United States
Environmental Protection
Agency
Environmental Research
Laboratory
Corvallis OR 97330
EPA-600 3 79-073
July 1979
Research and Development
Identification of
Fecal Indicator
Bacteria
Isolates from an
Ice-Covered River
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
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This document is available to the public through the National Technical Informa-
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EPA-600/3-79-073
July 1979
IDENTIFICATION OF FECAL INDICATOR BACTERIA
ISOLATES FROM AN ICE-COVERED RIVER
by
Elena B. Sparrow
Charlotte V. Davenport
Ronald C. Gordon
Arctic Environmental Research Station
Con/all is Environmental Research Laboratory
College, Alaska 99701
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
US. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Pro-
tection Agency would be virtually impossible without sound scientific data on
pollutants and their impact on environmental stability and human health.
Responsibility for building this data base has been assigned to EPA's Office
of Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).
The primary mission of the Corvallis Laboratory is research on the ef-
fects of environmental pollutants on terrestrial, freshwater, and marine
ecosystems; the behavior, effects and control of pollutants in lake and river
systems; and the development of predictive models on the movement of pollu-
tants in the biosphere. CERL's Arctic Environmental Research Station extends
the primary mission to the cold-climate environment; and develops and demon-
strates pollution control technology for cold-climate regions.
This report describes the generic composition of the total coliforms,
fecal coliforms, and fecal streptococci isolated with the membrane filter
technique from sample stations on an ice-covered river downstream from a major
source of domestic pollution.
James C. McCarty
Acting Director, CERL
111
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ABSTRACT
The membrane filter technique was used to enumerate the total coliform
(TC), fecal coliform (FC), and fecal streptococcus (FS) populations at seven
sample stations on an ice-covered river downstream from a major source of
domestic pollution. From each membrane filter population (m-TC, m-FC, and
m-FS), 210 typical colonies (30 per station) were selected for verification
and biochemical differentiation of the component genera. The 210 m-TC iso-
lates were Klebsiella pneumonias (46.2%), Esaherichia coli (20.5%), Entero-
baoter sp. (18.6%), other total coliforms (5.2%), and 9.5% which did not
verify as total coliforms. Among these m-TC cultures, 114 were verified as
fecal coliforms (gas production in EC broth at 44.5°C). These 114 fecal
coliforms were principally K. pneumonias (53.5%) and E. coli (35.1%). In
contrast, the 210 m-FC cultures were predominantly E. coli (77.6%), with K.
pnewnoniae (10.0%), other fecal coliforms (3.8%), and those not verified as
fecal coliforms (8.6%). Of the 210 m-FS isolates, 167 were identified as
enterococci, with 165 being Streptococcus faecalis biotypes. The results
suggested the majority of these indicator bacteria originated from warm-
blooded animal feces. Also, within each population, no overall differences in
low temperature survival of the component genera were noted. However, the
evidence does suggest that generic selectivity of the m-TC and m-FC techniques
biases determination of the FC population composition, and that the m-FC
technique underestimates the FC population density. Portions of this work
were presented at the 78th Annual Meeting of the American Society for Micro-
biology, Las Vegas, NV, 14-19 May, 1978 [abstract n83].
1v
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CONTENTS
Page
Foreword iii
Abstract iv
Tables vi
1. Introduction 1
2. Conclusions and Recommendations 2
3. Materials and Methods 3
4. Results 4
5. Discussion 9
References 11
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TABLES
Number
1. Membrane Filter Densities and Percent Verification of Indicator
Bacteria From Each Sample Station 5
2. Identification of Verified Total Coliform Isolates From Each Sample
Station 6
3. Identification of Verified Fecal Coliform Isolates From Each Sample
Station 7
4. Identification of the Enterococcus Group Isolates From Each Sample
Station 8
v1
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SECTION 1
INTRODUCTION
Although indicator bacteria densities are used as a measure of the proba-
ble presence of enteric pathogens in water, some of the genera or biotypes
comprising the total coliform (TC) and fecal coliform (FC) populations can be
found in a variety of environments (1, 2) and may enter surface waters from
nonfecal habitats (3, 4, 5). Therefore, important factors in relating indi-
cator bacteria to the presence of enteric pathogens are the influence of
specific waste sources on the composition of the population entering the
aquatic environment (3), and the genera or biotype survival in this"environ-
ment (6, 7). The IMViC (indole, methyl red, Voges-Proskauer, and citrate)
patterns have been widely used to define biotypes within the coliform popula-
tion, but lack both generic (2) and source (8, 9) specificity. Thus, as
proposed by Dufour and Cabelli (3) differentiation of coliforms at the generic
level would probably provide more valuable information than IMViC biotypes.
Although emphasis has been placed on the coliforms, the fecal streptococcus
(FS) group is also valuable in water pollution studies because some biotypes
are associated with specific pollution sources (10, 11). Therefore, survival
of FS biotypes in the aquatic environment should also be examined.
While the aquatic environment was noted as being generally unfavorable
for maintaining viability of most enteric bacteria (6), water temperature
appears to be a factor of major importance influencing survival of these
microorganisms (7, 12, 13). Davenport et at. (.12) recently examined the
persistence of the TC, FC, and FS populations at sample stations downstream
from a major source of domestic pollution. These authors found a high survi-
val rate and suggested that indicator bacteria populations manifest the great-
est resistance to viability loss under natural river conditions when the water
has a temperature of 0°C and is ice covered. In the current investigation,
these indicator bacteria populations were further examined. Isolates obtained
from membrane filters on day five of the survival study (12) were differenti-
ated biochemically to determine the generic composition of the TC, FC, and FS
populations and to examine the relative survival characteristics of the compo-
nent genera under low temperature conditions.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
1. The environmental conditions which prevailed during this study, along
with the absence of industrial waste sources in the area, precluded the
presence of enteric microorganisms in the river from sources other than
domestic waste effluents.
2. The coliforms isolated from the river were predominantly fecal coliform-
positive (from warm-blooded animal feces) with Esohevichia ooli and
Klebsiella pneumoniae as the most frequent isolates, while the principal
fecal streptococcus was Streptococcus faecalis which is generally preva-
lent in domestic wastes.
3. Among the component genera of the fecal indicator bacteria populations,
no overall survival differences in the aquatic environment were observed
during this study. Thus, to clearly define whether or not there is
differential survival among the component genera, it may be necessary to
biochemically differentiate a large number of isolates from any specific
sample station.
4. The fecal coliform-positive total coliforms from this domestic waste
source have essentially the same generic composition that others have
found associated with some industrial wastes.
5. The membrane filter technique for enumerating fecal coliforms appears to
have a generic selectivity which suggests this technique may frequently
underestimate the fecal coliform population density.
RECOMMENDATIONS
1. All fecal coliform-positive coliform bacteria (either m-FC or EC posi-
tive) should continue to be considered valid fecal coliforms indicative
of a potential health hazard.
2. Before discarding the methodology currently used for fecal indicator
bacteria enumeration, there needs to be a sound basis for recommending
the use of new or modified techniques.
3. New or modified techniques for enumerating fecal coliforms should mini-
mize the problem of underestimating the fecal coliform population.
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SECTION 3
MATERIALS AND METHODS
The TC, FC, and FS cultures used in this study were collected from seven
sample stations located on the Tanana River near Fairbanks, Alaska, downstream
from a major domestic pollution source. River characteristics, sample station
locations, flow time measurements between stations, domestic pollution sources,
field sampling techniques, membrane filter techniques (m-TC, m-FC, and tn-FS),
and isolate verification methods have been described (12).
Verified TC and FC cultures were streaked on Endo agar (BBL) to obtain
well-isolated, typical colonies. These colonies were transferred to Trypti-
case soy agar (BBL) slants for maintenance, and to improved Enterotubes (Roche
Diagnostics, Division of Hoffman-LaRoche Inc., Nutley, NO) for identification.
The cultures were identified using the Encise II system (Roche Diagnostics)
and supplemental tests as required. The verified FS cultures were streaked on
KF streptococcal agar (BBL), and well-isolated, typical colonies were trans-
ferred to brain heart infusion broth (BBL) for identification and to brain
heart infusion agar (BBL) slants for maintenance. The identification scheme
described by Geldreich (14), with the addition of growth in 40% bile (15), was
used to differentiate FS biotypes and identify the isolates showing reactions
typical of the enterococcus group.
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SECTION 4
RESULTS
The TC, FC, and FS populations at seven sample stations downstream from a
major source of domestic pollution were enumerated with membrane filter tech-
niques. For each population, 30 typical colonies per station (210 total) were
selected from the membrane filters for verification. The verified isolates
were then biochemically differentiated to determine the generic composition of
the populations persisting in the ice-covered river. Table 1 shows that
station T-700 downstream through T-100 encompassed a river reach having a mean
flow time of 7.1 days with a range of 0.8 to 1.9 days between sample stations.
It also shows that the indicator bacteria populations exhibited rapid de-
creases in density during the first 2.9 days with a generally slower rate of
decrease thereafter.
Among the 210 m-TC isolates, six genera in the family Entevobacteriaceae
were identified (Table 2). Klebsiella pneumoniae (46.2%) was the predominant
total coliform isolated and was found, along with Eschevichia ooli (20.5%) and
Entevobaater sp. (18.6%), at all sample stations. The other three genera
which were verified as total coliforms represented 5.2% of the cultures and
were isolated only occasionally, while 9.5% of the isolates did not verify as
total coliforms. Further examination of the verified total coliforms revealed
that 114 were fecal coliforms as defined by growth and gas production in EC
broth at 44.5°C (16). Among the 114 EC-positive cultures, K. pneimoniae
(53.5%) and E. coli (35.1%) were found at all sample stations with Entero-
bacter agglomerans (10.5%) at five stations.
The 210 m-FC cultures were verified as fecal coliforms by being positive
in EC broth. Table 3 shows E. coli (77.6%) was the predominant isolate and
was the only m-FC isolate found at all stations. K. pneimoniae (10.0% of the
isolates) was found at all but the T-700 station. The other fecal coliforms
were isolated infrequently and were 3.8% of the cultures. FC-negative iso-
lates made up 8.6% of the m-FC cultures.
Only 167 of the 210 m-FS isolates exhibited biochemical reactions typical
of the enterococcus group. Identification of the 167 enterococcus cultures
showed that 165 were streptococcus faecalis and that two were streptococcus
faeaim (Table 4). S. faecaUs subsp. faecalis was found at all sample sta-
tions and represented 74.8% of the enterococcus group. Together, S. faecalis
subsp. zymogenes, S. faecalis subsp. liquefaciens, atypical S. faecalis and S.
faeaium comprised 7.8% of the enterococci. The remaining 17.4% were identifed
as S. faecalis, but they peptonized litmus milk, did not hydrolyze gelatin and
were not beta hemolytic. These isolates may have been S. faecalis subsp.
zymogenes which lost the hemolytic character as a result of serial transfer in
the laboratory since this subspecies may peptonize litmus milk and may or may
not liquify gelatin (.17).
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TABLE 1. RIVER FLOW TIMES BETWEEN STATIONS, AND MEMBRANE FILTER DENSITIES OF
INDICATOR BACTERIA AT EACH STATION*
Sample
station
T-700
T-600
T-500
T-400
T-300
T-200
T-100
Days mean
flow time
between
stations
1.9
1.0
1.3
1.1
1.0
0.8
Indicator
100 ml of
Total
col i forms
6,200
1,800
700
500
230
180
200
bacteria
densities per
river water sample**
Fecal
col i forms
2,200
430
100
200
100
68
90
Fecal
streptococci
78
65
11
29
17
13
14
* Samples collected on day five of the survival study (12).
** Mean of three replicate aliquots.
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TABLE 2. IDENTIFICATION OF VERIFIED TOTAL COLIFORM ISOLATES FROM EACH SAMPLE STATION*
Genus and species
Encherichia aoli.
Klebsiella pneumonias
Enterobacter cloacae
Enterobacter agglomerana
Citrobacter freundii
Serrat-ia l-iquefaaiens
Proteus morgani-i
Total isolates verified
as TC
Total isolates verified
as FC
Reaction
in EC
broth at
44.5°C
positive
negative
positive
negative
negative
positive
negative
negative
positive
negative
negative
Total
isolates
verified
40
3
61
36
19
12
8
6
1
3
1
190
114
Number of
T-700
9
1
5
7
1
3
1
0
0
0
_0
27
17
T-600
1
0
14
5
3
2
1
0
0
0
_0
26
17
isolates from each sample station
T-500
6
0
10
4
0
3
2
1
0
1
_0
27
19
T-400
6
0
12
4
2
0
1
2
0
0
_0
27
18
T-300
5
0
7
7
4
2
1
0
0
1
_0
27
14
T-200
10
2
5
3
1
2
2
2
0
1
_]_
29
17
T-100
3
0
8
6
8
0
0
1
1
0
_0
27
12
* 210 colonies subcultured from membrane filters: 30 from each sample station, 10 from each replicate
aliquot.
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TABLE 3. IDENTIFICATION OF VERIFIED FECAL COLIFORM ISOLATES FROM EACH SAMPLE STATION*
Genus and species
Esoheriohi-a coli
Klebsiella pneumoniae
Klebsiella ozaenae
Enterobacter cloacae
Enterobaater agglomerans
Serratia liquefaoiens
Total isolates verified
Total
isolates
verified
163
21
1
1
5
1
192
Number of isolates from each sample
T-700
27
0
0
0
3
0
30
T-600
24
3
1
0
0
0
28
T-500
19
7
0
0
1
0
27
T-400
22
3
0
0
0
1
26
T-300
26
2
0
0
0
0
28
station
T-200
24
1
0
0
0
0
25
T-100
21
5
0
1
1
0
28
* 210 colonies subcultured from membrane filters: 30 from each sample station, 10 from each replicate
aliquot.
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TABLE 4. IDENTIFICATION OF THE ENTEROCOCCUS GROUP ISOLATES FROM EACH SAMPLE STATION*
00
Genus and species
Streptococcus faecal-is
subsp. faeoaUs
subsp. zymogenes
subsp. liquefaciens
other*
Atypical Streptococcus
faecalis**
Streptococcus faeaium
Total isolates verified
Total
isolates
verified
125
7
3
29
1
2
167
Number of isolates from each sample
T-700
23
0
1
2
0
1
27
T-600
19
1
0
5
1
0
26
T-500
20
1
0
7
0
0
28
T-400
17
1
0
3
0
0
21
T-300
15
0
0
5
0
0
20
station
T-200
9
2
1
7
0
0
19
T-100
22
2
1
0
0
1
26
* Litmus milk peptonized, gelatin not hydrolyzed, not beta hemolytic.
** Starch hydrolyzed.
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SECTION 5
DISCUSSION
The environmental conditions under which the study was conducted were
described in an earlier report (12), and precluded the presence of enteric
microorganisms from sources other than domestic waste effluent. The generic
composition of the m-TC, m-FC, and m-FS populations provided additional con-
firmation for the domestic waste origin of these enteric bacteria. Overall,
E. ooli was the most frequently isolated coliform, followed by K. pneumoniae,
and Enterobaater sp. (Tables 2 and 3). In addition, the majority of the
isolates were EC-positive which indicated that these coliforms were predomin-
antly from warm-blooded feces (8, 9). Furthermore, 5. faeoalis biotypes were
the principal FS cultures obtained from the Tanana River (Table 4); these
biotypes have been noted as the predominant fecal streptococci in domestic
wastes (14).
An earlier report (12) showed continuously decreasing TC, FC, and FS
population densities as length of time in the river increased. However, the
results in (Tables 2, 3, and 4) revealed no overall differences in persistence
among the component genera of the populations. These observations suggest
population viability loss as a whole may account for the decreasing fecal
indicator bacteria densities in the low temperature receiving water. This is
in general agreement with the observations of McFeters et al. (6) at warmer
temperatures. Although no overall differences in component genera persistence
were noted, the number of isolates examined was small and the generic distri-
bution did fluctuate between stations. This suggests that biochemical differ-
entiation of a large number of isolates from any specific sample station may
be required to clearly define whether or not there is differential survival
among the component genera.
The generic composition of the EC-positive total coliforms isolated in
the present study (Table 2) was compared with the reported composition of the
EC-positive total coliform populations entering the aquatic environment from
sources other than domestic wastes. Dufour and Cabelli (.4) showed that 45% of
the Klebsiella isolates from a textile finishing plant effluent were EC-
positive, while 63% of the Klebsiella isolates (all K. pneumonias) were EC-
positive in the present study. Downstream from a pulp mill, Huntley et al.
(.5) found that the EC-positive isolates consisted chiefly of Klebsiella (60%,
predominantly K. pneumoniae) and E. coli (35%). The EC-positive total coli-
form cultures in the current study also proved to be mostly K. pneumoniae
(54%) and E. coli (35%).
Fecal coliforms are considered to be a heterogeneous group with E. ooli
and Klebsiella as principal components (3). However, Geldreich (18) cited a
report (L. A. Vinogradova, Hyg. Sanlt. 36:157, 1971) in which E. ooli isolates
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from rivers in northern latitudes did not ferment carbohydrates at elevated
temperatures, and suggested the elevated temperature test may not be reliable
in the far north. In contract, 203 of the 206 E. coli cultures isolated from
the Tanana River (approximately 65° North Latitude) during this study fer-
mented lactose with gas production at 44.5°C (were EC-positive). Also, the
validity of K. pneumoniae as an indicator of fecal pollution is being ques-
tioned, even though it is a common human and animal intestinal tract inhabi-
tant (19). Part of the problem in establishing the sanitary significance of
K. pneumoniae was resolved recently when Bagley and Seidler (1) demonstrated
that some EC-positive strains give negative results with the m-FC technique.
These authors concluded that FC-positive strains (either m-FC or EC-positive)
are valid fecal coliforms indicating a potential health hazard. The fecal
coliforms isolated during the Tanana River study were a heterogeneous group
(Tables 2 and 3) with K. pneumoniae the principal EC-positive isolate from
the m-TC population and a relatively minor component of the m-FC population.
Colonies manifesting the color reactions described by Bagley and Seidler (1)
as EC-positive/m-FC-negative were generally present on the m-FC membranes
suggesting that K. pneumoniae may have comprised a larger percentage of the
fecal coliform population in the Tanana River than revealed by the m-FC tech-
nique. The m-FC technique and EC test were both developed to differentiate
between coliforms of fecal and nonfecal origin (20). However, the apparent
generic selectivity of the m-FC techniques suggests that technique frequently
underestimates the fecal coliform population density. This may partially
explain why the FC population densities previously reported in this survival
study (12) were invariabily lower with the m-FC technique than with the
multiple-tube method.
It was pointed out previously (21) that various coliform media and pro-
cedure combinations may show different generic selectivity patterns, and
there appears to be a growing concern that fecal coliforms enumerated with
the m-FC technique do not adquately indicate the probable presence of enteric
pathogens. Despite these methodology problems, fecal coliforms continue to
be, as noted by Dutka (22), "...one of the most important indicators of
potential health hazard due to fecal pollution." Thus, before discarding the
tools which have had such a significant role in improving and maintaining
human health, there must be a sound basis for recommending the use of new or
modified techniques.
10
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12. Davenport, C. V., E. B. Sparrow, and R. C. Gordon. Fecal Indicator
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12
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Identification of Fecal Indicator
from an Ice-Covered River
Bacteria Isolates
5. REPORT DATE
July 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Elena B. Sparrow, Charlotte V. Davenport, and Ronald C.
Gordon
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
U. S. Environmental Protection Agency
Arctic Environmental Research Station
College, Alaska 99701
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U. S. Environmental Protection Agency
Corvallis Environmental Research Laboratory
200 S. W. 35th Street
Corvallis, Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
inhouse
4. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
Portions of this work were presented at the 78th Annual Meeting of the American
Society for Microbiology, Las Vegas, NV, 14-19 May, 1978
16. ABSTRACT
The membrane filter technique was used to enumerate the total coliform (TC), fecal coli
form (FC), and fecal streptococcus (FS) populations at seven sample stations on an ice-
covered river downstream from a major source of domestic pollution. From each membrane
filter population (m-TC, m-FC, and m-FS), 210 typical colonies (30 per station) were
selected for verification and biochemical differentiation of the component genera. The
210 m-TC isolates were fO£eb-6-ce£&i pneumonias. (46.2%), E4c.ke/u£.kia c.oHJi (20.5%), EnteJio-
bacteA. sp. H8.6/0, other total coliforms (5.2%), and 9.5% which did not verify as tot-
al coliforms., Among these m-TC cultures, 114 were verified as fecal coliforms (gas pro
duction in EC broth at 44.5°C). These 114 fecal coliforms were principally K. pn&won-
UUL (53.5%) and E. cote (35.1%). In contrast, the 210 m-FC cultures were predominantly
E. coli (77.635), with K. pneumc^uae (10.0%), other fecal coliforms (3.8%), and those
not verified as fecal coliforms (8.6%). Of the 210 m-FS isolates, 167 were identified
as enterococci, with 165 being SiteptococcoA &a.e£.aJtM> biotypes. The results suggested
the majority of these indicator bacteria originated from warm-blooded animal feces.
Also, within each population, no overall differences in low temperature survival of the
component genera were noted. However, the evidence does suggest that generic selectiv-
ity of the m-TC and m-FC techniques biases determination of the FC population composi-
tion, and that the m-FC technique underestimates the FC population density.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Coliform Bacteria, Enterobacteriaceae,
Escherichia coli, Klebsiella,
Microorganisms, Stream Pollution,
Streptococcus
Enterobacter, Entero-
coccus, Fecal Coliform,
Fecal Indicator Bacteria,
Fecal Streptococcus, Low
Temperature Survival,
Membrane Filter Tech-
nigue. Total Coliform
06/F, M
08/H, L
19. SECURITY CLASS (This Report)
Release to public
21. NO. OF PAGES
19
20. SECURITY CLASS (Thispage)
22. PRICE
EPA Form 2220-1 (R»v. 4-77) PREVIOUS EDITION is OBSOLETE
13
699-320
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