Detection of Total Coliforms, Escherichia coli, and

APPLED AND ENVRONMENTAL MCROBOLOGY, Feb. 1993, p. 380-388 0099-2240/93/020380-09$02.00/0 Copyright 1993, American Society for Microbiology Vol. 59, No. 2 Evaluation of Commercial Presence-Absence Test Kits for Detection of Total Coliforms, Escherichia coli, and Other ndicator Bacteria JAMES A. CLARK'* AND ABDUL H. EL-SHAARAW2 Laboratory Services Branch, Water Quality Section, 125 Resources Road, Etobicoke, Ontario M9P 3V6,1 and National Water Research nstitute, Burlington, Ontario L7R 4A6,2 Canada Received 29 July 1992/Accepted 12 November 1992 Evaluations of several commercial presence-absence () test kits were performed over a 6-month period in 1990 by using the Ontario Ministry of the Environment (MOE) test for comparison. The general principles of the multiple-tube fermentation technique formed the basis for conducting the product evaluations. Each week, a surface water sample was diluted and inoculated into 25 99-ml dilution blanks for each of three dilutions. The inoculated dilution blanks from each dilution series were randomly sorted into sets of five. Three of these sets were inoculated into the test kits or vice versa, as required. The other two sets were passed through membrane filters, and one set of five membrane filters was placed onto m-endo agar LES to give replicate total coliform counts and the other set was placed onto m-tec agar to give replicate fecal coliform results. A statistical analysis of the results was performed by a modified logistic transform method, which provided an improved way to compare binary data obtained from the different test kits. The comparative test results showed that three of the four commercial products tested gave very good levels of recovery and that the fourth commercial product gave only fair levels of recovery when the data were compared with the data from MOE tests and membrane filter tests. bottles showing positive results after 18 h of incubation that were subcultured immediately in ECMUG tubes frequently could be confirmed as containing total coliforms, fecal coliforms, or Escherichia coli after 6 h of incubation; thus, the total incubation time was only 24 h. The presence of anaerogenic coliforms and Aeromonas spp. and presumptive positive occurrences were also recorded for consideration as possible indicators of deteriorating water quality. A limited number of split-sample analyses were performed with drinking water samples for two of the commercial test kits; the results showed that the level of indicator organism recovery was equivalent to that of the MOE test. Examination of bacteriological water samples to determine whether the quality of the water is acceptable for drinking and other domestic purposes has traditionally been done by most-probable-number (MPN) procedures or the membrane filter (MF) technique (1). nterest in presenceabsence () methods for determining the microbiological quality of drinking water has increased dramatically in the last 10 years. This methodology became officially available on 31 December 1990, when the Total Coliform Rule, promulgated by the U.S. Environmental Protection Agency, became effective (10). The new regulation changed the manner of reporting total coliforms from numbers per 100 ml to the presence or absence of total coliforms in 100 ml of sample. Prior to the promulgation of the new regulation, several studies were done to compare the test with the MF technique and the MPN procedure (otherwise known as the multiple-tube fermentation method). These studies essentially demonstrated that the test was equivalent to or better than the MF and MPN techniques for detecting total coliform organisms (2, 12, 15, 16). About the same time, an alternative test procedure for detecting total coliforms and Escherichia coli was being investigated (8, 9). One part of the test involved the substrate o-nitrophenyl-p-d-galactopyranoside; when this substrate was acted upon by an enzyme produced by total coliform bacteria, it released o-nitrophenyl, giving a yellow color to the broth medium. The other part of the test involved the * Corresponding author. 380 substrate 4-methylumbelliferyl-o-D-glucuronide. When this compound was acted on by the enzyme 13-glucuronidase produced by E. coli, a bright blue fluorescent color appeared in the broth medium when the preparation was viewed in the dark with long-wavelength UV light (11). The test systems were first combined in a commercial product known as Colilert, which was marketed by Access Analytical Systems. A similar product, known as Coliquik, was developed and marketed by the Hach Company. Several comparative studies were performed both in the United States and in Canada to determine whether the Colilert test detected total coliforms and E. coli as well as the Standard Methodsfor the Examination of Water and Wastewater MF, MPN, and procedures do (5, 8). Although equivalent results were not always obtained, the authors of the studies generally concluded that the Colilert test was a satisfactory alternative to the other Standard Methods tests used for detection of total coliforms. Other studies, in which both the Colilert and Coliquik tests were compared with the Standard Methods MF procedures used for detection of total coliforms and fecal coliforms, generally revealed good agreement among the tests used for detection of total coliforms, but the authors of these studies found that the two commercial tests were inferior to the Standard Methods fecal coliform procedure when colonies growing on mfc medium were identified as E. coli (3, 14). n this study we compared the Ministry of the Environment (MOE) test with the Colilert and Coliquik tests, as well as the total coliform and fecal coliform MF tests. n addition, as the Hach Company has also produced two other

VOL. 59, 1993 less expensive test kits (the Hach Disposable test kit and the Hach Vial or Tube test kit), both of these test kits were included in the comparative evaluation. The latter test kits were similar to the MOE test and contained a lactosebased medium supplemented with bromocresol purple to indicate lactose fermentation by coliform bacteria; the media in these test kits also contained the 4-methylumbelliferyl-,B- D-glucuronide reagent to determine the presence of 3-glucuronidase from E. coli. MATERALS AND METHODS test kits. The four commercial test kits mentioned above were purchased for analysis of 100-ml samples and were evaluated over a 6-month period starting at the end of May 1990. The Colilert and Coliquik test reagents were in powder form in screw-cap glass tubes and plastic pillows, respectively. Each reagent was added to a 100-ml sample, which was shaken to dissolve the powder. Each Hach Disposable test kit consisted of a plastic bottle containing about 50 ml of triple-strength medium to which 100 ml of a sample was added. Each Hach Vial test kit consisted of a small plastic vial that held about 25 ml of concentrated medium, which was added to 100 ml of a sample; the preparation was then shaken to mix the contents. Because of certain time and space limitations, only two commercial test kits could be compared with the MOE test and the MF methods used for detection of total coliforms and fecal coliforms at any one time. Four groups of test evaluations were done in the Central (Toronto) Laboratory, Etobicoke, Ontario, Canada, and one group of test evaluations was done in the London Regional Laboratory, London, Ontario, Canada. Test protocol. Although the test was intended primarily for examination of samples obtained from distribution systems, these types of samples rarely produce positive test results except on certain unpredictable occasions. This attribute makes distribution systems poor sources for samples when new tests or media must be evaluated in a short period of time. For this reason, a surface water sample was used as a source of indicator organisms. A sample for the Toronto laboratory was usually collected each Monday from a local river, the Humber River, in a sterile 300-ml plastic bottle containing sodium thiosulfate. This sample was used for analyses on the day of collection and then was refrigerated and used the following day for another set of analyses. Samples for the London Regional Laboratory were collected weekly from various locations that were polluted by sewage or food wastes. The general principles of the multiple-tube fermentation technique were used to dilute out to extinction the indicator organisms in each sample (1). Each week, three appropriate decimal dilutions of the surface water sample were prepared, and 1-ml volumes of each dilution were inoculated into 25 99-ml sterile buffered dilution blanks, as shown in Fig. 1. Each dilution blank then became a 100-ml sample for inoculation into one of the test kits or filtration through an MF. The inoculated dilution blanks or samples from each 25-bottle dilution series were randomly sorted into sets of five. One set of five samples was inoculated into five bottles used for one of the test kits; the second and third sets were treated similarly for the other two test kits. The fourth set of five 100-ml samples was passed through five MFs, each of which was placed onto m-endo agar LES (Difco); the fifth set was treated in a similar manner, and the membranes were placed onto m-tec agar (Difco). COMMERCAL TEST KTS 381 Surface water sample 10 ml lml 0 90ml 0 4-99ml dilution dilution imil ml blank lml blank o 0 0 25 bottles 99 m dlton 1 in 10 1 in 1 00 bilankson blanks dilution ~ ~ dilution ~~~~~ o o 0 Pick sets of 5 bottles at random from each dilution series and rearrange in sets of 5 as indicated 0 0 000 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0 00 00 0 00 00 00 0 PAPA PAMFMF PAPA PAMFMF PAPA PAMFMF 1 2 3 TC FC 1 2 3 TC FC 1 2 3 TCFC FG. 1. Diagramatic representation of the dilution procedure used for inoculation of 100-ml volumes into test kits and through MFs. TC, total coliforms; FC, fecal coliforms. The whole procedure was repeated for the other two dilution series, so that each test kit and MF parameter consisted of five bottles or filters that were inoculated with 100-ml volumes of a sample over three decimal dilutions. Each of the operations described above was randomized as much as possible. The m-endo agar LES plates were incubated at 350C for 20 to 22 h (1), and the m-tec agar plates were incubated at 44.50C for 20 to 22 h (7). Any red colony with a metallic sheen on m-endo agar LES was considered a total coliform colony, and any yellow or yellow-brown colony on m-tec agar was considered a fecal coliform colony. The test bottles were incubated at 350C and were examined over a 3-day period for presumptive positive tests (4).- MF test confirmation. Total coliform and fecal coliform counts were determined to establish the relative number of target organisms that were initially inoculated into each dilution blank, but the plates were also scored on the basis of the presence or absence of the target organisms. No colony confirmation procedure was performed with the initial set of comparative tests, but later any plates containing only one or two colonies had their colonies removed for confirmation in ECMUG broth; the preparations were incubated at either 35 or 44.50C depending on the original isolation medium. EGMUG broth contained 37 g of EC medium (Difco) per liter plus 0.075 g of 4-methylumbelliferyl-o-D-glucuronide reagent (Hach) per liter. Following incubation, ECMUG tubes exhibiting growth with or without gas were checked for fluorescence. However, because limited numbers of fecal coliform colonies were isolated in ECMUG broth for E. coli confirmation, this parameter was not included in the statistical comparison of MF and results. test confirmation. test bottles were incubated at 350C and examined over a 3-day period for presumptive

382 CLARK AND EL-SHAARAW Positive ECMUG tube 350C 44.50C Turbidity Gas Gas and Turbidity Gas Gas and only positive fluorescent only positive fluorescent positive positive Total Escherichia ±Fecal i coliform 11 coli coliform 1 Streak MacConkey Streak nutrient gelatin agar for isolated agar for isolated colonies colonies l l l Red or pink Colourless Gelatin and Gelatin positive lactose positive lactose negative oxidase or negative colonies colonies positive and oxidase l negative Anaerogenic Aeromonas Presumptive coliform sp. result? FG. 2. Procedure and interpretation of results for confirmation tests. positive results; the bottles which showed presumptive positive results were then subjected to confirmation tests (4). The bottles were examined as soon as 18 to 21 h after inoculation, but they were also examined at 24, 28, 48, and 72 h, depending on the test kit being evaluated. nocula (0.05 ml) from each bottle showing evidence of a positive result were transferred into two tubes containing ECMUG broth; one of these tubes was incubated at 35 C, and the other was incubated at 44.5 C. The ECMUG broth tubes inoculated with material from positive 18- to 21-h bottles were read after 6, 24, and 48 h of incubation. All other tubes were read after 24 and 48 h. The indicator groups distinguished by the confirmatory tests included total coliforms, fecal coliforms, E. coli, Aeromonas spp., and anaerogenic coliforms. Figure 2 shows how we interpreted the ECMUG broth tube results. Gas production at 35 C was scored as a positive result for total coliforms; gas production at 44.5 C was scored as a positive fecal coliform result. Gas production was indicated by the presence of effervescence at 6 h and gas accumulation in the small inverted tubes after 24 to 48 h. UV fluorescence at either temperature was considered a positive result for E. coli on the basis of the results of a previous in-house study; in this study the researchers used Enterotubes (Hoffman-LaRoche Ltd., Vaudreuil, Quebec, Canada), and E. coli was isolated and identified from 97% of 36 samples which produced gas and fluorescence in ECMUG broth at either 35 or 44.5 C or at both 35 and 44.5 C. Tubes exhibiting only turbidity after 48 h of incubation were subcultured onto MacConkey and nutrient gelatin agar plates to determine the presence of anaerogenic coliforms or aeromonad types of organisms. With some bottles, appropriate identification of the bacteria was not possible, and these bottles were labelled presumptive. Statistical analysis. The results of each of the four test kits were compared with the results of the MOE and MF tests for the total coliform and fecal coliform parameters only. The E. coli parameter was not included as fecal coliform colonies were not always identified following MF tests. Also, although results were determined for anaerogenic coliforms, Aeromonas spp., and presumptive tests, no statistical analysis was performed. These parameters were considered less important because of their lower frequency of occurrence. n other studies workers have used a variety of statistical techniques to compare the results of MF and tests (2, 3, 5, 8, 12, 14-16). n this study, the use of a statistical procedure which tested whether the proportion of positive samples from one set of bacteriological test kit results was constant for all of the bacteriological test kit results was considered more appropriate. The procedure used to provide an evaluation of the performance of the and MF tests was a procedure described by Cox (6). n 1970, Cox published a treatise on the analysis of binary data (i.e., data arranged in two classes, either present or absent). The format used in this study involved calculations of a modified logistic transform associated with the jth ( = 1, 2..., k) bacteriological test, that is, Zj = log {(rj + 0.5)/[(nj - rj) + 0.5]}, where rj is the number of positive samples and nj is the total number of samples tested. The addition of 0.5 to the numerator and denominator on the right side of the equation ensures that Zj is defined for all rj values, including rj = 0 and rj = nj. The data for each test group were arranged in a k x 2 contingency table, with rj and nj - r, being the elements of the jth row of such a table. As part of the calculation, the approximate variance of Zj was determined by using the following equation: Vj = (nj + 1) (nj + 2)/nj (rj + 1) [(nj - rj) + 11. Before the results of any two bacteriological tests were compared, an overall statistical test for homogeneity or for the equality of k bacteriological tests (k > 2) was made, so that k Z 21Vj - k :2 l/vj, and j = 1 where Z = ( Zj/Vj )/± 1/Vj. j=1 j= APPL. ENVRON. MCROBOL. The distribution of D is approximately a chi-square distribution with k - 1 degrees of freedom, and the chi-square table was used for determining significance. For k = 2, this test reduced to the case of comparing two of the bacteriological test kit results (say i andj), so that Dji = (Zj - Zi)21(V + vi). Since Dji has a chi-square distribution with a single degreeof freedom, the statistic Wii = (Zj - Zi)J/\Vj+Vi was computed for every pair (, i) of bacteriological tests, and the resulting data were compared with the quartiles of the standard normal distribution to determine the significance of paired comparisons of the various bacteriological tests for either total coliforms or fecal coliforms. As noted previously, MF results were determined on a basis for the purpose of applying the test statistics described above. Routine drinking water sample analysis. The Hach Vial test kit was chosen in 1991 for an additional study of drinking water samples obtained from municipal distribution systems. Each week, several sample submissions from various municipalities were chosen for parallel analyses. A 100-ml volume of a sample was analyzed by using the MOE test medium, and another 100 ml of sample from the same bottle was analyzed by using the Hach Vial test kit. The results for all of the samples that produced presumptive positive test results were confirmed by using the procedure described previously (4). The parallel analyses were performed in the Central (Toronto) Laboratory, the London Regional Laboratory, and the Thunder Bay Regional Laboratory. The Thunder Bay Regional Laboratory also had some Hach Disposable test bottles that were available to perform parallel analyses on some drinking water samples.

VOL. 59, 1993 COMMERCAL TEST KTS 383 TABLE 1. Comparison of the number of indicator bacterial groups isolated from test kits and in the MF test No. of the following indicator groups isolated: Homogeneity tests (D)a Test No. of Type of group tests test Total Fecal E. coli Anaerogenic Aeromonas Total Fecal coliforms coliforms coliforms spp Presumptve coliforms coliforms A 150 MOE 78 39 36 6 9 14.888b 12.060b Colilert 80 35 35 7 2 6 Coliquik 53 27 27 7 7 11 MF 83 53 c c c B 300 MOE 143 71 60 8 1 10 0.280 1.236 Hach 144 63 55 14 3 3 Disposable Hach 141 62 56 12 7 2 Vial MF 138 61 4 C 90 MOE 45 24 23 4 2 0.297 0.542 Colilert 43 21 19 4 10 Hach 42 20 20 3 1 Disposable MF 45 21 1 D 105 MOE 57 43 41 3 1.687 0.996 Colilert 49 40 41 4 6 Hach 56 37 36 3 1 2 Vial MF 57 37 E 150 MOE 112 84 94 3 1 5 36.924b 15.795b Colilert 112 70 74 6 1 8 Coliquik 76 51 60 8 19 14 MF 122 77 a See text for explanation. b Significantly different at P < 0.01. c _, additional tests were not done on MF colonies to distinguish these indicator groups. RESULTS test kit evaluation. The results of the comparative evaluation of the commercial test kits are shown in Table 1 along with the results of the homogeneity tests done on the recovery data obtained for total coliforms and fecal coliforms for each of the five evaluation studies. Although a five-tube, three-dilution procedure was used throughout to achieve extinction of the target organisms (i.e., few or no total coliforms in the highest dilution), the total number of MF or positive samples from each set was determined for comparative purposes; this was done instead of displaying the data in an MPN format. For statistical analysis, the proportion of one set of positive samples was compared with the proportion of another set of positive samples by using a modified logistic transform procedure. This procedure simplified the overall evaluation and was used for comparisons of levels of target organism recovery in both and MF tests. The first test comparisons performed were comparisons between the Colilert and Coliquik commercial tests and the MOE and MF tests. The results shown for test group A in Table 1 are the results obtained after inoculation of 150 test bottles for each test procedure or kit over a 10-week period. The MF test for total coliforms gave the highest level of recovery; 83 MFs had one or more sheen colonies present following filtration of the inoculated dilution blanks. The Colilert test produced 80 confirmed positive total coliform results from 95 bottles that gave yellow o-nitrophenyl-3-dgalactopyranoside reactions. The MOE test produced 78 confirmed positive total coliform results from 93 presumptive positive bottles. The Coliquik test (53 confirmed positive total coliform results from 78 presumptive positive bottles) gave a much lower level of recovery than the other test kits; its recovery rates were about 60% for total coliforms and 50% for fecal coliforms when the data were compared with the data for the MF technique. The fecal coliform results exhibited considerable variation; the MF test (53 positive results) gave a much higher level of recovery than the other test kits. Again, the level of recovery obtained with the Coliquik test (only 27 positive results) was much lower than the levels of recovery obtained with the other two test kits. When the first group of results was tested for homogeneity, significant D values were obtained for test group A (Table 1) for both total coliforms and fecal coliforms. When an additional analysis of the test group A results was done in a pair-wise manner (Table 2), the low total coliform recovery value obtained for the Coliquik test (53 positive results) was very significantly different (i.e., Wji statistic) from the other and MF test results. For the fecal coliform pair-wise comparison, the MF result (53 positive results) was very significantly different (P < 0.01) from the Coliquik result (27 positive results) and only significantly different (P < 0.05) from the Colilert result (35 positive results). Although the level of recovery of fecal coliforms in the MOE test (39 positive results) was only slightly higher than the levels of recovery obtained with the other two test kits, mathe-

384 CLARK AND EL-SHAARAW APPL. ENVRON. MCROBOL. Test No. of TABLE 2. Statistical analysis of the performance of the procedures Paired comparison value (W[i) for total coliform recovery with: Paired comparison value (Wji) for fecal coliform recovery with: group tests~ Testlecomparedi Hach Hach Col iet Coliquk Mtet Hach Hach Colilert Colteqstik MF test Disposable Vial MFtest qte test Disposable Vial tes test test ~tes test test test test A 150 MOE -0.230 2.886a -0.577 0.534 1.660-1.742 Colilert 3.110a -0.347-2.264b Coliquik _3.444a -3.338a B 300 MOE 0.408-0.082 0.163 0.903 0.783 0.883 Hach Disposable 0.490 0.245 0.201 0.100 Hach Vial 0.245 0.100 C 90 MOE 0.297 0.000 0.445 0.513 0.513 0.689 Colilert -0.297 0.148 0.000 0.177 Hach Disposable 0.445-0.177 D 105 MOE 1.098 0.000 0.138 0.421 0.848 0.848 Colilert -1.098-0.096 0.427 0.427 Hach Vial -0.137 0.000 E 150 MOE 0.000 4.227a -1.385 1.610 3.785a 0.807 Colilert 4.227a -1.385 2.222b -0.807 Coliquik -5.435a -3.009a a Significantly different at P < 0.01. b Significantly different at P < 0.05. matically it was not significantly different from the MF test result. n the second group of tests, we compared the Hach Disposable bottle and Hach Vial tests with the MOE and MF tests. The results for test group B (Table 1) revealed no significant differences among the tests for the total coliform recovery and among the tests for fecal coliform recovery when either the homogeneity tests or the pair-wise comparisons were used (Table 2). As the total coliform results obtained with the Hach Disposable and Hach Vial tests were minimally different from one another, each of these tests was compared with the Colilert test, which was clearly better than the Coliquik test (Table 1, test group A). n the third group of tests (Table 1, test group C), we compared the Colilert and Hach Disposable tests with the MOE and MF tests. No significant differences were observed among the tests in levels of total coliform or fecal coliform recovery. n the last series of tests done in the Toronto laboratory we compared the Colilert and Hach Vial tests with the MOE and MF tests. The results for test group D (Table 1) revealed no significant differences in levels of total or fecal coliform recovery. Table 1 also shows the results for test group E from a comparative evaluation done in the London laboratory. As we found for test group A (Table 1), the Coliquik test results revealed the lowest levels of recovery of total coliforms and fecal coliforms. The values obtained were significantly lower than the values obtained with the other or MF tests for test group E (Table 2). The higher level of recovery of E. coli than fecal coliforms in the London laboratory resulted from the presence of more fluorescence-positive ECMUG broth tubes at 35 C than at 44.5 C. This type of response was not observed as frequently in the Toronto laboratory and resulted in a large difference (20 E. coli confirmations) between the results for the MOE test and the results for the Colilert test in the London laboratory. ndicator group isolation frequencies. The isolation frequencies for each indicator group were determined (Table 3) at different initial response times (i.e., color change and/or effervescence in the appropriate medium within 18 to 28, 48, or 72 h). The fecal coliform and E. coli parameters had the shortest response times since the test kits that detected these parameters produced positive results usually 98 to 100% of the time with only 1 day of incubation (i.e., 18 to 28 h). The MOE test (96 to 98% positive results) was slightly slower, and the percentages of fecal coliforms and E. coli that were detected within 48 to 72 h were higher with this test than with the other test kits. For the Colilert and Coliquik tests the level of detection of total coliform isolates was 95% with response times of 18 to 28 h, whereas for the Hach Disposable and Hach Vial tests the average level of detection was 90% for the same period. The MOE test was slower, having a level of total coliform detection for the period from 18 to 28 h of 86%; a greater percentage of total coliform isolates was detected after 48 to 72 h. The Coliquik test detected anaerogenic coliforms, Aeromonas spp., and the presumptive group at higher percentages than the other test kits. The anaerogenic coliforms and Aeromonas spp. organisms were detected more frequently after 48 and 72 h than after 18 to 28 h with the Hach Disposable and Hach Vial tests. All test kits had a small proportion of their positive test results which could be categorized only as presumptive, and these test results occurred at all of the time periods (i.e., 18 to 28, 48, or 72 h). Although the fluorescent 4-methylumbelliferyl-p-D-glucuronide reaction was not an indicator organism parameter for isolation frequency, the results of this reaction were also recorded on the basis of response time for each of the test kits. The Colilert, MOE, and Hach Vial tests had the highest levels of fluorescent reactions (87 to 88%) in the time period from 18 to 28 h; the levels of response were lower in the Hach Disposable and Coliquik tests (68 to 78%). For each of the test kits fluorescent responses occurred at 48 and 72 h, but less than one-half of these responses were confirmed as E. coli responses when the preparations were transferred to ECMUG broth. Conversely, E. coli fluorescent reactions occurred in confirmatory ECMUG broth tubes which had been inoculated from presumptive test bottles in which no fluorescent reactions occurred during the initial incubation period in the test bottles. Response time of ECMUG broth tubes. The results of

VOL. 59, 1993 COMMERCAL TEST KTS 385 TABLE 3. solation frequencies and percentages of indicator bacteria determined according to the time required for an initial response in the test solation frequencies (%) at the following test kit Total no. of tests ndicator group response times: isolates No.ate 18 to 28 h 48 h 72 h MOE 795 Total coliforms 376 (86) 47 (11) 12 (3) 435 Fecal coliforms 255 (98) 6 (2) 261 E. coli 244 (96) 8 (3) 2 (1) 254 Anaerogenic coliforms 13 (62) 8 (38) 21 Aeromonas spp. 1 (50) 1 (50) 2 Presumptive 10 (34) 13 (45) 6 (21) 29 Fluorescent' 194 (87) 21 (9) 8 (4) 223 Colilert 495 Total coliforms 270 (95) 14 (5) 284 Fecal coliforms 164 (99) 2 (1) 166 E. coli 167 (99) 2 (1) 169 Anaerogenic coliforms 13 (62) 7 (33) 1 (5) 21 Aeromonas spp. 2 (67) 1 (33) 3 Presumptive 16 (55) 7 (24) 6 (21) 29 Fluorescenta 143 (88) 10 (6) 9 (6) 162 Coliquik 300 Total coliforms 123 (95) 4 (3) 2 (2) 129 Fecal coliforms 78 (100) 78 E. coli 85 (98) 2 (2) 87 Anaerogenic coliforms 11 (73) 4 (27) 15 Aeromonas spp. 22 (85) 4 (15) 26 Presumptive 13 (52) 9 (36) 3 (12) 25 Fluorescenta 51 (68) 18 (24) 6 (8) 75 Hach Disposable 390 Total coliforms 170 (91) 11 (6) 5 (3) 186 Fecal coliforms 83 (100) 83 E. coli 75 (100) 75 Anaerogenic coliforms 12 (70) 3 (18) 2 (12) 17 Aeromonas spp. 2 (67) 1 (33) 3 Presumptive 2 (50) 2 (50) 4 Fluorescenta 47 (78) 12 (20) 1 (2) 60 Hach Vial 405 Total coliforms 175 (89) 9 (4) 13 (7) 197 Fecal coliforms 99 (100) 99 E. coli 92 (100) 92 Anaerogenic coliforms 9 (60) 1 (7) 5 (33) 15 Aeromonas spp. 2 (25) 3 (38) 3 (37) 8 Presumptive 3 (75) 1 (25) 4 Fluorescenta 72 (87) 11 (13) 83 a Fluorescent reaction in bottle. inoculation of confirmatory ECMUG broth tubes (Fig. 2) with 0.05-ml inocula from positive bottles are shown in Table 4. The 6-h results were usually recorded between 3:30 and 4:30 p.m. and consisted of observations of growth, gas, or effervescence and fluorescence in ECMUG broth tubes that had been inoculated between 8:30 and 9:30 a.m. on the same day. Because equivalent numbers of confirmatory ECMUG broth tubes were not inoculated from each of the test kits, the results for the different time periods were converted to percentages of total positive tubes. The percentages determined at 6 and 24 h were related to the numbers of tubes that gave final responses of either gas TABLE 4. Results of confirmatory ECMUG broth tube tests at different times and with different incubation temperatures following inoculation from positive bottles Gas production Fluorescence ncubation at 35 C ncubation at 44.5 C ncubation at 35 C ncubation at 44.5 C %of tubes % oftubes ~~~% of tubes % of tubes test kit producing gas No. of tubes producng gas No. of tubes go. of tubes frescenc No. of tubes after: producing after: producing after: producing after: producing gas gas fluorescence fluorescence 6 24 48 6 24 48 6 24 48 6 24 48 h h h h h h h h h h h h MOE 61 93 100 435 72 97 100 261 82 94 100 247 71 96 100 243 Colilert 45 81 100 284 48 97 100 166 77 94 100 161 57 96 100 159 Coliquik 28 74 100 129 28 100 100 78 59 94 100 79 43 95 100 74 Hach Disposable 48 91 100 186 60 96 100 83 87 100 100 70 73 100 100 74 Hach Vial 58 91 100 197 68 99 100 99 82 97 100 89 68 99 100 90

386 CLARK AND EL-SHAARAW APPL. ENvRON. MCROBOL. TABLE 5. Comparison of the MOE test with the Hach Vial and Hach Disposable bottle tests for analysis of drinking water samples No. of the following indicator groups recovered: Laboratory No. of tests Type of test Total Fecal Anaerogenic Aeromonas coliforms coliforms. col coliforms spp. Presumptive Toronto 1602 MOE 38 8 7 3 5 11 Hach Vial 37 8 6 2 6 20 London 356 MOE 1 1 2 Hach Vial 1 2 1 29 Thunder Bay 150 MOE 3 2 3 Hach Vial 3 4 6 Thunder Bay 115 MOE 7 2 1 Hach Disposable 7 1 1 or fluorescence (or both) after 48 h at temperatures of 35 and 44.5 C, respectively. For example, Tables 3 and 4 show that of 795 MOE tests, 435 produced ECMUG broth tubes that were gas positive at 35 C and 247 produced tubes that produced fluorescence; when equivalent numbers of inoculated ECMUG broth tubes were incubated at 44.5 C, 261 tubes produced gas and 243 tubes produced fluorescence. nocula from MOE and Hach Vial bottles resulted in an average level of confirmed gas or effervescence responses of 60% with the ECMUG broth tubes within 6 h at 35 C (Table 4); inocula from Colilert and Disposable test bottles resulted in lower levels of confirmed responses (45 and 48%, respectively). At 44.5 C, the MOE, Hach Disposable, and Hach Vial test bottles gave levels of confirmed responses of more than 60%, but for the most part the results represented cultures that also responded at 35 C. nocula from positive Coliquik tests gave the lowest level of confirmed responses (28% for the 6-h period). After 24 h, the MOE, Hach Disposable, and Hach Vial tests produced confirmed test results in more than 90% of the EC medium tubes at 35 C, and the Colilert and Coliquik tests produced confirmed test results in 81 and 74% of the tubes, respectively. EC medium tubes incubated at 44.5 C produced confirmed positive results for fecal coliforms in more than 95% of the tubes by the end of the 24-h period for all of the test kits. The fluorescent responses shown in Table 4 appeared more quickly than the gas responses. More than 80% of the ECMUG broth tubes that were incubated at 35 C and inoculated from MOE, Hach Disposable, and Hach Vial test kits were positive after 6 h. A lower level of fluorescent response was observed with the Colilert and Coliquik tests, but even with these two tests, the fluorescent responses (77 and 59%, respectively) were better than the gas responses (45 and 28%, respectively). A comparison of the fluorescent responses at 35 C with the fluorescent responses at 44.5 C showed that more fluorescent tubes occurred after 6 h at 35 C. By 24 h, 94% or more of the tubes at both 35 and 44.5 C had become positive, and the remainder became fluorescent within 48 h. The numbers of fluorescent tubes at 35 and 44.5 C were quite similar to each other and to the numbers of tubes positive for gas production at 44.5 C, in contrast to the higher number of gas-positive tubes at 350C. Drinking water sample analyses. A total of 2,108 splitsample, analytical tests were done on municipal drinking water samples in the Toronto laboratory and two regional laboratories by using the Hach Vial test medium and the MOE test medium. The Thunder Bay Regional Laboratory also performed split-sample analyses by using a set of 115 Hach Disposable bottles along with the MOE test medium. The results of these analyses are shown in Table 5. No statistical analysis was performed on this data as the variation in parameter recovery levels for total coliforms, fecal coliforms, and E. coli was very minimal for all of the parallel test analyses. The largest variation occurred with the Hach Vial test, which produced more presumptive positive tests; however, none of the usual indicator groups was isolated or confirmed. This type of result was more prevalent in the London Regional Laboratory than in the other two laboratories. DSCUSSON The primary purpose of this study was to evaluate the various commercial test kits which have been marketed recently to detect the presence of total coliforms and E. coli. A secondary purpose of this study was to determine whether confirmed results for total coliforms, fecal coliforms, or E. coli could be obtained within 24 h by the MOE test procedure to make this test comparable to the Colilert and Coliquik test kits, which according to the manufacturers can detect total coliforms and E. coli within 24 h. Our experiments were performed over a period of several months. We compared two test kits at a time, and the Colilert, Coliquik, Hach Disposable, or Hach Vial test was compared with the routine MOE procedure and the total coliform MF test by using m-endo agar LES and with the fecal coliform test by using m-tec agar. The results of the first group of evaluations (Table 1, test group A) done in the Toronto laboratory showed that the Coliquik test kit was significantly lower than the other test procedures for recovering total coliforms (Table 2). A similar result was obtained for test group E when a series of test (Tables 1 and 2) was performed in the London laboratory. n the Toronto laboratory both the Colilert and Coliquik tests kits gave significantly lower values than the MF test for recovery of fecal coliforms, but in the London laboratory, only the Coliquik test gave significantly lower levels of fecal coliform recovery than the other test procedures. n both laboratories, the Coliquik test gave higher levels of presumptive positive unconfirmed tests and Aeromonas spp. recovery than the other test kits. Although the data are not shown in Table 1, certain trials with the Coliquik test kit produced results equivalent to the results obtained with other test procedures, but in other trials, the Coliquik test gave lower levels of recovery of total and fecal coliforms. Other studies have shown that the Coliquik test is equivalent to or better than the Colilert test (3, 14), but whether another lot number would have produced better levels of recovery in our laboratory was not determined. Because of its low level of recovery, the Coliquik test was not included in other test comparisons.

VOL. 59, 1993 The next group of evaluations (Table 1, test group B) involved comparisons between the Hach Disposable and Hach Vial test kits and the MOE and MF tests. No significant differences were observed between the tests for total coliform recovery or the tests for fecal coliform recovery. Consequently, in the third and fourth evaluation groups we compared the MOE and Colilert tests with the Hach Disposable test kits and then with the Hach Vial test kits (Table 1, test groups C and D, respectively). The levels of recovery for both the total coliform and fecal coliform parameters were quite similar for all of the and MF test comparisons. Overall, when the low Coliquik test results were excluded, no significant difference was found between the tests for total coliform recovery. A significant difference was found with the Colilert test for fecal coliform recovery, but only in test group A in the Toronto laboratory. As a result, the Hach Disposable, Hach Vial, and Colilert test kits had levels of recovery that for all practical purposes were essentially the same as the levels of recovery for the MOE test for the total coliform and fecal coliform parameters. Because provisions were not made early in the study for confirming that fecal coliform colonies on m-tec agar were E. coli colonies, a statistical comparison among the various test kits was not done. For the analyses performed in the Toronto laboratory, most of the differences among the test kits used for E. coli recovery were minimal; the exception was the Coliquik test kit. However, in the London laboratory large differences between both the Colilert and Coliquik test kits and the MOE test were observed. Workers in the London laboratory also reported a higher level of recovery of E. coli with all of the test kits as a result of the fact that more ECMUG broth tubes produced gas and fluorescence at 35 C than at 44.5 C. n fact, no growth occurred in most of the tubes incubated at 44.5 C, even though the parallel inoculated tubes incubated at 35 C gave good gas and fluorescent responses. This unexpected observation was not anticipated, and the organisms in the 35 C ECMUG broth tubes were not identified more specifically. As workers at the London laboratory collected their samples mainly from previously chlorinated sewage effluent water, we considered the possibility that injured E. coli cells which were not able to express their characteristic growth at 44.5 C were present in these samples. Other studies have shown that stressed organisms are not recovered efficiently on the usual growth media, such as m-endo agar LES, but require a less inhibitory medium, such as m-t7 (1). Whether differences in the MOE and Colilert tests were the result of differences in medium ingredients, the sample source, or inaccurate confirmation of the presence of E. coli in ECMUG broth tubes at 35 C will require further study. The isolation frequencies for the indicator bacterial groups (Table 3) were determined according to the time required to produce an acid reaction or an acid and foam reaction in the bottle test. Although most of the initial responses occurred within 24 h, all test kits produced presumptive positive tests from which total coliforms and fecal coliforms were recovered after 24 to 28 h. With the test kits in which the broth was used, it was not unusual for presumptive positive tests to show up even after 72 h of incubation. The data in Table 3 demonstrate the need for extended incubation periods for bottles and the need for the use of confirmation tests for all bottles giving a positive coliform test to increase the level of recovery of E. coli. The E. coli recovery values reported in Table 3 represent the results from ECMUG broth tube tests, whereas the fluorescent COMMERCAL TEST KTS 387 values represent what the level of E. coli recovery would have been if only bottle fluorescence had been used to indicate the presence of E. coli. As these values show, the fluorescent response in bottles was always less than the EC medium fluorescent response in ECMUG broth tubes. Samples from which fecal coliforms and E. coli were recovered usually produced presumptive positive tests in 98% or more of the samples within 18 to 24 h. For total coliforms, the Colilert and Coliquik tests gave levels of recovery of 95% within this same time period, whereas the average level of recovery for the Hach Disposable and Hach Vial tests was 90%. The level of recovery for the MOE test was the lowest level recorded (86%), even after 28 h of incubation. Other indicator groups, such as anaerogenic coliforms, and Aeromonas spp. were for the most part recovered within 48 h, although with the Hach Disposable and Hach Vial tests on several occasions these organisms were isolated after 72 h of incubation. Colilert and Coliquik advertisements make the claim that these products simultaneously detect, identify, and confirm total coliforms and E. coli in the same container within 24 h or less, with a single inoculation and with no additional confirmation test. The advertising claims are essentially correct when the results for a large number of samples are considered, but as with most biological systems, identification of organisms by using simplified procedures does not always result in correct identification. As shown in this study, 5% or more of the test kits took 48 h or more to produce an initial response, and in a number of samples only Aeromonas spp. were detected by confirmation tests; in other samples, organisms other than typical coliforms were responsible for positive o-nitrophenyl-,-d-galactopyranoside tests. f ECMUG broth tube tests had not been used, E. coli would not have been detected in a number of samples. n an attempt to speed up the identification and confirmation of total coliforms, fecal coliforms, and E. coli by the MOE test, confirmation tests were performed with a pipetting device equipped with sterile disposable tips which delivered 0.05-ml inocula from the bottles into ECMUG broth tubes rather than with just loopfuls of inoculum. test preparations inoculated on the previous day either in the morning or in the early afternoon were removed from the incubator the following morning, and inocula from presumptively positive tests were transferred into ECMUG broth tubes by 9:00 to 9:30 a.m. By 3:00 to 3:30 p.m. of the same day, tubes that produced fluorescence at either 35 or 44.5 C were considered to have E. coli present, and tubes that produced effervescence at 35 C when they were shaken were considered to have total coliforms present; tubes that produced effervescence at 44.5 C were considered to have fecal coliforms present (Table 4). n effect, this procedure permitted confirmation of total coliforms, fecal coliforms, and E. coli within 24 to 28 h for approximately 60% of the samples from which total and fecal coliforms were ultimately isolated, and in the case of E. coli the average level of response was 80% for the test kits in which broth was used. n the case of the Colilert and Coliquik tests, the confirmation response was slower, presumably because the organisms had to readjust their enzyme systems to working in a more enriched medium. Each of the EC medium tubes that produced effervescence within 6 h contained a sizable quantity (>10%) of gas in the inverted tube by the following day; if only weak fluorescence was detected within 6 h, much stronger fluorescence was present after 24 h. With 40% or more of the tubes, obvious gas production was not observed until after 24 h of incuba-

388 CLARK AND EL-SHAARAW tion, and with fluorescence about 20% or more of the tubes were not sufficiently fluorescent to be detected until after 24 h. Surprisingly, fluorescence was detected earlier when the preparations were incubated at 35 C than when they were incubated at 44.5 C (Table 4). ndicator organisms were detected and recovered much less frequently from drinking water samples than from the diluted river and sewage samples which were used in the first part of the study. However, except for more frequent presumptive positive results with the Hach Vial test, both Hach test kits recovered indicator organisms in drinking water samples in numbers almost identical to the numbers recovered by the MOE test procedure. The results for parallel samples were not always identical, but the overall levels of recovery of indicator organisms in the test procedures were similar. Another consideration which should be taken into account when a test kit is chosen for routine use is the cost per test. When our evaluation tests were done in 1990, the cost (in Canadian dollars) of the Access Analytical Systems Colilert test was about $8.00 to $9.00 per test depending on the quantity ordered, and the cost of the Coliquik test was about $7.00 per test. The costs of the Hach Disposable and Hach Vial tests were about $4.00 and $2.00, respectively. When the MOE test was made up in-house by using broth medium, the cost was about $1.00 per test. ncreased usage and production of these test kits may lower their costs in the future. Although the Colilert and Coliquik test kits may be easier to use than the test kits containing a lactose-based medium, the former are more expensive and do not have the flexibility for detecting and isolating other water quality indicator groups. Besides total coliforms and E. coli, the lactose type of tests can detect the potential presence of other indicator organisms, such as Aeromonas spp., Staphylococcus or Micrococcus spp., Clostridium perfringens, and fecal streptococci (4). Aeromonas spp. and Staphylococcus or Micrococcus spp. often produce a distinctive off-yellow color in presumptive positive bottles. Familiarization with this color reaction or the bright yellow color and foam reaction that occur in the presence of total coliforms or E. coli can often assist a technician or operator to speculate on the indicator group that will be identified by confirmatory tests. The presence of total coliforms, fecal coliforms, or E. coli is well recognized as an indication of unsafe or poor water quality for which corrective measures should be taken. These measures include increased chlorination or water main flushing or a combination of both of these actions. Less well recognized is the possibility that an increased frequency of presumptive positive tests and/or the isolation of Aeromonas spp., Staphylococcus spp., and other indicator organisms is a sign that the water quality is deteriorating even though members of the coliform group have not been detected. Although confirmatory tests are required to detect and differentiate these other indicator organisms, these tests are relatively simple and easy to perform (4). The additional information provided by these tests should help operators of water works to determine when simple corrective measures should be initiated before a serious water quality incident occurs. Otherwise, more costly and embarrassing corrective measures, such as the measures that accompany the issuance of a boil water order and public notification of unsafe water quality, could be necessary. ACKNOWLEDGMENTS APPL. ENVRON. MCROBOL. We thank Colleen Cotter and the staff of the Thunder Bay Regional Laboratory and Bill Kutas and the staff of the London Regional Laboratory for performing regional laboratory analyses for this study. We thank Steve Debreceni, Rosa Lee, ngrid Bonkowski, and the staff of the Central Laboratory for their assistance with analytical tests and preparation of the manuscript. REFERENCES 1. American Public Health Association. 1989. Standard methods for the examination of water and wastewater, 17th ed. American Public Health Association, Washington, D.C. 2. Bancroft, K., E. T. Nelson, and G. W. Childers. 1989. Comparison of the presence-absence and membrane filter techniques for coliform detection in small, nonchlorinated water distribution systems. Appl. Environ. Microbiol. 55:507-510. 3. Clark, D. L., B. B. Milner, M. H. Stewart, R. L. Wolfe, and B. H. Olson. 1991. 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National field evaluation of a defined substrate method for the simultaneous enumeration of total coliforms and Escherichia coli from drinking water: comparison with presence-absence techniques. Appl. Environ. Microbiol. 54:1003-1008. 9. Edberg, S. C., and M. M. Edberg. 1988. A defined substrate technology for the enumeration of microbial indicators of environmental pollution. Yale J. Biol. Med. 61:389-399. 10. Federal Register. 1989. Drinking water: national primary drinking water regulations; total coliform proposed rule. Fed. Regist. 54:27544-27567. 11. Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol. 43:1320-1329. 12. Jacobs, N. J., W. L. Zeigler, F. C. Reed, T. A. Stukel, and E. W. Rice. 1986. Comparison of membrane filter, multiple-fermentation-tube, and presence-absence techniques for detecting total coliforms in small community water systems. Appl. Environ. Microbiol. 51:1007-1012. 13. 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