Comparison of m-endo LES, MacConkey, and Teepol Media

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1979, p /79/ /08$02.00 Vol. 38, No. 3 Comparison of m-endo LES, MacConkey, and Teepol Media for Membrane Filtration Counting of Total Coliform Bacteria in Water W. 0. K. GRABOW* AND MARTELLA DU PREEZ National Institute for Water Research, Council for Scientific and Industrial Research, Pretoria 0001, South Africa Received for publication 2 April 1979 Total coliforn counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, sand filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction. The total coliform count is one of the most useful indicators of water pollution (2, 9, 19, 28, 33, 44, 47). Although various members of the coliform group of bacteria may multiply in environments other than the gastrointestinal tract, they are excreted in large numbers in the feces of warm-blooded animals and their presence in water is associated with fecal pollution (4, 19). An important feature of coliform bacteria is that they are detectable by relatively simple, rapid, and cheap techniques. Unfortunately, however, a wide variety of techniques which differ in accuracy and reliability are being used to evaluate coliforms in water (5, 22, 34). Consequently, results from different laboratories can hardly be compared, the implementation of water quality standards is of limited value, and unreliable techniques may even have far-reaching health inplications (5, 6, 22). In this study, counts obtained by means of three growth media commonly used in standard membrane filtration (MF) tests for total coliforms in water have been compared. The results contribute to information urgently needed for the standardization of coliform techniques (5, 6, 16, 22). The media involved were m-endo LES 351 agar, used in countries such as the United States (2), Canada (33), and West Germany (3), Teepol media, used in Britain (9), and MacConkey agar, used in South Africa (22, 44) and Canada (33). MATERIALS AND METHODS MF techniques. Laboratory procedures complied with the specifications of the American Public Health Association (2), Department of Health and Social Security, London, England (9), and the South African Bureau of Standards (44). Sartorius filter holders, Gelman GN-6 membranes (pore size, 0.45 um), and disposable plastic petri dishes (diameter, 65 mm) with loose-fitting lids were used. All counts are expressed as the average of tests done in triplicate. Incubation was in a circulating air incubator at C for 20 to 24 h. Media. m-endo agar LES (code ; Difco Laboratories) was obtained commercially. Membrane-enriched Teepol broth (0.4 ET) was prepared as specified by the Department of Health and Social Security (9) and contained 40 g of peptone (Difco), 6 g of yeast extract (Difco), 30 g of lactose (Merck & Co., Inc.), 50 ml of a 0.4% aqueous solution of phenol red, 4 ml of Teepol 610 (British Drug Houses), and 1,000 ml of distilled water. Membranes were either incubated on pads saturated with this medium or on the medium solidified with 1.5% agar. MacConkey agar was pre-

2 352 GRABOW AND DU PREEZ pared by the following formula (26): 15 g of agar (Difco), 20 g of peptone (Difco), 10 g of lactose (Merck), 5 g of bile salts (Difco no. 3), 5 g of sodium chloride, 0.12 g of bromocresol purple, and 1,000 ml of distilled water. Water samples analyzed. Samples of settled wastewater, biofilter effluent, activated sludge effluent, and a mixture of biofilter and activated sludge effluents before and after chlorination to a total chlorine content of about 4 mg/liter, followed by sand filtration, were collected at the Daspoort wastewater purification works in Pretoria, South Africa (29). The Apies River was sampled shortly before and after the discharge of secondary treated wastewater (humus tank effluent, unchlorinated) from the Daspoort purification works (27). Samples were also collected from the following stages in an experimental 4,500 m3/day multiple-barrier wastewater reclamation plant (Stander plant) in Pretoria (25): influent (secondary treated wastewater) (DR1); after lime treatment at ph levels ranging from 9.6 to 11.4 (DR3); after quality equalization in a pond with mean residence time of about 10 h (DR5); after sand filtration (DR11); after breakpoint chlorination (DR12); after active carbon treatment (DR13); after final chlorination (DR14); and after ozonation (DR15). The latter was at times used for disinfection instead of breakpoint chlorination. Bottles used for collecting samples of chlorinated water contained sodium thiosulfate for dechlorination (2). Samples were collected during the period from December 1976 to January They were homogenized in a Sanyo mixer for 4 min at a speed selector setting of 4 (26) and processed within 3 h after collection. Identification of coliform-like bacteria. Membranes with 20 to 50 well-spaced coliform-positive colonies were selected. All coliform-positive colonies were picked from these membranes and purified on the same medium for identification by means of the commercial API 20E system (30, 40). The IMVic (indole, methyl red, Voges-Proskauer, citrate) (2) and cytochrome oxidase (15) tests were done additionally on all these isolates. Names of isolates in Table 3 which do not appear in the 8th edition of Bergey's Manual of Determinative Bacteriology are used by the manufacturers of the API test system (Analytab Products Inc., Plainview, N.Y.). APPL. ENVIRON. MICROBIOL. RESULTS Coliform counts on wastewater and river water. Average total coliform counts obtained on MacConkey, Teepol, and Endo agars for samples collected at the Daspoort wastewater purification works are listed in Table 1. MacConkey and Endo agars were compared in tests on 70 of these samples. Endo agar yielded the highest average count for each sampling point (Table 1). The highest count for 51 (73%) of the 70 samples was recorded on Endo agar, whereas Mac- Conkey agar yielded the highest count for 19 (27%) of the samples. MacConkey, Teepol, and Endo agars were compared in tests on 22 samples of secondary effluent before and after chlorination and of the river upstream and downstream of the discharge from the purification works (Table 1). Endo agar yielded the highest average count for each of the sampling stations, followed by Teepol and MacConkey agars. Endo, Teepol, and MacConkey agars had the highest counts for 17 (77%), 3 (14%), and 2 (9%) of the 22 samples, respectively. The difference in counts on different media was relatively constant for all sampling points, which indicates that the quality of water had no significant effect on the ability of the media to support the production of colonies which conform to the definition of total coliforms. Coliform counts on samples from the Stander wastewater reclamation plant. All the media concerned were compared in various combinations in total coliform tests on samples from different treatment stages of the Stander wastewater reclamation plant. Table 2 shows that, apart from a few minor exceptions, the highest average count was recorded on Endo agar, followed by Teepol agar, Teepol broth, and MacConkey agar. Endo and MacConkey agars were compared in tests on 90 samples (Table 2). Endo agar yielded the highest count for 56 (62%) of the 90 samples, and MacConkey agar yielded the highest count for 34 (38%). However, Endo agar had 0 counts for 12 of the samples, and MacConkey agar had 0 counts for only 4. MacConkey agar, Teepol broth, and Teepol agar were compared in tests on 96 samples of the Stander plant (Table 2). All three media had 0 counts for six of the samples. Teepol agar, Teepol broth, and MacConkey agar had the highest counts for 49 (55%), 38 (42%), and 3 (3%) of the remaining 90 samples, respectively. Teepol agar yielded 0 counts for a total of five samples, and MacConkey agar and Teepol broth each for seven samples. All four media were compared in tests on another 53 samples of the Stander plant. One sample had 0 counts on all media. Endo agar, Teepol agar, Teepol broth, and Mac- Conkey agar had the highest counts for 48 (92%), 4 (8%), none (0%), and none (0%) of the remaining 52 samples, respectively. The large differences in coliform counts for various series of tests on DR3 (Table 2) are due to variations in lime dosage which were introduced for experimental purposes at the time when these counts were done (25). The lime dosage also affected coliform counts in the next two treatment units, namely, the quality equalization pond (DR5) in which a relatively high ph level was maintained for about 10 h (25) and the sand filters (DR1l). Samples for each series of comparative coliform tests on DR3, DR5, and DR1l were taken when lime dosage was at a

3 VOL. 38, 1979 TABLE 1. Source of sample TECHNIQUES FOR TOTAL COLIFORMS 353 Total coliform counts for wastewater and river water obtained by means of membrane filtration using different growth media No. of Total coliform count/100 mla samples sape MacConkey agar Teepol agar m-endo LES agar Settled wastewater x 1o5; (50 to 340) 253 x 105; (90 to 450) x X Biofilter effluent x 104; (350 to 520) 178 x 104; (36 to 730) x X Activated sludge x 103; (80 to 360) 209 x 103; (110 to 390) effluent x 103 X 103 Effluent before x 103; (24 to 500) 200 x 103; (70 to 700) x chlorinationb X x 103; (250 to 852 x 103; (270 to 918 x 103; (390 to 1,800) 1,900) X 103 2,200) x 103 X 103 Effluent after 11 1,173; 0-6,500 2,316; 0-9,800 chlorinationc 5 778; 190-2,000 1,710; (250 to 5,000) 2,286; 630-6,000 River before x 103; (6 to 83) x 54 x 103; (5 to 190) x discharged x 103; (7 to 60) x 92 x 103; (15 to 213) x 129 x 103; (12 to 360) x River after discharge' x 103; (51 to 983 x 103; (25 to 5,400) 2,000) x 103 X x 103; (8 to 400) x 208 x 103; (20 to 600) 342 x 103; (33 to 1,100) i03 X103 X 103 a Each value indicates average, followed by range of counts. b Mixture of Daspoort biofilter and activated sludge effluents. Mixture described in footnote b after chlorination and sand filtration. d. e Apies River upstream and downstream of secondary treated discharge from Daspoort purification works. TABLE 2. Source of sample Total coliform counts for samples from various stages in a wastewater reclamation plant, obtained by means of membrane filtration with various growth media No. of Total coliform count/100 mla sape samples MacConkey agar Teepol broth Teepol agar m-endo LES agar Intake (DR1)b x 103; (13 to 191 x 103; (16 to 800) x 103 1,020) x x 103; (17 to 213 x 103; (43 to 243 x 103; (65 to 320) x ) x ) x x 103; (50 to 393 x 103; (170 to 385 x 103; (180 to 618 x 103; (300 to 590) x ) x ) x 103 1,280) x 103 After lime ; 0-3, ; 0 to 3,800 treatment (DR3) 14 3,077; 33-19,000 3,296; 60-17,000 4,276; 20-28, x 103; (22 to 127 x 103; (35 to 149 x 103; (79 to 215 x 103; (130 to 100) X ) x ) x ) x 103 After quality ; 0-1, ; 0-3,100 equalization 16 2,595; 0-17,200 2,760; 0-14,500 3,069; 0-20,800 (DR5) 8 42 x 103; (24 to 72 x 103; (45 to 73 x 103; (15 to 134 x 103; (72 to 88) x ) x ) x ) X 103 After sand filtration ; 0-1, ; 0-8,200 (DR1l) 16 2,309; 0-9,200 2,743; 0-10,200 2,976; 0-19, x 103; (0.8 to 17.3 x 103; (5.6 to 21.9 x 103; (6.9 to 34.7 x 103; ( ) x ) x ) x 103 to 76.0) x 103 After ozonation 7 4; ; ; 0-14 (DR15) 8 6; ; ; ; 0-92 After carbon 28 41; ; ; treatment 14 61; ; ; ; (DR13) a Each value indicates average, followed by range of counts. b The raw water intake was activated sludge effluent.

4 354 GRABOW AND DU PREEZ constant level. The seven samples of DR3 in which all four media were compared (Table 2) were taken during a period of low-lime dosage (operational ph in lime treatment unit about 9.6), whereas the 17 samples of DR3 in which MacConkey and Endo agars were compared were taken during a period of high-lime dosage (operational ph level in lime treatment unit about 11.2). In comparative tests on 49 samples of water taken after intermediate chlorination (DR12) and final chlorination (DR14), MacConkey and Endo agars yielded positive results for five and two samples, respectively. In tests on another 24 samples of chlorinated water, Teepol agar, MacConkey agar, and Teepol broth yielded positive results for four samples, two samples, and one sample, respectively. All four media were compared in tests on 14 other samples of chlorinated water, but none of them yielded positive results. The general quality of the chlorinated water compared favorably with that of conventional drinking water supplies, and coliforms were recorded on occasions when chlorination was intentionally inefficient for research purposes (23). The same applied to ozonation (23). The results of tests on chlorinated and ozonated water indicate that Teepol and MacConkey agars tended to yield positive results more often than did Endo agar. However, the difference between positive and negative results was generally one or a few colonies, and the number of samples which yielded these differences were not enough to warrant statistically significant conclusions. Apart from the uncertainty about chlorinated and ozonated water, the results of tests on the other samples from the Stander plant showed that the treatment of the water had no significant effect on the relative differences in coliform counts on the different media (Table 2). The following data on the highest counts for individual samples irrespective of source were derived from the above-mentioned tests, as well as additional tests which were excluded for statistical reasons from Tables 1 and 2 because they were done on a series of tests comprising fewer than five samples. Teepol broth had higher counts than MacConkey agar for 131 samples, whereas MacConkey agar had higher counts than Teepol broth for 30 samples. Teepol agar had higher counts than Teepol broth for 93 samples, whereas Teepol broth had higher counts than Teepol agar for 59 samples. Endo agar had higher counts than Teepol agar for 81 smples, whereas Teepol agar had higher counts than Endo agar for 12 samples. Endo agar had higher counts than MacConkey agar for 179 APPL. ENVIRON. MICROBIOL. samples, whereas MacConkey agar had higher counts than Endo agar for 54 samples. Identification of coliform-like isolates. Isolates recorded as coliform positive were identified in tests on 13 samples of water in which MacConkey, Teepol, and Endo agars were used as growth media. The samples comprised two of Daspoort secondary-treated wastewater, three of the Apies River before the Daspoort discharge, one ofthe Apies River after the Daspoort discharge, two of the Daspoort secondary treated wastewater after chlorination and sand filtration, one of the raw water intake (DR1), three of the effluent of the lime treatment unit (DR3), and one of the effluent of the ozonation unit (DR15) in the Stander wastewater reclamation plant. The identities of the 871 isolates are combined in Table 3, since there was no significant difference in the relative numbers of species isolated from samples of different sources. Among the 225 isolates from Mac- Conkey agar, 116 (45%) were true coliforms (Escherichia coli and species of Klebsiella, Enterobacter, and Citrobacter), whereas 135 (53%) were Aeromonas hydrophila and the other 4 (2%) were various other species. The 275 isolates from Teepol agar consisted of 149 (54%) true coliforms, 114 (41%) A. hydrophila, and 12 (4%) other species. The 341 coliform-like colonies picked from membranes incubated on Endo agar comprised 186 (55%) true coliforms, 138 (40%) A. hydrophila, and 17 (5%) other species. General features of the media. Colonies which conformed to the definition of coliforms were generally easy to recognize on all the media. The golden-green metallic sheen of the colonies on Endo agar was occasionally difficult to identify. Differentiation of yellow coliform colonies from pink and light red colonies on Teepol media was at times uncertain, particularly on crowded membranes. Using pads saturated with Teepol broth proved time consuming, tedious and inconvenient. The pads cannot be prepared in advance and stored for immediate use like the agar-based media. The most important disadvantage of the saturated pads was that they tended to dry out during incubation, which often interfered with counts. One advantage of Endo agar is that the medium does not require sterilization (2). MacConkey agar has to be autoclaved for 15 min (44), and Teepol broth has to be steamed for 30 min on 3 successive days (9). On the other hand, Endo agar is a complex medium, and it is advisable to only use good quality media from reliable commercial suppliers (16). MacConkey and Teepol media are easily prepared from basic ingredients in the laboratory. Teepol medium was cheaper than

5 VOL. 38, 1979 TABLE 3. TECHNIQUES FOR TOTAL COLIFORMS 355 Identity ofpositive isolates in total coliform tests on 13 water samples of various origins, using membrane filtration and three different growth media Positive isolates on the following medium: Isolate MacConkey agar Teepol agar Endo agar No. %a No. %a No. %a E. coli C. freundii C. diversus 2 <1 Citrobacter Spp.b 1 <1 1 <1 K. pneumoniae K. ozaenae <1 K. rhinoscleromatis 2 <1 Klebsiella spp.b 1 <1 E. aerogenes 2 <1 2 <1 E. cloacae Enterobacter spp.b 1 <1 Erwinia herbicola (Enterobacter agglomerans) Total for the above typical coliforms Y. enterocolitica 1 <1 1 <1 Shigella dysenteriae 1 <1 Shigella Spp.b 1 <1 Serratia liquefaciens <1 S. rubidaea 1 <1 1 <1 S. marcescens 3 <1 Alcaligenes faecalis 2 <1 Alcaligenes Spp.b 4 1 Flavobacterium meningosepticum 2 <1 1 <1 F. odoratum 1 <1 Pasteurella multocida 1 <1 1 <1 Proteus inconstans 1 <1 P. paucimobilis 2 <1 A. hydrophila a Each value indicates percent of total isolates. b Species not identifiable by available techniques. available supplies of MacConkey and Endo media. DISCUSSION The comparative coliform tests were done on samples of water collected from a wide variety of sources. In these samples, the numbers of coliforms and bacteria which may interfere with coliform counts varied from very high to 0, and they included coliforms exposed to the river environment, conventional wastewater purification and advanced tertiary processes such as lime treatment, sand filtration, active carbon treatment, and disinfection by means of ozone and chlorine. Endo agar yielded the highest average coliform counts for all these samples (Tables 1 and 2). Teepol agar generally had higher counts than Teepol broth, whereas the lowest counts were usually recorded on MacConkey agar. Teepol broth was omitted from many comparative tests, because using the saturated pads proved inconvenient and time consuming, the pads tended to dry out, and the agar-based Teepol medium proved to generally yield higher counts. A similar experience has been reported for pads saturated with liquid Endo (38) and MacConkey (22) media. The slightly higher cost of agar-based media is justified by more reliable results, convenience and saving in time (22, 33, 38) İdentification of 871 isolates from 13 samples of various origins showed that A. hydrophila was the species most frequently present among colonies which conformed to the definition of total coliform bacteria on MacConkey, Teepol, and Endo agars (Table 3). Endo agar proved slightly more specific than Teepol agar, and much more specific than MacConkey agar, for species of Escherichia, Citrobacter, Klebsiella, and Enterobacter, which is the group of primary interest in total coliform tests (37).

6 356 GRABOW AND DU PREEZ Most probable number (MPN) tests were excluded from this evaluation of total coliform methods, since evidence has been presented that MF is the technique of choice for general purposes (11, 20, 22, 34). The advantages of MF are: it gives more accurate counts (11, 22, 34); it gives a direct count, whereas MPN evaluations are based on statistical estimates with inherent errors (9, 35); it yields results within 16 to 24 h and MPN evaluations only after 48 to 96 h; it yields valuable additional information on organisms such as Aeromonas and Pseudomonas species (22) and even pathogens like Yersinia enterocolitica (32, 42); colonies can easily be picked from membranes for further identification; larger volumes of water are being tested in standard MF tests and the volume can easily be increased extensively if necessary; organisms such as Clostridium perfringens (9) and coliphages (43) may interfere with MPN evaluations; MF tests are cheaper than MPN evaluations (20, 22), and their performance is less cumbersome and time consuming; the petri dishes used for MF take up less incubator space than the racks with tubes required for MPN evaluations; and MF may conveniently be applied under field conditions (48). MPN evaluations yield higher counts for chlorinated effluents than standard MF techniques without enrichment procedures (35, 39). If for any particular reason the higher counts on chlorinated effluents are required, the addition of relatively simple enrichment procedures to MF techniques will yield counts equivalent to those of MPN evaluations (9, 35). The significance of these higher counts, which are attributed to the inclusion of stressed coliforms (35), is uncertain. In chlorinated effluents, pathogens are presumably stressed to the same extent as coliforms. There is no evidence that these stressed pathogens are of any health significance, since they may not be able to survive host defense mechanisms. Standard MF tests for total coliforms have rarely if ever failed to prove the microbiological (including virological) safety of properly treated drinking water (1, 8, 17, 22, 23, 24). The additional labor and cost of enrichment procedures are therefore not regarded necessary for general purposes and routine analysis of drinking water supplies (2, 22, 33). MPN evaluations remain useful for tests on highly turbid samples which clog membranes. However, the turbidity of drinking waters should be well below levels which may affect the efficiency of MF techniques (18). Proposals to exclude cytochrome oxidase-positive organisms, mainly Aeromonas and Pseudomonas species, from total coliform counts (3, 37) should be considered with caution. The total APPL. ENVIRON. MICROBIOL. coliform count should not be regarded as a specific indicator of fecal pollution. The fecal coliform count primarily serves this purpose (10). Even if an oxidase test is included, the total coliform count cannot meet this requirement, since many oxidase-negative coliforms multiply in the water environment and are not of fecal origin (4, 10). The total coliform count should be regarded primarily as an indicator of the sanitary quality of drinking water (1, 17). Properly designed, operated, and controlled drinking water plants consistently produce water which is free of total coliforms per 100-ml sample when tested by standard techniques (1, 17, 18, 21, 24). A positive total coliform test and the presence of lactose-negative bacteria which overgrow membranes and obscure coliforms, indicate inefficient treatment, secondary contamination, or aftergrowth, none of which should be tolerated (1, 7, 21, 46). Coliforms isolated from drinking water should immediately be identified to establish possible fecal origin. In addition to their indicator value, bacteria which yield a positive coliform test may themselves constitute health hazards and should therefore also not be tolerated in drinking water. K. pneumoniae, which readily multiply in various water environments, constitute an opportunistic pathogen of increasing importance (4). A. hydrophila, which primarily multiply in water, are not only pathogenic to fish and various other animals, but may also infect humans (14, 31). Gram-negative bacteria which multiply in highly purified water such as distilled water cause serious problems in hospitals (13). Bacteria involved in standard coliform tests may also carry hazardous plasmids which are transferable among gram-negative organisms. These plasmids include resistance factors which confer on their hosts resistance to antimicrobial drugs, disinfectants, and various other agents (27, 28), whereas others may code for enterotoxin production which turns normally harmless bacteria such as E. coli into a serious pathogen (12, 29, 41). In view of these considerations, the oxidase requirement would unnecessarily increase the cost, labor, time, and technical know-how needed for the coliform test and at the same time limit its sensitivity, efficiency, and reliability. Evaluation of the above advantages and disadvantages of available techniques for total coliforms indicates that the method of choice for routine analysis of drinking water is MF, using good quality membranes such as those used in this study (36, 45) and incubation at C for 22 to 24 h on m-endo LES agar without enrichment procedures or an oxidase test. This technique is not only reliable but also the most

7 VOL. 38, 1979 appropriate for laboratories in small communities or developing countries with limited funds, laboratory facilities, and trained staff. ACKNOWLEDGMENTS Thanks are due to Irmela G. Middendorff and J. S. Burger for skillful technical assistance, to N. P. Nicolle, Chief Chemist of the Pretoria Municipality, for permission to sample the Daspoort purification works and the Apies River, and to 0. W. Prozesky and L. S. Smith for their advice. This paper is published with the approval of the Director of the National Institute for Water Research. LITERATURE CITED 1. Allen, M. J., and E. E. Geldreich, Jr Evaluating the microbial quality of potable waters, p In C. W. Hendricks (ed.), Evaluation of the microbiology standards for drinking water. U.S. Environmental Protection Agency, Washington, D.C. 2. American Public Health Association Standard methods for the examination of water and wastewater, 14th ed. American Public Health Association, Inc., Washington, D.C. 3. Aurand, K., U. 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