appl. environ. microbiol. 19

1983, 45(2):484.Appl. Environ. Microbiol. McFetersM W LeChevallier, S C Cameron and G A from drinking water.recovery of coliform bacteria New medium for improved

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Vol. 45, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p. 484-4920099-2240/83/020484-09$02.00/0Copyright C6 1983, American Society for Microbiology

New Medium for Improved Recovery of Coliform Bacteriafrom Drinking Water

MARK W. LECHEVALLIER, SUSAN C. CAMERON, AND GORDON A. McFETERS*Department of Microbiology, Montana State University, Bozeman, Montana 59717

Received 21 June 1982/Accepted 26 October 1982

A new membrane filter medium was developed for the improved recovery ofinjured coliforms from drinking water. The new medium, termed m-T7, consists of5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 mlof Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g ofbromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilledwater. Additional selectivity may be obtained by aseptically adding 0.1 ,ug ofpenicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endoagar. m-T7 agar also recovered an average of43% more verified coliforms from 67surface and drinking water samples than did the standard m-Endo membrane filtertechnique. From drinking water, m-T7 agar recovered nearly three times morecoliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gavefalse-negative reactions, whereas >70% of the typical yellow colonies from m-T7agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolatedon m-T7 agar belonged to one of the four common coliform genera: Escherichia,17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The resultsdemonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolationof injured coliforms from drinking water.

The 15th edition of Standard Methods for theExamination ofWater and Wastewater (1) speci-fies two methods for the microbiological analy-sis of potable water, the most-probable-numbermethod and the membrane filter (MF) method.The MF technique has gained wide acceptancebecause the procedure is simple, rapid, andprecise and gives definitive results. However,factors such as turbidity (16, 19, 22, 28), highnumbers of noncoliform bacteria (9, 10, 19, 21,24, 29, 42, 43), and membrane filter type (33, 41)may severely influence the sensitivity of theprocedure. In addition, the medium specified foruse with the MF technique, m-Endo agar or m-Endo LES agar, has several shortcomings, in-cluding: (i) low recoveries of injured coliforms(13, 31-33); (ii) poor detection and differentia-tion of coliforms from noncoliforms (13, 14, 39);and (iii) uncertainty about the availability ofhigh-quality basic fuchsin (E. Geldreich, person-al communication). As a result, many investiga-tors have proposed alternative most-probable-number or MF methods (12, 15, 25, 26).However, an MF medium has not been found tobe clearly superior to m-Endo agar and capableof isolating injured coliforms from drinking wa-ter.

Recently, we reported that some laboratory

procedures currently used in water analysis mayfurther reduce the recovery of injured coliforms(32). Included in that report was the observationthat the majority of selective media used toisolate gram-negative bacteria recovered 30% orless of the injured coliforms. However, Tergitol7 agar recovered between 71 and 100% of theinjured coliforms tested.

Tergitol 7 agar was first introduced by Chap-man in 1947 (8). The medium produced a con-sistent and characteristic colonial morphologywith Escherichia coli, Enterobacter aerogenes,and other gram-negative bacteria. Tergitol 7(sodium heptadecyl sulfate) also inhibited manygram-positive bacteria, including Staphylococ-cus aureus, Bacillus subtilis, and Bacillus cereus(36). This medium was later modified to include0.04% 2,3,5-triphenyltetrazolium chloride andwas recommended as the medium of choice forthe quantitative detection of E. coli in rat fecesand drinking water (27, 38). In more recentstudies, Tergitol 7 medium (without 2,3,5-tri-phenyltetrazolium chloride) recovered slightlymore E. coli from chlorinated waters than dideither Teepol 610 or sodium lauryl sulfate medi-um (25).

In this report, we describe a further modifica-tion of Tergitol 7 agar to improve its selective

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NEW COLIFORM MEDIUM 485

TABLE 1. Formulation of m-T7 mediuma

Ingredientb Amt per liter ofdistilled water

Difco Proteose Peptone no. 3 ......... 5 gYeast extract .................... 3 gLactose .......... .......... 20 gTergitol 7 (25% solution) ............. 0.4 mlPolyoxyethylene ether W-1 ........... 5 gBromthymol blue.................... 0.1 gBromcresol purple................... 0.1 gAgar ....... ............. 15 g

a The medium was autoclaved at 121°C for 15 min,and final pH was adjusted aseptically to 7.4 with 0.1 NNaOH. Additional selectivity may be obtained byaseptically adding 0.1 ,ug of penicillin G per ml to themedium after autoclaving. Media prepared with peni-cillin G should be used within 1 week when stored at40C.

b All ingredients were manufactured by Difco Labo-ratories except polyoxyethylene ether W-1 and brom-cresol purple, which were manufactured by SigmaChemical Co., and Tergitol 7, which was obtainedfrom Baker Chemical Co.

and differential properties. This modified medi-um, termed m-T7, was superior to m-Endo agarfor recovering coliforms from drinking water.

MATERIALS AND METHODS

Study area and sample collection. Drinking watersamples were collected from the distribution system oftwo small communities near Bozeman, Mont. The firstdrinking water system serviced 2,000 residents andconsisted of both surface water and well water thatwas only intermittently chlorinated. The second sys-tem serviced 8,000 residents and consisted of a net-work of eight unchlorinated wells. Raw water sampleswere collected from the East Gallatin River approxi-mately 0.5 mile (0.8 km) downstream from the outfallof a sewage treatment plant. Laboratory-chlorinatedsamples were prepared in two ways: (i) surface waterwas treated with 1.0 mg of free residual chlorine perliter (average pH, 7.7; average temperature, 10°C),prepared daily from stock solutions of bleach; (ii)chlorinated distribution water (free residual chlorine,0.5 to 1.2 mg of chlorine per liter; average pH, 7.6;average temperature, 10°C) was mixed with 10o sur-face water. Both preparations were dechlorinated withsodium thiosulfate (0.008%) after 10 min of contact (1).Water samples were collected in sterile 2-liter poly-

propylene containers with (drinking water) or without(surface water and samples for laboratory chlorina-tion) added sodium thiosulfate (0.008%) (1). Sampleswere placed on ice and transported to the laboratorywithin 1 h and analyzed within 5 h after collection.Medium development. A number of selective and

differential compounds were tested to determinewhich agents allowed the recovery of stressed coli-forms. Coliforms were injured by placing washedcultures of E. coli, Klebsiella pneumoniae, Enterobac-ter cloacae, or Citrobacter freundii (10' colony-form-ing units/ml) in diffusion chambers (33) that wereimmersed in Bozeman chlorinated drinking water, aspreviously published (6, 31-33, 43, 45). Injury was

determined as the percent difference between thenumber of colony-forming units on the nonselectivemedium (TLY) and the selective medium (TLY-D)(32). The recovery of injured coliforms was evaluatedin the presence of the following selective agents:Tergitol 7 (J. T. Baker Chemical Co.), Tergitol 4,Tergitol 8, polyoxyethylene ether W-1, and Triton X-100 (all from Sigma Chemical Co.) in PLY agar (DifcoProteose Peptone no. 3, 5.0 g; lactose, 20 g; yeastextract, 3.0 g; and agar, 15 g, per liter of distilled water[Table 1]). Differential agents that were tested includ-ed: bromthymol blue (sodium salt; Sigma), bromcresolpurple (Difco), eosin B, eosin Y, methylene blue,analine blue, and janus green (all from Baker) andwere added to PLY agar. In all cases coliforms wereenumerated by the spread plate technique and incubat-ed at 35°C for 24 h (1).Medium preparation and microbiological analysis.

The formulation and preparation instructions for m-T7agar are shown in Table 1. Occasionally, high numbersof gram-positive bacteria in drinking water (29) pro-duced a false-positive reaction. Additional selectivitywas obtained by aseptically adding penicillin G (0.1,ug/ml; Sigma) to the medium after autoclaving. Stocksolutions of 0.01 mg of penicillin G per ml were filtersterilized, using a 0.22-p.m membrane filter (MilliporeCorp.), and frozen in 2.5-ml amounts. When m-T7medium was prepared, one 2.5-ml vial of the antibioticwas added to 250 ml of tempered agar. Penicillin Gmay be stored frozen for 6 months, and preparedplates may be stored at refrigerator temperature for upto 1 week provided excessive drying does not occur (1,18, 37).Medium performance was evaluated by filtering

three replicates of each sample dilution, using six MFtechniques. The standard and resuscitation MF tech-niques (labeled m-Endo and m-Endo + lauryl tryptosebroth [LTB], respectively) were conducted accordingto established procedures, using Millipore HC-typefilters and m-Endo agar and LTB (both from Difco).Millipore HC-type membrane filters were used be-cause the surface pore size (2.4 ,um) has been indicatedas optimum for recovery of injured fecal coliforms (32,41). In the third MF technique, plates prepared withapproximately 7 ml of m-Endo agar were overlaid with3 ml of lactose agar (Difco) immediately before use(labeled m-Endo + lactose agar). A single-step andresuscitation MF procedure (labeled m-T7 and m-T7 +PLY, respectively) were developed with m-T7 agar.PLY broth rather than LTB was used to saturatesterile resuscitation pads (Millipore Corp.) on whichmembrane filters were preincubated. The last MFtechnique compared in this study used anaerobic incu-bation of m-T7 agar plates (labeled m-T7 A) in a Gas-Pak (BBL Microbiology Systems) anaerobe jar. Com-parisons between m-T7 agar and Tergitol 7 agar(Difco) were also made. Volumes sampled were 100 mlfor drinking water, 0.1 ml for surface water, and 0.1 to50 ml for laboratory-chlorinated water samples. Be-cause widely varying dilutions were used for differentwater samples, counts are tabulated on a per-filterbasis and are represented irrespective of dilution.

All smooth, yellow, convex colonies on m-T7 agarand typical green-sheen colonies on m-Endo agar werecounted with the aid of a dissecting microscope (x15to 20). At least 20%o of the colonies were transferredfrom the membrane filter into tubes of LTB for deter-

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486 LECHEVALLIER, CAMERON, AND McFETERS

mination of gas production. Positive LTB tubes werenot routinely transferred to brilliant green bile brothbecause of the inhibitory nature of this medium (14),but all gas-producing isolates from m-T7 agar wereidentified with the API-20E system (Analytab Prod-ucts).

Quality control and statistical comparisons. A qualityassurance program, as outlined in Standard Methods(1) and Microbiological Methods for Monitoring theEnvironment (3), was used throughout the course ofthis study. Performance of media and sterility controlswere determined on a per-lot or a per-batch basis.Materials used during each experiment were checkedfor sterility. The temperatures of autoclaves and incu-bators were monitored on a per-use basis.

Statistical comparisons were made by using thepaired t-test.

RESULTS

To ensure the growth of injured coliforms on

m-T7 agar, various selective ingredients were

examined to determine the optimum level whichpermitted high recoveries of stressed organisms.A variety of surface-active agents were initiallytested, including Tergitol 4, Tergitol 7, Tergitol8, polyoxyethylene ether W-1, Triton X-100, andTween 80 (Table 2). Optimum concentrationswere determined as the highest level of surface-active agent which permitted 90 to 100% recov-

ery of injured cells. The optimum concentrationof Triton X-100 was 0.001%, that of Tween 80was 0.1%, and that of Tergitol 7 was 0.01%.Tergitol 4 was very inhibitory to injured cellseven at the 0.001% level, whereas Tergitol 8 andpolyoxyethylene ether W-1 were not inhibitoryat the concentrations tested.The selectivity of the surfactants at concentra-

tions determined to be "noninjurious" to coli-forms was evaluated by determining the percentinhibition of standard plate count (SPC) bacteriafrom contaminated river water. Tergitol 7 inhib-ited 55% of SPC bacteria; polyoxyethylene etherW-1, 54%; Tergitol 8, 7%; and Triton X-100, 7%.Tween 80 showed no inhibition (Table 2). Acombination of Tergitol 7 and polyoxyethyleneether W-1 exhibited a 55% inhibition of SPCbacteria, whereas m-Endo agar showed 78%inhibition.A variety of differential agents were evaluated

as indicators of lactose fermentation (Table 2).Compounds were either noninjurious and poorin differentiating lactose fermentation (eosin B)or fair to good in differentiating lactose fermen-tation but very inhibitory to injured coliforms(eosin Y, erythrosin, methylene blue, analineblue, janus green). Bromthymol blue and brom-cresol purple were not inhibitory to injuredcoliforms at the 0.01% level and gave gooddifferential reactions when used in combination.In addition, the combination of the two dyes

provided additional inhibition of noncoliformbacteria (Table 2).

Occasionally, strains of Staphylococcus sp.and Micrococcus sp. produced false-positivecoliform reactions on m-T7 agar. Incorporationof 0.1 ,ug of penicillin G per ml (final concentra-tion) aspetically added to the medium after auto-claving prevented the growth of these orga-nisms. Parallel tests of m-T7 agar with andwithout penicillin G gave equivalent recovery ofboth laboratory-injured coliforms and coliformsfrom nine surface and drinking water samples(data not shown).To evaluate the efficiency of m-T7 agar for

recovery of injured coliforms, cultures of E.coli, K. pneumoniae, Enterobacter cloacae, andC. freundii were stressed (>90% injury) in drink-ing water. Depending on the organism tested, m-T7 agar recovered 86 to 99% more coliformsthan did m-Endo agar (data not shown). Inaddition, 67 water samples were analyzed withm-T7 and m-Endo agars by various MF tech-niques. Overall, m-T7 agar recovered signifi-cantly more (P < 0.05) coliforms than did eitherthe standard m-Endo or the m-Endo with LTBresuscitation techniques (Fig. 1 and 2). For allwaters tested, m-T7 agar recovered 43% morecoliforms than did m-Endo agar and 36% morecoliforms than did m-Endo agar with LTB resus-citation. In one instance, m-T7 agar recovered17 confirmed coliforms, whereas m-Endo agarrecovered none (Fig. 1 and 2). From the 44drinking water samples analyzed, m-T7 agarrecovered 2.7 times more coliforms than did them-Endo resuscitation technique and nearly threetimes more coliforms than did the standard m-Endo technique (Table 3). From drinking watersamples, m-T7 agar recovered more verifiedcoliforms than any of the other five MF tech-niques (Table 3).The single-step m-T7 agar technique is the

easiest method for analysis of drinking water,but other methods (m-T7 + PLY and m-T7 A)were used to compare the effectiveness of thesingle-step technique. Addition of a resuscita-tion step to m-T7 agar generally did not increaseoverall coliform recoveries, but the resuscitationtechnique did recover more coliforms than m-T7agar from laboratory-chlorinated surface watersamples.Anaerobic incubation of m-T7 agar plates was

used as a means of inhibiting SPC bacteria, sincethe majority of SPC bacteria in drinking waterare obligate aerobes (21, 29). This technique waseffective for surface water and chlorinated sur-face water, where high densities of "back-ground" bacteria existed (Table 3). However,anaerobic incubation of m-T7 agar proved to beinadequate in recovering more coliforms fromdrinking water samples than the standard m-

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TABLE 2. Effect of various indicator/selective agents on inhibition of SPC bacteria and recovery of injuredE. coli

%Of ISP %Of

Indicator/selective agent' injured inhib- Indicator/selective agent1' injured SPCcoliform itedb coliform inhib-

recovered" ie II recoveredb itedc

Surfactants (%)Tergitol 4

0.0010.0050.01

Tergitol 70.010.050.1

Tergitol 80.010.050.1

Triton X-1000.0010.0050.01

Tween 800.10.51.0

Polyoxyethylene ether W-10.050.10.5

Polyoxyethylene ether W-1(0.5) + Tergitol 7 (0.01)

85 NDd85 ND60 ND

92 5572 ND33 ND

105 ND88 ND95 7

100 773 ND29 ND

104 073 061 0

95 51100 51102 54

97 55

Indicators (%)Bromthymol blue

0.010.05

Bromthymol blue (0.01) +bromcresol purple (0.01)

Bromcresol purple0.0050.010.05

Eosin B0.0010.0050.01

Eosin Y0.0010.0050.01

Erythrosin0.0010.0050.01

Methylene blue0.0010.0050.01

Analine blue0.0010.0050.01

Janus green (0.01)

m-Endo agar

113 3792 87

99 58

114 32103 2390 75

103 27102 19117 23

86 4924 8237 85

83 7417 949 99

88 3276 5376 55

64 ND25 ND16 ND

14 ND

53 74a The basal medium contained PLY agar.I Between 90 and 999o of the coliforms were injured.I Percent inhibition of SPC bacteria in Gallatin River water was calculated relative to the nonselective PLY

counts.d ND, Not done.

Endo agar technique. A lactose agar overlaymethod was used as another way of resuscitatinginjured coliforms since previous research hasindicated that LTB may inhibit as many as 79oof injured coliform organisms (32). The overlaytechnique recovered significantly more (P <0.05) coliforms than did the LTB resuscitationmethod when only chlorinated surface watersamples were tested. For surface water and

drinking water there was no statistical differencein coliform recoveries between the methods.

Initial studies compared coliform recoveriesof Tergitol 7 agar with m-T7 agar. m-T7 agarrecovered 2.2 times more coliforms from 19surface and drinking water samples, comparedwith Tergitol 7 agar (9.69 and 4.45, respective-ly). Moreover, only 49.7% of the 169 presump-tive coliform isolates on Tergitol 7 agar pro-

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70

60

50

r'.I-E

.

0

040J Sof equality

0

.30

20 /0

20

0

10 * /

0 1 0 20 30 40 50 60

m-EndoFIG. 1. Comparison of coliform recoveries from water samples on m-Endo and m-T7 agars. Counts are

represented as relative values.

duced gas in LTB. Because of low coliformrecoveries and low verification rates, furtherevaluation of Tergitol 7 agar was not continued.Nearly 240 nonyellow, background colonies

from m-T7 agar were tested for false-negativecoliform reactions. These colonies were inocu-lated into LTB and tested for gas productionafter 48 h at 35°C incubation. Less than 0.5%(one coliform) proved to be a false-negative fromm-T7 agar. This organism was identified asSerratia rubidaea by the API-20E system. Inaddition, up to 10 coliform colonies from eachtechnique and sample were verified for gas pro-duction by inoculating tubes of LTB. Over 930yellow coliform colonies from m-T7 agar werechecked for gas production; of these, 658(70.6%) were positive. Colonies picked from m-Endo agar had a confirmation rate slightly lowerthan that ofm-T7 agar; 69.6% of the 860 coliformcolonies tested produced gas in LTB.A total of 295 coliforms isolated on m-T7 agar

have been identified by the API-20E system(Table 4). The predominate genera were Esche-

richia (17.6%), Klebsiella (21.7%), Enterobacter(32.2%), and Citrobacter (17.3%). About 5% ofthe isolates were Aeromonas, Serratia, and Pro-teus species and 7.5% were not identified. Over90% of the coliforms isolated on m-T7 agar gavetypical green-sheen colonial reactions when re-streaked on m-Endo agar.

DISCUSSIONThe many problems associated with current

m-Endo-type medium formulations prompted usto develop a new medium capable of accuratelyenumerating coliform densities from drinkingwater. One of the most important considerationsis the inhibitory nature of the selective ingredi-ents in m-Endo-type agar. Coliforms in the envi-ronment may be stressed by exposure to avariety of factors, including chlorine and otherdisinfectants, heat, freezing, acid mine drainage,transition metals, sunlight, and UV light (2, 5, 6,17, 23). Previous research has shown that vari-ous medium formulations commonly used for



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0

0

.

40 *

/E/ E

30

E /line of equality

20

10~~~~~~~

* jr

0 10 20 30 40 50 80 70

m-Endo LTBFIG. 2. Comparison of coliform recoveries from water samples on m-Endo agar with LTB resuscitation and

m-17 agar. Counts are represented as relative values.

water analysis, containing more than 0.05% bilesalts or deoxycholatae, were highly inhibitory toinjured coliforms (32). M-Endo agar contains0.1% deoxycholate plus 0.005% sodium laurylsulfate and has been shown to inhibit as many as70%o of the injured coliforms (32). Because ofinadequate techniques to measure injury, theextent of injured coliforms in drinking water islargely unknown, although some reports esti-mate that coliforms in aquatic environments are

recovered with efficiencies of 10%o or less (6, 13,31, 32). The results of this study indicated thattwo-thirds of the coliforms present in drinkingwater samples were injured.The second problem associated with m-Endo-

type agar formulations is the inability to distin-guish coliforms from noncoliforms. Coliformsare differentiated from other bacteria on m-

Endo-type media by the production of a darkcolony with a metallic green sheen (1). In one

study, nearly 25% of the nonsheen backgroundcolonies on m-Endo LES agar produced gas inm-LAC broth and were identified as Citro-bacter, Enterobacter, Escherichia, or Klebsiella

species (14). Such occurrences of false-negativecoliforms on m-Endo agar are particularly dis-tressing since these organisms would not beinterpreted as an indication of potentially con-

TABLE 3. Comparison of m-Endo and m-T7techniques

Relative coliform counts

Medium Drinking Surface Chlorinatedwater water surface water

(n = 44) (n = 11) (n = 12)

m-Endo 1.41 27.8 13.6m-Endo + LTB pad 1.48 32.3a 14.2m-Endo + LA overlayb 1.27 32.8a 22.6am-17 3%.a 39.9a 20.6am-T7 + PLY padC 3.85a 40.3a 25.2am-T7 A 0.57 37.4a 22.8a

a Significantly greater value compared with m-Endo(P < 0.05).

b LA, Lactose agar.c PLY pad, Base composition of m-T7 containing

Difco Proteose Peptone no. 3, lactose, and yeastextract.

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TABLE 4. Identification of coliforms isolated on m-T7 agar

Organism ~~No. of timesOrganism | isolateda

Escherichia coli 52 17.6Enterobacter agglomerans 46 15.6Klebsiella pneumoniae 64 21.7Citrobacter freundii 51 17.3Enterobacter aerogenes 7 2.4Enterobacter cloacae 42 14.2Aeromonas spp. 7 2.4Serratia rubidaea 1 0.3Serratia liquefaciens 2 0.7Proteus morganii 1 0.3Not identified 22 7.5

a Coliforms were isolated from surface water,chlorinated surface water, and drinking water (seetext).

taminated drinking water. Several outbreaks ofsalmonella and poliomyelitis virus have beenreported in drinking water which had no or lowcoliform levels, possibly because injured coli-forms did not grow on m-Endo agar or becausethe indicator organisms failed to produce a typi-cal green-sheen colony (4, 30, 35, 40).The accuracy and sensitivity of the MF tech-

nique are greatly influenced by the efficiency ofthe verification procedure used. As many as56% of the typical green-sheen colonies on m-Endo agar may not produce gas in lactose broth(13, 14, 20). These false-positive reactions on m-Endo-type agar have been attributed to sheenproduction by slow lactose fermenters (20),gram-positive bacteria (20), and synergistic reac-tions producing a sheen by two noncoliforms(39). Recently, however, methods used for theconfirmation of gas production have been attrib-uted to cause low verification rates (14, 34).Factors known to influence the rate and amountof gas production in lactose-containing mediainclude nutritional composition (7, 11), amountof buffer (14, 34), medium volume, and Durhamtube size (18).An additional problem with m-Endo-type me-

dia is the availability and quality of basic fuchsin(Geldreich, personal communication). Repro-ducibility of water sampling data depends heavi-ly on the consistent quality and adequate supplyof all ingredients in the specified medium formu-lations. Recently, changes in quality and solubil-ity of dyes in m-Endo-type agar have beennoted. It is unknown at this time how mucheffect dye quality has on coliform detection.The results of this report have demonstrated

that there is an effective alternative to m-Endoagar for the analysis of coliforms in drinkingwater. Steps taken in the formulation of m-T7agar have ensured that the medium is selective

while minimizing the inhibition of stressed coli-forms. The effectiveness of m-T7 agar is evi-denced by a 43% overall increase in verifiedcoliform counts from all samples tested (Fig. 1).High recoveries of laboratory-injured coliformsas well as a threefold increase in coliform recov-eries from drinking water samples were alsoobserved (Table 3). In addition to yielding highcoliform counts, m-T7 agar also had a false-negative rate of <0.5%. Only 1 nonyellow colo-ny of 239 tested proved to be a coliform.

In this study both m-Endo and m-T7 agars hadhigh rates of false-positive coliforms. However,preliminary data of ongoing studies indicatethat, of the 30% coliform colonies not producinggas in LTB, nearly 80% of the 145 isolates testedwere o-nitrophenyl-,-D-galactopyranoside posi-tive and cytochrome oxidase negative. At pres-ent, 29 of these organisms have been identified:66% were Enterobacter agglomerans; 17%, E.coli; 10%, K. pneumoniae; 3%, C. freundii; and3%, Enterobacter cloacae. It is possible thatyellow-pigmented bacteria may occasionally becounted as false-positive coliforms, but withexperience pigmented organisms and coliformsproducing a yellow acid reaction on m-T7 agarcan easily be distinguished.There was no problem with excessive back-

ground colonies on m-T7 agar with the 44 drink-ing water samples examined in this study; how-ever, there was some crowding of colonies onthe filters of both types of medium when surfacewater samples containing sewage effluent wereexamined. The data in Table 3 indicate that thesingle-step MF technique may be used with m-T7 agar for analysis of coliforms in all waters,but anaerobic incubation may facilitate the re-covery of coliforms from highly contaminatedsurface waters.Most of the coliforms isolated on m-T7 agar

were identified as members of one of the fourcommonly accepted coliform genera (Table 4). Itis not possible to determine from these datawhether m-T7 agar was biased in the types ofcoliforms detected. However, over 90% of thecoliforms isolated on m-T7 agar gave typicalgreen-sheen reactions when restreaked on m-Endo agar. It can be concluded that the differ-ence in recoveries between the two media is dueto injured coliforms not capable of growing onm-Endo agar and not due to atypical or unusualcoliform isolates.Although this report has demonstrated the

superiority of m-T7 agar to m-Endo agar in therecovery of coliforms from Montana drinkingwater, additional testing needs to be done tofully evaluate the effectiveness of m-T7 agar inother regions. In addition, other problems asso-ciated with the MF technique, such as efficiencyof verification procedures, effects of particulates



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and SPC organisms on recovery of indicatorbacteria, and factors relating to aquatic injury,require further investigation to make the indica-tor concept a more reliable predictor of potablewater quality.

ACKNOWLEDGMENTS

We thank Theresa Ramirez, Kathy Collins, Marie Martin,Kelly Kimball, and Bruce Lapke for technical assistance. Thesuggestions and comments of Donald A. Schiemann are alsogreatly appreciated.

This study was supported by funds from the MicrobiologicalTreatment branch of the Drinking Water Research Division,U.S. Environmental Protection Agency, Cincinnati, Ohio(grant R80709201).

LITERATURE CITED

1. American Public Health Association. 1980. Standard meth-ods for the examination of water and wastewater, 15th ed.American Public Health Association, Washington, D.C.

2. Beuchat, L. R. 1978. Injury and repair of gram-negativebacteria, with special consideration of the involvement ofthe cytoplasmic membrane. Adv. Appl. Microbiol.23:219-243.

3. Bordner, R., and J. Winter (ed.). 1978. Microbiologicalmethods for monitoring the environment. U.S. Environ-mental Protection Agency, Cincinnati, Ohio.

4. Boring, J. R., III, W. T. Martin, and L. M. Elliott. 1971.Isolation of Salmonella typhimurium from municipal wa-

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