APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1987, p. 1246-1250 0099-2240/87/061246-05$02.00/0 Copyright C) 1987, Americn Society for Microbiology Vol. 53, No. 6 Improved Membrne Filtrtion Medi for Enumertion of Totl Coliforms nd Escherichi coli from Sewge nd Surfce Wterst TIMOTHY A. FREIER AND PAUL A. HARTMAN* Deprtment of Microbiology, Iow Stte University, Ames, Iow 50011 Received 9 September 1986/Accepted 9 Mrch 1987 Two medi were developed tht llowed both totl coliform count nd n Escherichi coli count to be determined on the sme medium fter 24 h of incubtion t 35 C. The new medi were tested long with two stndrd medi on 10 surfce wter nd 7 sewge smples. The experimentl medi yielded equivlent or higher counts reltive to the stndrd medi nd recovered more specificlly the desired indictor groups s determined by colony identifiction. Current membrne filtrtion methods hve been criticized for their mny shortcomings (for review, see reference 15). Severl investigtors hve suggested tht Escherichi coli be used s n indictor becuse it is specific for fecl pollution nd provides more ccurte indictor-to-pthogen rtio thn totl or fecl coliform enumertion methods (4, 7, 9). Incresed ttention is being given to the recovery of injured indictor bcteri (3, 5, 6, 19, 20, 22, 23). One of the mjor fctors cusing reduced recoveries of injured bcteri is the selective gent(s) tht is used. Monensin is n ionophore tht selectively inhibits ll grm-positive bcteri while it llows growth of injured grm-negtive bcteri (25). Tergitol 7, surfctnt, hs similr effect (17, 19). Recently, very sensitive nd specific method for the detection of P-glucuronidse production by E. coli ws developed (10, 15, 25). Over 95% of E. coli hydrolyze nonfluorescent substrte, 4-methylumbelliferyl-p-D-glucuronide (MUG), to produce 4-methylumbelliferone, which fluoresces under long-wve UV light. Approximtely 50% of Shigell spp., some Slmonell spp., nd few strins of Yersini enterocolitic re the only other members of the fmilies Enterobctericee nd Vibrioncee tht produce this enzyme (12, 14, 18, 21, 25, 29). The purpose of this study ws to develop n improved membrne filtrtion method by using medi tht contin monensin, Tergitol 7, nd MUG. The gol ws the enumertion of totl coliforms nd E. coli on single medium incubted t 35 C with improved recovery of injured cells nd improved selective specificity when compred with stndrd membrne filtrtion medi nd methods. MATERIALS AND METHODS Medi. MUG ws obtined from Hch Co. (Lovelnd, Colo.). Monensin (90 to 95% pure) ws obtined from Sigm. All medi were prepred with 1.5% gr, sterilized, tempered to 47 C, nd poured in pproximtely 3-ml volumes into petri pltes (dimeter, 47 mm). Peptone-Tergitol-glucuronide (PTG) gr ws prepred s described previously (5). Peptone glucuronide (PG) gr ws prepred exctly s PTG gr, except tht the Tergitol 7 (J. T. Bker Chemicl Co., Phillipsburg, N.J.) ws omitted. PG gr ws poured into stndrd petri pltes (dimeter, 100 mm) for the isoltion nd purifiction of colonies. Enriched * Corresponding uthor. t Journl pper no. J-12389 of the Iow Agriculture nd Home Economics Experiment Sttion, project 2678. 1246 luryl sulfte-nline blue (ELSAB) gr ws prepred s described by Wright (31). Tergitol 7 gr (m-t7) ws prepred initilly s described previously (19); lter, it ws obtined from Difco Lbortories (Detroit, Mich.). m-fc broth bse nd m-endo broth MF were obtined from Difco nd prepred with 1.5% gr. m-fc gr ws used without rosolic cid. The formultion of lctose-monensin-mug (m-lmm) gr, which ws utoclved for 15 min t 121 C (finl ph, 7.2), ws s follows: Tryptone (Difco), 5.0 g; yest extrct, 2.5 g; lctose, 10.0 g; bromocresol purple, 80.0 mg; K2HPO4, 3.3 g; KH2PO4, 1.0 g; MUG, 100.0 mg; gr, 15.0 g; monensin, 38.0 mg (in 10 ml of 95% ethnol); distilled wter, 1,000.0 ml. Tergitol-monensin-MUG (m-tmm) gr ws prepred exctly s m-lmm, except tht 0.25 ml of Tergitol 7 per liter ws dded before steriliztion. m-tec gr ws prepred s described previously (8). Strch mpicillin (SA) gr (24) ws used for the presumptive identifiction ofaeromons hydrophil. A modified stndrd plte count medium (m-spc) ws mde by the method of Tylor nd Geldreich (28). All dilutions were mde in 0.1% peptone wter. Smple collection. Smples were collected in sterile 200-ml glss screw-cp bottles. Surfce wter smples were collected from lkes, strems, nd rivers in centrl Iow. Sewge smples were collected from vrious tretment stges t the Ames, Iow, nd the Nevd, Iow, wstewter tretment plnts, both of which process residentil nd light industril wstes, including moderte quntity of surfce wter runoff. The sewge effluents were not chlorinted. An dditionl sewge smple ws collected from lgoon serving smll residentil community. The smples were tested to determine the pproximte coliform nd E. coli levels nd were stored overnight t 4 C. The next dy (within 28 h of collection), the smples were subjected to extensive comprtive studies. Medi comprison. On the bsis of preliminry experiments reported below (MUG incorportion), PTG, m-lmm, nd m-tmm grs were selected for comprison with m- Endo nd m-fc grs in more extensive tests. A totl of 10 surfce wter smples nd 7 sewge smples were collected over 7-month period. Fifteen filters were prepred from ech of severl pproprite dilutions. The filters were plced on triplicte pltes of the three tril nd two stndrd medi. The pltes were incubted t 35 C, except for m-fc pltes, which were incubted in 44.5 C circulting wter bth for 18 to 24 h. Typicl lctose-positive rections were counted s drk blue colonies on m-fc gr, green colonies with
VOL. 53, 1987 TABLE 1. Geometric men coliform counts obtined on three medi before nd fter correction for A. hydrophil No. of coliforms/100 ml fter growth on: Smple m-endo m-lmm m-tmm Sewge' 6.6 x 106 4.3 x 106 5.5 x 106 Sewgeb 5.2 x 106 4.1 x 106 5.2 x 106 Surfce wter' 9.3 x 103 9.1 X 103 9.3 x 103 Surfce wterb 5.6 x 103 8.5 x 103 9.1 X103 Uncorrected counts. b Corrected counts fter subtrction of the proportionte number of lctosepositive A. hydrophil s determined for ech smple. ' Significnt increse over m-endo count (P < 0.1). metllic sheen on m-endo gr, nd yellow colonies on m-lmm nd m-tmm grs. Fluorescent colonies were detected by plcing the pltes under long-wve UV light. To enhnce fluorescence, n bsorbnt filter pd ws plced in the cover of dish, 3 to 5 drops of undiluted mmonium hydroxide (30% NH3) were plced on the pd, nd the plte to be counted ws plced over it for 10 to 15 s. Colony identifiction. Isolted colonies were picked from the most pproprite dilution of ech medium type. For ech smple, five lctose-positive, MUG-positive colonies (presumptive E. coli); five lctose-positive, MUG-negtive colonies (presumptive coliforms); nd five lctose-negtive, MUG-negtive colonies (presumptive noncoliforms) were chosen t rndom from m-lmm nd m-tmm grs. From m-endo nd m-fc grs, five lctose-positive nd five lctose-negtive colonies were chosen t rndom from ech medium type. Five MUG-positive nd five MUG-negtive colonies were picked t rndom from PTG gr pltes. Colonies picked from the vrious medi were struck for isoltion on pltes of PG gr. The pltes were incubted for 18 to 24 h t 35 C nd then were exmined under white nd UV light. Pure cultures were tested for the production of cytochrome oxidse. Oxidse-positive cultures were struck on pltes of SA gr, nd the pltes were incubted for 24 h t 30 C nd were then flooded with Lugol iodine solution. Colonies tht were cpble of growth on mpicillin nd tht exhibited zones of clering (mylse positive) were recorded s A. hydrophil. Oxidse-positive cultures tht were negtive for growth or mylse production on pltes of SA gr were inoculted for identifiction in Rpid NFT strips (Anlytb Products, Plinview, N.Y.). Oxidse-negtive cultures were inoculted in API 20E (Anlytb) strips. Chlorintion study. To determine recoveries of chlorineinjured cells on vrious medi, the method of Cmper nd McFeters (3) ws used, except E. coli B ws injured t chlorine concentrtion of 2.6 mg/liter for 6 min, nd the diluent used ws 0.1% peptone wter. Dilutions of control nd injured cells were dispersed in 100-ml peptone blnks; MEDIA FOR COLIFORMS AND E. COLI 1247 drwn through membrne filters; nd plced on m-spc, m-fc, m-endo, PTG, m-t7, m-lmm, nd m-tmm grs. All pltes were incubted t 35 C for 24 h, except tht m-fc gr pltes were incubted t 44.5 C in circulting wter bth. This experiment ws repeted three times. Sttisticl nlysis. The medi comprison dt nd the chlorintion study dt were nlyzed by nlysis of vrince. Nturl logrithmic trnsformtions of the observed counts were nlyzed in rndomized complete block design (27). Differences in the men recoveries on the vrious medi were compred by using the Student t test. RESULTS MUG incorportion. The optiml concentrtion of MUG for incorportion into m-lmm nd m-tmm gr ws 100,ug/ml. Fluorescent rections on m-tec, ELSAB, m-t7, m-endo, nd m-fc grs were difficult to observe, even t MUG concentrtion of 200 jig/ml. MUG-positive colonies on PTG were bright nd esy to detect t MUG concentrtion of 50,ug/ml, s reported by Dmre et l. (5). During the preliminry experiments, severl colonies with vrious mor, phologies were picked from the experimentl medi to be Grm stined. Grm-positive colonies were never isolted from ELSAB, m-tec, m-t7, m-lmm, or m-tmm grs. A smll number of pinpoint colonies on PTG gr were grm positive; however, these usully were not visible until incubtion periods exceeded 24 h. ELSAB nd m-tec grs were eliminted from further studies becuse of low recoveries nd the poor visuliztion of the MUG rection on these medi (dt not shown). Recoveries on m-t7 gr contining MUG were excellent, but the MUG rections were poor. Recovery comprisons. Coliform recoveries on m-endo, m-lmm, nd m-tmm grs re shown in Tble 1. PTG gr ws differentil only for E. coli, not for totl coliforms; therefore, PTG gr counts were not included in the totl coliform results. Becuse of the lrge numbers of A. hydrophil recovered on m-endo gr, the totl coliform counts were corrected to show the number of true coliforms tht were recovered (Tble 1). m-tmm coliform counts were significntly higher thn m-endo counts for surfce wters. The recoveries of fecl coliforms on m-fc gr nd E. coli on PTG, m-lmm, nd m-tmm grs re shown in Tble 2. PTG nd m-lmm grs yielded significntly greter recoveries of E. coli thn totl fecl coliforms on m-fc gr for surfce wter smples. Species distribution. Species distributions of lctosepositive bcteri isolted from ech medium differed s shown in Tbles 3 nd 4. On m-lmm nd m-tmm grs higher percentge of the gener normlly clssified s coliforms ws recovered. Percentges of A. hydrophil isolted on the five medi re TABLE 2. Geometric men counts of fecl coliforms nd E. coli on four medi Smple m-fc (no. of Lc'/ No. of MUG'/100 ml fter growth onb: 100 ml)" PTG m-lmm m-tmm Sewge 3.5 x 105 5.1 x 105c 3.5 x 105 3.8 x 105 Surfce wter 9.7 x 102 1.7 x 103'c 2.1 x 103d 1.1 X 103 " Chrcteristic blue colonies were counted s fecl coliforms. bchrcteristic blue hlos under long-wve UV light were counted s E. coli. c Significnt increse over m-fc count (P < 0.1). d Significnt increse over m-fc count (P < 0.05).
1248 FREIER AND HARTMAN APPL. ENVIRON. MICROBIOL. Orgnism TABLE 3. Lctose-fermenting orgnisms isolted from smples of surfce wter on four medi No. of isoltes (%) fter growth on: m-endo m-fc m-lmm m-tmm Aeromons hydrophil 14 (32.5) 0 (0) 2 (2.3) 2 (2.2) Citrobcter mlonticus 1 (2.3) 0 (0) 0 (0) 1 (1.1) Citrobcter freundii' 6 (14.0) 0 (0) 8 (9.1) 6 (6.7) Enterobcter erogenes 0 (0) 0 (0) 0 (0) 1 (1.1) Enterobcter gglomerns 0 (0) 1 (2.0) 3 (3.4) 12 (13.5) Enterobcter cloce 2 (4.7) 0 (0) 9 (10.2) 5 (5.6) Enterobcter sp. 0 (0) 0 (0) 1 (1.1) 0 (0) Escherichi coli 11 (25.6) 47 (92.2) 54 (61.4) 48 (53.9) Klebsiell oxytoc 4 (9.3) 0 (0) 6 (6.8) 5 (5.6) Klebsiell pneumonie 3 (7.0) 3 (5.9) 3 (3.4) 7 (7.9) Providenti sturtii 1 (2.3) 0 (0) 0 (0) 0 (0) Serrti fonticol 1 (2.3) 0 (0) 1 (1.1) 0 (0) Serrti liquefciens 0 (0) 0 (0) 1 (1.1) 1 (1.1) Serrti rubide 0 (0) 0 (0) 0 (0) 1 (1.1) Coliform (s defined by Gvini et l. [11]). shown in Tble 5. A slightly higher percentge of A. hydrophil ws recovered from sewge smples thn from surfce wter smples on m-lmm nd m-tmm grs. However, percentges of A. hydrophil were much lower on m-lmm nd m-tmm grs thn on m-endo gr or PTG gr. No A. hydrophil were recovered on m-fc gr. The use of the MUG rection to identify E. coli ws specific (dt not shown). Only one MUG-positive non-e. coli ws isolted during this study. It ws typed s Shigell sonnei nd ws isolted from sewge sludge digestor smple. Of 224 E. coli isolted nd identified from surfce wters, 7.7% were MUG negtive nd 9.8% were lctose negtive. Sewge smples yielded 11.2% MUG-negtive nd 12.6% lctose-negtive E. coli of the 129 isoltes identified from this source. Chlorine injury study. The bilities of seven different medi to recover chlorine-injured cells re shown in Tble 6. On m-fc gr significntly lower number of cells ws recovered compred with the other six medi even before chlorintion (P < 0.05). The highest counts were obtined on m-spc gr, which is nonselective medium. As expected, on m-endo nd m-fc grs smll percentges of injured cells were recovered (40 nd 24%, respectively). On PTG, TABLE 4. Lctose-fermenting orgnisms isolted from smples of sewge on four medi Orgnism No. (%) of isoltes fter growth on: m-endo m-fc m-lmm m-tmm Aeromons hydrophil 6 (18.3) 0 (0) 2 (3.3) 3 (5.0) Centers for Disese 1 (3.0) 0 (0) 0 (0) 1 (1.7) Control enteric group 17 Citrobcter sp. 1 (3.0) 0 (0) 0 (0) 0 (0) Citrobcterfreundii 1 (3.0) 1 (3.2) 8 (13.1) 7 (11.7) Enterobcter erogenes 1 (3.0) 0 (0) 0 (0) 1 (1.7) Enterobcter gglomerns 1 (3.0) 0 (0) 2 (3.3) 0 (0) Enterobcter cloce 4 (12.1) 0 (0) 6 (9.8) 3 (5.0) Escherichi coli 5 (15.2) 24 (77.4) 36 (59.0) 32 (53.3) Kluyver sp. 0 (0) 0 (0) 1 (1.6) 1 (1.7) Klebsiell oxytoc 8 (24.3) 0 (0) 3 (5.0) 2 (3.3) Klebsiell ozene 1 (3.0) 0 (0) 1 (1.6) 0 (0) Klebsiell pneumonie 3 (9.1) 5 (16.2) 2 (3.3) 9 (15.0) Serrti liquefciens 0 (0) 0 (0) 0 (0) 1 (1.7) Serrti oderifer 1 (3.0) 0 (0) 0 (0) 0 (0) Shigell sonnei 0 (0) 1 (3.2) 0 (0) 0 (0) Coliform (s defined by Gvini et l. [11]). m-t7, m-lmm, nd m-tmm grs, significntly greter numbers of injured cells were recovered (P < 0.05). DISCUSSION Incresed recoveries of totl coliforms were obtined on m-tmm gr when compred with m-endo gr when surfce wter smples were exmined. Incresed recoveries of fecl coliforms were obtined from surfce wters on PTG nd m-lmm grs when compred with m-fc gr nd from sewge on PTG gr. The surfce wter smples probbly contined higher number of injured cells thn the sewge smples. A significntly different spectrum of bcteril species ws recovered on the monensin-contining medi (m-lmm nd m-tmm) in comprison with the Tergitol 7-contining medium (PTG gr; dt not shown) nd the stndrd medi. The most importnt difference ws the recovery of A. hydrophil; lrge proportions of coliform-positive colonies on m-endo gr nd totl colonies on PTG gr were A. hydrophil. Aeromons spp. usully re excluded from the coliform group (11) becuse they re not normlly found in the feces of helthy humns nd would not, therefore, indicte fecl pollution (1). A. hydrophil cn lso multiply in the environment (2). A method to enumerte specificlly Aeromons spp. in the presence of closely relted bcteri hs been developed (26). Becuse m-lmm nd m-tmm grs recovered low numbers of eromonds, bckground interference ws reduced, nd the counts tht were obtined reflected more ccurtely the snitry history of the wter smple. A greter diversity of smple types nd loctions should be exmined to determine if this incresed specificity is universl nd consistent. TABLE 5. Numbers nd percentges of lctose-positive nd -negtive A. hydrophil isolted from surfce wters nd sewge from five different medi Medium No. (%) of A. hydrophil from: Surfce wters Sewge m-endo 45 (52.3) 34 (50.8) m-fc 0 (0) 0 (0) PTG 16 (18.2) 17 (28.8) m-lmm 5 (3.6) 9 (9.5) m-tmm 5 (3.6) 10 (10.6)
VOL. 53, 1987 TABLE 6. Recoveries of chlorine-injured E. coli B on seven medi Count/100 ml: Medium Before After % Injured chlorintion chlorintion-b cells recoveredc m-spc 7.1 x 109" 1.2 x 107de 100.0 m-endo 6.8 x 109' 4.8 x 106 39.9 m-fc 4.1 x 109 2.9 x 106 24.3 PTG 6.0 x 109d 7.3 x 106d 60.8 m-t7 6.8 x 109d 9.0 x 106d.e 75.3 m-lmm 6.1 x 109d 7.6 x 106d 63.7 m-tmm 6.8 x 109d 7.6 x 100'f 63.3 Arithmetic mens of three runs. b Treted with 2.6 mg of chlorine per liter for 6 min t 25 C. c (Count on selective medi/count on m-spc) x 100. d Very significnt increse over m-fc count (P < 0.05). every significnt increse over m-endo count (P < 0.05). f Significnt increse over m-endo count (P < 0.1). When E. coli re enumerted on PTG, m-lmm, or m- TMM grs, pltes should be exmined for fluorescent colonies, even if the totl number of colonies is too numerous to count. If E. coli is present s smll frction of the coliform popultion of smple, seprte dilutions must be prepred for the totl coliform nd E. coli counts. The membrnes should be exmined fter 22 to 24 h of incubtion. Cre should be tken not to count fluorescent pseudomonds, which pper s smll, green-glowing colonies under long-wve UV light. The use of NH40H ids in distinguishing between colonies of fluorescent pseudomonds nd true MUG-positive colonies. All colonies with ny yellow or green colortion under white light should be counted s coliforms on m-lmm nd m-tmm grs. Noncoliforms will pper smller nd purple. It hs been recommended tht 0.1% peptone wter rther thn diluent contining potssium or sodium be used in conjunction with monensin-contining medi (25). An importnt dvntge of using one of the three new medi tested in this study is tht no resuscittion step is needed, thus reducing time nd expense. The bility to enumerte E. coli (fecl coliforms) t 35 C, rther thn t 44.5 C, increses recovery of injured cells without the use of low-temperture resuscittion step (16). The determintion of E. coli directly provides more ccurte indiction of fecl pollution thn do determintions of fecl coliforms bsed on their bility to grow t high tempertures. High counts of thermotolernt Klebsiell spp. hve been reported in wters tht were not polluted by fecl mteril (4, 30). The recovery of chlorine-injured cells on m-lmm nd m-tmm grs ws slightly below tht on m-t7 gr, lthough the difference ws not sttisticlly significnt for this set of experiments. Unfortuntely, becuse of the formultion of m-t7 gr, lctose-fermenting colonies produced lrge quntities of cid, nd the MUG rection ws often difficult nd sometimes impossible to detect. The fluorescence of 4-methylumbelliferone is highly dependent on ph (13). More work needs to be done to determine the efficcy of the use of these new medi in recovering indictor cells in chlorinted tp wters. For enumertion of both totl coliforms nd E. coli on single medium t 35 C, m-lmm nd m-tmm grs should be used side by side on representtive smples to determine which medium is best for specific smple ctegory. The use of these medi elimintes the need for criticlly controlled MEDIA FOR COLIFORMS AND E. COLI 1249 44.5 C incubtor nd the need for two membrnes nd medi for ech smple (one for totl coliforms nd nother for fecl coliforms) nd lso offers incresed specificity. ACKNOWLEDGMENTS This study ws supported by grnt from the Iow High Technology Council. The skilled technicl ssistnce of Andrew K. Benson is gretly pprecited. LITERATURE CITED 1. Americn Public Helth Assocition. 1985. Stndrd methods for the exmintion of wter nd wstewter, 16th ed. Americn Public Helth Assocition, Wshington, D.C. 2. Burmn, N. P., nd J. S. Colbourne. 1977. 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