, the benefit of preliminary LT enrichment is negligible. The advantage in recovery is less than that of direct BGB verification, and what advantage there is amounts to only 0.7% of samples verified. Similar conclusions were reached by Evans et al. (2) using direct verification inoculation to another selective medium, m-endo-based (m-lac) broth. However, direct BGB verification perhaps corresponds more closely to past definition of the coliform group, and this approach uses an additional gram-negative selective agent not employed in the MF culturing. MF overgrowth. The occurrence of MF culture overgrowth during routine testing has proved to be a substantial problem ranging from about 5 to 20% of specimens submitted from various sample sources (Table 4). Recovery of coliforms [BGB(+) by definition] from overgrowth is best done by direct inoculation of overgrowth to BGB medium. As indicated in Table 5, direct BGB verification, relative to an initial LT enrichment step, increases recovery from overgrown cultures by 7% of total specimens. The meaning of overgrowth, where direct BGB verification is negative, has not been defined. A number of factors may result in regrowth of organisms in a water system or in a sample container which could cause MF overgrowth but would not necessarily have sanitary significance. Bacterial populations of <5/ml

8 460 HSU AND WILLIAMS could result in overgrowth if a large proportion of the population were capable of growth on MF media. Taking these factors into account, it still seems advisable to consider overgrowth as an indicator of potential water supply problems based on the following considerations. (i) Some overgrown organisms (particularly pseudomonads) may be antagonistic to coliform growth. (ii) A comparison study of standard plate counts with overgrowth occurrence for 500 routine water supply samples showed 75% of overgrown MF cultures also gave plate counts of >2,000/ml (unpublished work). (iii) As indicated by direct BGB verification, coliform recovery from overgrown cultures is about three times more frequent than for cultures without overgrowth (Table 4). Sample holding time effects. Although there are no known correlations between holding times and water supply construction or operational aspects which could affect testing results, relatively large numbers of samples (Table 6) were included in sample holding time statistics in hopes that the size of the sampling would tend to average out any such correlation within each holding time group. Nearly all samples included in this data were transported to the laboratory without special preservation or temperature control. Figure 2 illustrates some definite trends in overgrowth occurrence with respect to increasing sample holding time. There were consistent substantial increases in percent overgrowth occurrence for each longer holding time. This trend was similar for each type of sample source shown, and very few exceptions to this type of trend were found in individual monthly statistics for the 12 months included in the total group. Overall, the percentage of drinking water specimens showing overgrowth increased an average of about 5% of total samples for each 24 h of sample holding time. A more rapid increase is apparent for chlorine-neutralized swimming pool samples. Obviously, any interpretation of the sanitary significance of an overgrowth test result must also consider the sample holding time. MF counts are expected to change with increasing sample holding times, and federal guidelines (7) recommend that holding times be limited to less than 30 h. In fact, for any individual sample, counts may be continually increasing or decreasing at rates dependent on a com- APPL. ENVIRON. MICROBIOL. plex combination of factors including temperature, ph, chemical content, and coliform strains present. Although it indicates that testing should be done as soon as possible, this expected behavior does not provide a basis for any specific sample holding time limit. Preliminary work on coliform population decreases in minimal nutrient media at near neutral ph indicates that the time to complete die-off (i.e., MF count of <1/100 ml) for strains investigated is more a matter of days than hours. Changes in counts with time shown in Table 7 illustrate this point and suggest that cyclical dieoff and regrowth patterns may occur over periods of several days for some members of the coliform group. The percentage of positive test results for routine testing did not exhibit regular increases or decreases with increasing sample holding times. Figure 3 shows a lack of any consistent trends for up to 72 h of sample holding time. The overall percentages of positive samples for all holding times remain relatively constant for each sample source included: municipal distribution, %; private water supplies, %; and swimming pools, %. For monitoring programs where investigation and corrective action are prompted by any positive MF count per 100 ml of sample, these data do not support any specific limit. For this type of response to any positive test results, the preceding data imply that the coliform indicator system is generally effective to at least 72 h of sample holding time. LITERATURE CITED 1. American Public Health Association Standard methods for the examination of water and wastewater. American Public Health Association, Inc., New York. 2. Evans, T. M., R. J. Seidler, and M. W. LeChevallier Impact of verification media and resuscitation on accuracy of the membrane filter total coliform enumeration technique. Appl. Environ. Microbiol. 41: McCarthy, J. A., and J. E. Delaney Membrane filter studies. Water Sewage Works 105: McCarthy, J. A., J. E. Delaney, and R. J. Grasso Measuring coliforms in water. Water Sewage Works 108: McCarthy, J. A., H. A. Thomas, Jr., and J. E. Delaney Evaluation of the reliability of coliform density tests. Am. J. Public Health 48: Thomas, H. A., Jr Statistical analysis of coliform data. Sewage Ind. Wastes 27: U.S. Environmental Protection Agency Manual for the interim certification of laboratories involved in analyzing public drinking water supplies. Environmental Protection Agency publication no. 600/ Office of Monitoring and Technical Support, U.S. Environmental Protection Agency, Washington, D.C.