APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 341-348 0099-2240/78/0036-0341$02.00/0 Copyright i 1978 American Society for Microbiology Vol. 36, No. 2 Printed in U.S.A. Fecal Coliform Elevated-Temperature Test: a Physiological Basis WILLIAM S. DOCKINS AND GORDON A. McFETERS* Department of Microbiology, Montana State University, Bozeman, Montana 59717 Received for publication 14 March 1978 The physiological basis of the Eijkman elevated-temperature test for differen- tiating fecal from nonfecal coliforms was investigated. Manometric studies indi- cated that the inhibitory effect upon growth and metabolism in a nonfecal coliform at 44.50C involved cellular components common to both aerobic and fermentative metabolism of lactose. Radioactive substrate incorporation experi- ments implicated cell membrane function as a principal focus for temperature sensitivity at 44.5°C. A temperature increase from 35 to 44.50C drastically reduced the rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecal coliforms were essentially unchanged. In addition, relatively low levels of nonfecal coliform ,B-galactosidase activity coupled with thermal inactivation of this enzyme at a comparatively low temperature may also inhibit growth and metabolism of nonfecal coliforms at the elevated temperature. The coliform group of bacteria has been de- fined as "all the aerobic and facultative anaero- bic, gram-negative, nonsporeforming, rod- shaped, bacteria that ferment lactose with gas formation within 48 hours at 35C" (1). This group has long been used as an indicator of fecal contamination of natural waters. Because water- borne coliforms can originate from sources other than the intestinal tract of warm-blooded ani- mals, such as from the soil or the surface of vegetation and insects (7, 8), it is desirable to determine the source of these organisms before attempting to relate them to fecal contamination and the potential presence of pathogens. A num- ber of different procedures have been devised to separate fecal from nonfecal coliforms; however, the most successful and widely accepted tech- nique involves incubation of coliforms at ele- vated temperatures. The first elevated-temperature test proposed by Eijkman (5) differentiated fecal from nonfecal coliforms by the ability of fecal coliforms to ferment glucose in a glucose-peptone broth at 460C. The current and most widely used ele- vated-temperature procedure relies upon fecal coliform fermentation of lactose in a highly buffered medium, with the production of hydro- gen and carbon dioxide at 44.50C. These im- proved procedures are largely the result of the early work done by Perry and Hajna (10, 11, 21) and more recently by Geldreich et al. (6, 7). These workers and Mishra et al. (20) have re- ported a high correlation of gas production from lactose at 44.50C with coliforms that are nor- mally associated with fecal pollution. Some waterborne coliforms not normally con- sidered to be of fecal origin are able to ferment lactose at 44.50C (6, 12, 20), and under certain conditions these organisms may be present in relatively high numbers in water samples (2). These findings constitute a basis for controversy over the validity of the elevated-temperature test for indicating the potential presence of path- ogens in the aquatic environment. The wide- spread use and sanitary implications of the ele- vated-temperature procedure in detecting fecal water pollution stress the importance of deter- mining and understanding the physiological mechanisms involved in colifonn growth and metabolism at 44.50C. A knowledge of the bio- chemical basis would also be of value in the formulation of improved fecal coliforn assay procedures. The primary objectives of this in- vestigation were to locate the cellular site(s) of temperature sensitivity of nonfecal coliforms which might account for their failure to ferment lactose or glucose at 44.50C and to describe the physiological characteristics of coliforms insofar as they relate to the elevated-temperature test. MATERIALS AND METHODS Cultures. The enteric bacterial cultures used in these studies were obtained from the Montana State University (MSU) Culture Collection or the American Type Culture Collection or were isolated by membrane filtration from streams in the area of Bozeman, Mont. All organisms conformed to the coliform designation as defined by the American Public Health Association (1). Stream isolates were further differentiated by indole, methyl red, Voges-Proskauer, and citrate (IMViC) classification and by the ability of the orga- 341 Downloaded from https://journals.asm.org/journal/aem on 15 December 2021 by 171.224.229.25.
Transcript
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, p. 341-3480099-2240/78/0036-0341$02.00/0Copyright i 1978 American Society for Microbiology
Vol. 36, No. 2
Printed in U.S.A.
Fecal Coliform Elevated-Temperature Test: a PhysiologicalBasis
WILLIAM S. DOCKINS AND GORDON A. McFETERS*
Department ofMicrobiology, Montana State University, Bozeman, Montana 59717
Received for publication 14 March 1978
The physiological basis of the Eijkman elevated-temperature test for differen-tiating fecal from nonfecal coliforms was investigated. Manometric studies indi-cated that the inhibitory effect upon growth and metabolism in a nonfecalcoliform at 44.50C involved cellular components common to both aerobic andfermentative metabolism of lactose. Radioactive substrate incorporation experi-ments implicated cell membrane function as a principal focus for temperaturesensitivity at 44.5°C. A temperature increase from 35 to 44.50C drastically reducedthe rates of [14C]glucose uptake in nonfecal coliforms, whereas those of fecalcoliforms were essentially unchanged. In addition, relatively low levels of nonfecalcoliform ,B-galactosidase activity coupled with thermal inactivation ofthis enzymeat a comparatively low temperature may also inhibit growth and metabolism ofnonfecal coliforms at the elevated temperature.
The coliform group of bacteria has been de-fined as "all the aerobic and facultative anaero-bic, gram-negative, nonsporeforming, rod-shaped, bacteria that ferment lactose with gasformation within 48 hours at 35C" (1). Thisgroup has long been used as an indicator of fecalcontamination ofnatural waters. Because water-borne coliforms can originate from sources otherthan the intestinal tract of warm-blooded ani-mals, such as from the soil or the surface ofvegetation and insects (7, 8), it is desirable todetermine the source of these organisms beforeattempting to relate them to fecal contaminationand the potential presence of pathogens. A num-ber of different procedures have been devised toseparate fecal from nonfecal coliforms; however,the most successful and widely accepted tech-nique involves incubation of coliforms at ele-vated temperatures.The first elevated-temperature test proposed
by Eijkman (5) differentiated fecal from nonfecalcoliforms by the ability of fecal coliforms toferment glucose in a glucose-peptone broth at460C. The current and most widely used ele-vated-temperature procedure relies upon fecalcoliform fermentation of lactose in a highlybuffered medium, with the production of hydro-gen and carbon dioxide at 44.50C. These im-proved procedures are largely the result of theearly work done by Perry and Hajna (10, 11, 21)and more recently by Geldreich et al. (6, 7).These workers and Mishra et al. (20) have re-ported a high correlation of gas production fromlactose at 44.50C with coliforms that are nor-mally associated with fecal pollution.
Some waterborne coliforms not normally con-sidered to be of fecal origin are able to fermentlactose at 44.50C (6, 12, 20), and under certainconditions these organisms may be present inrelatively high numbers in water samples (2).These findings constitute a basis for controversyover the validity of the elevated-temperaturetest for indicating the potential presence ofpath-ogens in the aquatic environment. The wide-spread use and sanitary implications of the ele-vated-temperature procedure in detecting fecalwater pollution stress the importance of deter-mining and understanding the physiologicalmechanisms involved in colifonn growth andmetabolism at 44.50C. A knowledge of the bio-chemical basis would also be of value in theformulation of improved fecal coliforn assayprocedures. The primary objectives of this in-vestigation were to locate the cellular site(s) oftemperature sensitivity of nonfecal coliformswhich might account for their failure to fermentlactose or glucose at 44.50C and to describe thephysiological characteristics of coliforms insofaras they relate to the elevated-temperature test.
MATERIALS AND METHODSCultures. The enteric bacterial cultures used in
these studies were obtained from the Montana StateUniversity (MSU) Culture Collection or the AmericanType Culture Collection or were isolated by membranefiltration from streams in the area of Bozeman, Mont.All organisms conformed to the coliform designationas defined by the American Public Health Association(1). Stream isolates were further differentiated byindole, methyl red, Voges-Proskauer, and citrate(IMViC) classification and by the ability of the orga-
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nism to ferment lactose at 44.5°C in EC broth (DifcoLaboratories) with the production of gas. Those coli-forms capable of fermenting lactose with gas produc-tion at 44.5°C were termed fecal coliforms (1) andincluded Escherichia coli B (MSU culture collectionno. 164) and stream isolates of IMViC types ++--(five strains) and -+--. Coliforms unable to fermentlactose at the elevated temperature (nonfecal coli-forms) included Klebsiella pneumoniae ATCC 13883and stream isolates of IMViC types -+++, -+-+,and -+--. All cultures were maintained on nutrientagar (Difco) and stored at refrigeration temperatures.Manometric methods. Cells employed in experi-
ments with EC medium were grown in tryptic soybroth (Difco)-0.3% yeast extract-0.25% glucose (TSY)at room temperature and were aerated by shaking.The coliforms were harvested after 18 h by centrifu-gation at 3,000 x g (Sorvall, RC2-B), washed twice,resuspended in cold standard phosphate buffer (1),and standardized to 0.60 absorbance at 660 nm (A60)with a Varian Tectron model 635 spectrophotometer.
Cells used in experiments in which TSY broth wasthe medium used were grown without shaking at 35°Cin TSY broth. These cells were harvested after 18 h,washed once, and resuspended in cold phosphatebuffer to Ass of 0.75.
Single side-arm flasks (Gilson) were used in allmanometric experiments. In experiments measuring02 uptake, 0.5 ml of 40% KOH and a fluted filter paperwick were inserted into the center wells of each flask.Aliquots (4 ml) of the specified medium were added tothe main chamber, and 1 ml of a cell suspension wasplaced in the side arm of each flask. Control flasksunderwent similar preparation except that 1 ml ofphosphate buffer was added to the side arm in placeof the cell suspension. The flasks were attached to aGilson differential respirometer and allowed to equi-librate at 44.50C (±0.20C, measured with standardthermometer) for approximately 20 min. In experi-ments that were done anaerobically, the respirometerflasks were purged of 02 by shaking them for 15 minbefore the equilibration time as nitrogen gas waspassed through them. The 1-ml cell suspension in theside arm was then tipped into the main chamber, andthe manometer readings were taken at 10- or 15-mmintervals.
[14Cjglucose uptake. Coliforms used in ['4C]glu-cose uptake experiments were grown without shakingat 350C in TSY broth. The cells were harvested after12 h, washed once with phosphate buffer, and resus-pended in TSY broth to an Ass of 0.1 to 0.2. Flaskscontaining 60 ml of the cell suspension in TSY brothwere equilibrated at 35 or 44.50C in water baths. Atzero time 0.5 ml of uniformly labeled ['4C]glucose (0.5,uCi/ml, New England Nuclear Corp.) was added tothe suspension, and a sample was taken immediatelyand at timed intervals thereafter. Aliquots (3 ml) ofthe culture suspension were removed for biomass de-termination (Ass), and 10-ml aliquots were filteredthrough a 0.45-,um filter (Millipore Corp.) for cellularradioactive glucose uptake measurements. Filters werewashed with distilled water to remove extracellular['4C]glucose, dried for 15 min at 105°C, and placed inpoly Q scintillation vials (Beckman Instrument Co.).Toluene (4 ml) and 9 ml of Aquasol (New England
APPL. ENVIRON. MICROBIOL.
Nuclear) were added to each vial. Labeled carboncounts and external standards were measured for eachvial with a liquid scintillation counter (Beckman LSC100) set at 5% error. Background counts were deter-mined by filtering 10 ml of TSY broth and preparingthis sample in the same manner as described above.Because of the similarity of the external standardcounts, data were directly compared and expressed ascounts per minute per absorbance unit (cpm/A6w).The maximal rate of uptake during the first 40 min ofincubation was graphically calculated by determiningthe slope of the steepest sustained portion of the curve(counts per minute per A6s versis minutes).Temperature shift. Coliform organisms were
grown at 35°C with shaking in TSY broth (withoutglucose) containing sodium acetate at a concentrationof 1.0%. Cells were harvested after 18 h, washed twicewith cold phosphate buffer, and resuspended in freshTSY broth (without glucose). The A6s of this suspen-sion was adjusted to fall within the range of 0.1 to 0.2.The suspension was equally divided into two flaskswhich were placed into a 35°C water bath and allowedto equilibrate for approximately 20 min. Aliquots (10ml) of 10% lactose were added to each flask. The Assof each cell suspension was measured at 15-min inter-vals for 3 h. After 1 h one flask was removed from the35°C bath and placed at 44.5°C while the other flaskremained at 35°C.
,B-Galactosidase assay. Coliform organisms weregrown in TSY broth (without glucose) to which lactoseat a final concentration of 1.0% was added. Thesecultures, fully induced for ,B-galactosidase, were har-vested after 18 h, washed once, and resuspended incold phosphate buffer. The A6s of the cell suspensionswas adjusted to 1.25, and the cells were broken with aBronwill Biosonic IV sonic oscillator set at 90% ofmaximum intensity. Sonication time was 15 min andthe temperature of the suspension was not allowed toexceed 20°C. This method of sonication resulted inapproximately 98% cell death. The extracts were thencentrifuged at 3,000 x g for 5 min to remove remainingcells and larger cell fragments. The total protein con-tained in each extract was determined by the methodof Lowry et al. (17). All cell-free extracts were storedon ice until use.
f-Galactosidase activity was measured in the sonicextracts by a modified o-nitrophenyl-,8-D-galactopy-ranoside (ONPG) hydrolysis method (15,23). A 2.0-mlaliquot of 1.0 mM ONPG was added to 5 ml of theextract, and the mixture was incubated at a prescribedtemperature for exactly 10 or 20 min. At the end ofthe incubation period, 2.0 ml of 0.5 M NaCO3 wasadded, and A420 was measured. In all assays the ONPGused was equilibrated to the temperature of the assaybefore addition. Results are expressed as A420 perminute per milligram of protein, and each data pointis the average of three values.
Aldolase assays. The preparation procedure forsonic extracts used in the aldolase assays was the sameas that used for the f,-galactosidase assays except thattris(hydroxymethyl)aminomethane buffer (0.05 M,Sigma Chemical Co.) was substituted for phosphatebuffer. Aldolase assays were performed by a modifi-cation of the spectrophotometric method of Jaganna-than et al. (13). Aliquots (2 ml) of hydrazine sulfate
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(3.5 mM in 0.1 mM ethylenediaminetetraacetic acid)were placed into a cuvette and allowed to equilibrateto 35 or 44.5°C in a spectrophotometer equipped witha recorder and a jacketed cuvette holder attached toa circulating heater. Fructose 1,6-diphosphate (0.10ml, 12 mM) was then added, and the change in Assover a 3-min interval was measured and recorded.Data were expressed as the initial slope of the Assversus time per milligram of protein in the sonicextract (Ams per minute per milligram of protein).Aldolase assays were performed in triplicate, and eachdata point presented is the average of three initialslopes.
RESULTSThe results of manometric experiments in EC
broth utilizing both a fecal coliforrn isolate(IMViC ++--) and a nonfecal coliform isolate(IMViC-+++) are shown in Fig. 1 and 2. Thefecal coliform was capable of using lactose underboth fermentative and aerobic conditions at44.50C. The nonfecal coliform did not evolve gasfrom lactose at 44.50C in the respirometer underanaerobic conditions and furthermore did notmetabolize lactose aerobically, as evidenced bythe lack of oxygen uptake.
Respiration studies in TSY broth gave resultssimilar to those in EC broth (Fig. 3). Coliformscapable of a positive reaction in EC broth at theelevated temperature (44.50C), including anIMViC type ++-- isolate and E. coli B, showeda high respiration rate in TSY broth at 44.50C,whereas two nonfecal isolates (IMViC-+-+
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and -+++) demonstrated a miniimal rate ofrespiration at that temperature. The nonfecalorganisms, however, showed minimal respirationin TSY broth at 44.50C, in contrast to the lackOf 02 uptake that was observed in EC broth atthat temperature.Labeled glucose uptake studies were done to
examine the effect of the elevated temperatureupon bacterial transport functions (Table 1).Both nonfecal and fecal coliform isolates werecapable of incorporating [14C]glucose at 350C inTSY broth and the maximal rates of uptake(counts per minute per A6ws) at this temperaturewere similar for the organisms tested. At 44.50Cthe fecal coliform isolates transported [14C]glu-cose at a rate comparable to that observed at350C. In sharp contrast to this, nonfecal cohl-forms showed little uptake of the labeled glucoseat 44.50C compared with the maximal uptakerate at 350C. The rates of glucose incorporation(counts per minute per A6w, per minute) shownin Table 1 are not directly comparable betweeneach organism tested because different activitiesof labeled glucose were used in different experi-ments; however, the ratio of gluocose uptake atthe two temperatures can be directly comparedfor the various coliforms examined.A shift in growth temperature from 35 to
44.50C was followed where a nonfecal coliform(IMViC, -+++) was growing in TSY broth con-taining lactose. This experiment demonstrated
FIG. 2. Comparison of gas evolution rates (CO2 + H2) for fecal and nonfecal coliforms in EC broth at44.50C. Respirometer flasks were gassed with 100% nitrogen for 15 min to attain fermentation conditions.
that the high temperature stopped active celldivision (Fig. 4). The temperature of the shiftedsuspension equilibrated to 44.50C from 350Cwithin 10 min. The first observable change inthe growth rate occurred within 30 to 60 minafter the shift, and active growth of the culturegradually ceased between 30 and 90 min.A second differential effect of the elevated
temperature was observed which involved theenzyme f8-galactosidase of fecal and nonfecalcoliforms. f8-Galactosidase assays were done attemperature increments of approximately 5°Cthroughout the range of 10 to 45°C. The levelsof f1-galactosidase activity in sonic extracts offully induced fecal coliform cultures were con-
sistently higher than those seen in comparablepreparations from nonfecal coliforms (Fig. 5).Optimal activity of this enzyme in freshly pre-pared sonic extracts of fecal coliforms typicallyoccurred at 30 ± 2°C, and the activity decreasedrapidly as the temperature increased above 35to 380C. At 44.50C fecal coliform f3-galactosidaseactivity was 25 to 50% of the optimal activity;however, nonfecal coliform /3-galactosidase ac-tivity was generally low or not measurable atthat temperature. f8-Galactosidase activity wasmeasured over the 10 to 45°C temperature range
for two additional sonically disrupted culturesnot shown in Fig. 5. An E. coli B culture wassimilar to the two fecal coliforms shown but wasslightly lower than those organisms in the levelof enzyme activity that was observed. A nonfecalK. pneumoniae culture showed little fl-galacto-sidase activity at any of the assay temperatures.
Spectrophotometric assays for aldolase activ-ity were performed on sonic extracts of fecal andnonfecal coliforms to study the effect of theelevated temperature on this enzyme and todetermine whether the reaction kinetics wereaffected by increased temperatures in the samemanner as fB-galactosidase. Results of the aldo-lase assays for fecal coliforms (IMViC ++--and E. coli B) and nonfecal coliforms (IMViC-+++ and -+-+ isolates) indicate little differ-ence between the two types of coliforms (Table2), and the activity of that enzyme increasedwith temperature between 35 and 44.5°C regard-less of the coliform type.
DISCUSSIONAlthough the elevated-temperature test has
been used successfully for many years and sev-eral studies have addressed the sanitary signifi-cance of the procedure, the physiological basis
underlying the test has received little attention.Hendricks (12) examined several Enterobacterspecies using manometric techniques and sug-gested that the effects of the elevated tempera-ture are focused upon the formate dehydrogen-ase complex. He postulated that there are atleast two distinct biochemical types of Entero-bacter in the aquatic environment and that theresults of the elevated-temperature test de-
pended upon whether or not a coliform pos-sessed formate dehyrogenase activity at 44.50C.The manometric data and growth curves ob-
tained in this study indicate that the fecal coli-form phenotype is a manifestation of more thana temperature effect upon the activity of theformate dehydrogenase complex in coliform bac-teria. Although the elevated-temperature test isdefined in terms of fermentative gas productionfrom lactose at 44.50C, results of this studyindicated that aerobic metabolism of lactose atthis temperature also served to differentiate fe-cal from nonfecal coliforms. The fecal coliformstrains tested were capable of producing gas asan end product of the fermentation of lactose at44.5°C (Fig. 2), and they could take up atmos-pheric oxygen during the metabolism of lactoseunder aerobic conditions (Fig. 1). In contrast,nonfecal coliforms could not evolve gas fromlactose under fermentative conditions (Fig. 2),and, furthermore, lactose was not metabolizedaerobically, as evidenced by the lack of oxygenuptake at the elevated temperature (Fig. 1). Inaddition, nonfecal coliforms actively growing inlactose at 35°C were found to discontinue cell
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NOT SHIFTED (35C)
/ SHIFTED
(--.IL--- ---
I
07 35 C-44.5-C/
0 0.6
ED~~~~,0.5
0.4 -
0. /
30 60 90 120 150 180MINUTES
FIG. 4. Effect ofa 35 to 44.5°C temperature shift upon growth of a nonfecal coliform in TSY broth (withoutglucose) containing 1% lactose.
division within 1 h after a shift to 44.5°C (Fig.4). E. coli possesses two inducible, soluble, mem-brane-associated formate dehydrogenase com-plexes (24). One of these complexes is involvedin the evolution of carbon dioxide and molecularhydrogen from formate, and it is functional onlyunder anaerobic growth conditions (9, 22). Ifformate dehydrogenase is a target of the inhibi-tory effect of the elevated temperature uponnonfecal coliforms, the lack of gas production inEC broth at 44.5°C would be expected, and thiswas observed. However, aerobic metabolismwhich is conducted via metabolic pathways notinvolving formate dehydrogenase was also in-hibited at 44.50C. Therefore, whether or notformate dehydrogenase is affected by the ele-vated temperature, additional metabolic sites oftemperature sensitivity must also exist.There is much information in the literature
concerning the effects of temperature fluctua-tions upon the composition and function of theE. coli cell membrane. Marr and Ingraham (19)
and others (4) have shown that E. coli increasesthe ratio of unsaturated to saturated fatty acidsas growth temperature is increased and that therate of change in this ratio is greatest at temper-atures over 40°C. These closely regulatedchanges in the composition and the resultantphysical properties of the lipid phase of themembrane have been found to affect manymembrane-associated functions, including thelactose, f8-glucoside, and amino acid transportsystems (3). Data collected in this study impli-cate cell membrane involvement in the observedtemperature effects with respect to the expres-sion of the fecal coliform phenotype. The use ofradioactively labeled substrate demonstratedthat a temperature increase from 35 to 44.50Cdrastically reduced the rates of ['4C]glucose up-take in nonfecal coliforms while those of fecalcoliforms were essentially unchanged. We sug-gest that the inability of the nonfecal coliformsto incorporate ['4C]glucose or utilize lactose at44.5°C could be due in part to deleterious effects
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of the elevated temperature upon membranelipid synthesis or upon control mechanisms in-volved in lipid phase transitions in this group ofcoliform organisms.
Several factors led to the investigation of asecond elevated-temperature effect involving anaspect of lactose metabolism in nonfecal coli-forms. The nonfecal coliforms tested seemedcapable of metabolizing TSY broth at a minimallevel at 44.5°C, with evidence for this appearingin both the manometric experiments (Fig. 3) andin the [14C]glucose incorporation experiments(Table 1). In contrast, nonfecal coliforms werefound to gradually cease cell division after a 35to 44.5°C shift in incubation temperature whengrowing in TSY broth (without glucose) contain-ing lactose (Fig. 4) and were incapable of metab-ohlzing the lactose-containing EC broth at thattemperature (Fig. 1). The bile salts component
.30 r
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' .25
JE'2 .20c
0
49.1 5
.10-
0
.I0!
52:
, ---.---I.
0 24.-----------------30--- 40-10 20 30 40 50
TEMPERATURE CC)
FIG. 5. Effect oftemperature upon 13-galactosidaseactivity in sonically disrupted cell-free extracts offecal and nonfecal coliforms. Symbols: 0, fecal coli-form, IMViC ++--; U, fecal coliforn, IMViC-+--; 0, nonfecal coliforn, IMViC -+++; 0, nonfe-cal coliforn, IMViC -+-+.
ofEC medium alone exerted a marked inhibitoryeffect upon the respiration rate of fecal coliformsat 44.5°C; however, when combined with theother components of the highly buffered ECbroth this effect was essentially compensated forand was not likely an important consideration(unpublished data). After entry into the cell viaa specific permease-mediated transport system(14), the /-1,4 linkage of lactose is hydrolyzedvia the enzyme fi-galactosidase. Because thisenzyme is not used and in fact its synthesis isrepressed during the metabolism of glucose (18),it seemed possible that the additional inhibitoryeffect of the elevated temperature upon thenonfecal coliform may be focused upon this en-zyme. Our results with cell-free extracts showedthat the total 8-galactosidase activity was con-sistently greater in fecal coliforms than in nonfe-cal coliforms. By comparison, levels of aldolaseactivity, a constitutive enzyme chosen for itscentral position in carbon metabolism, were sim-ilar for fecal and nonfecal coliforms (Table 2). Inaddition, thermal inactivation of fi-galactosidasein both fecal and nonfecal coliforms appeared ata relatively low temperature compared withmost mesophilic enzymes. Generally, the reac-tion rates of these enzymes approximately dou-ble for each 100C (Qlo = 2) rise in temperatureuntil the temperature of optimal activity isreached. Beyond this temperature (usually 55 to600C) thermal inactivation results in a relativelyrapid loss of activity (16). Between 35 and 44.5°Cthe ,B-galactosidase-specific enzyme activity de-creased rapidly (Fig. 5) as evidenced by a Qlovalue of less than 1 (Table 2). In contrast, theactivity of fecal and nonfecal coliform aldolaseincreased between 35 and 44.50C in all cases (Qio> 1). The additive effect of the relatively lowthennal inactivation temperature and the lowlevels of,8-galactosidase activity at temperaturesover 35 to 380C resulted in virtually no meas-urable fi-galactosidase activity at 44.50C in thenonfecal coliforms tested. A dramatic decreaseof fl-galactosidase activity due to thermal inac-tivation at temperatures over 35 to 380C wasalso observed for fecal coliforms (Fig. 5), butthere was sufficient enzyme activity present at44.50C to allow the active metabolism of lactose.
TABLE 2. Comparison of temperature effects on aidolase and 13-galactosidase activities offecal andnonfecal coliforms
a Q,o, Activity at 44.50C per activity at 350C (10°C/9.5°C).
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This decrease in activity in fecal coliforms hasbeen indirectly observed by Warren et al. (25)who found that lowering the 44.5°C incubationtemperature by 1 or 20 resulted in significantlyfaster rates of ONPG hydrolysis.Two cellular sites of sensitivity to the elevated
temperature in nonfecal coliforms have beenidentified in this study. Although incubation at44.5°C may produce other effects deleterious tometabolism and growth of nonfecal coliforms,the inhibitory action upon cell membrane func-tion and f,-galactosidase activity may be mostimportant because of their initial position inlactose metabolism and the presence of thatgrowth substrate in the commonly used differ-ential media. Because of the heterogeneous na-ture of the coliform group of bacteria, it is quitepossible that our findings will not explain allphenotypes observed in response to the elevatedtemperature; however, our results seem to beindicative of the response of the majority ofthese organisms Methods for separating fecalfrom nonfecal coliforms could be developedbased upon the knowledge of these two temper-ature effects. One rapid spectrophotometrictechnique has been proposed by Warren et al.(25) and is based upon enzymatic hydrolysis of0.06 M ONPG by fecal coliforms at 44.50C. Al-though much work remains to be done beforethey could be adopted for general use, suchtechniques could prove to be not only more rapidbut more reliable than those relying upon gasproduction from lactose at 44.5°C.
ACKNOWLEDGMENTSWe thank Anne K. Camper and Susan B. Olson for tech-
nical aaaistance and John E. Schillinger and David G. Stuartfor their helpful discuions and review of this manuscript.
This research was supported by funds from the U.S. De-partment of the Interior under the Water Resources ResearchAct of 1964, Public Law 88-379 and administered through theMontana Joint Water Resources Research Center (grantOWRR A-099 Mont.).
LITERATURE CITED1. American Public Health Association. 1976. Standard
methods for the examination of water and wastewater,14th ed. American Public Health Assoc., Washington,D.C.
2. Bagley, S. T., and R. J. Seidler. 1977. Significance offecal colifonn-positive Klebsiella. Appl. Environ. Mi-crobiol. 33:1141-1148.
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