12.

13.

14.

15.

16.

V7,

18.

19,

20.

21.

22.

Davenport, C. V., E. B. Sparrow, and R. C. Gordon. Fecal Indicator
Bacteria Persistence Under Natural Conditions in an Ice-Covered River.
Appl. Environ. Microbiol., 32:527-536, 1976.

McFeters, G. A., and D. G. Stuart. Survival of Coliform Bacteria in
Natural Waters: Field and Laboratory Studies with Membrane-Filter Cham-
bers. Appl. Microbiol., 24:805-811, 1972.

Geldreich, E. E, Handbook for Evaluating Water Bacteriological Labora-
tories. EPA-670/9-75-006, U.S. Environmental Protection Agency, Cincin-
nati, Ohio, 1975. 195 pp.

Kenner, B, A., H. F. Clark, and P. W. Kabler. Fecal Streptococci, II.
Quantification of Streptococci in Feces. Am. J. Pub. Health, 50:1553-
1599, 1960.

American Public Health Association. Standard Methods for the Examination
of Water and Wastewater, 13th ed., 1971. p. 669.

Deibel, R. H., and H. W. Seeley, Jr. Family II. Streptococcacae fam.
nov. In: Bergey's Manual of Determinative Bacteriology, 8th ed., R. E.
Buchanan and N. E. Gibbons, eds. The Williams & Wilkins Co., Baltimore,
Maryland, 1974, pp. 490-509.

Geldreich, E. E. Microbiology of Water. J. Water Pollut. Contr. Fed.,
46:1355-1372, 1974.

Bordner, R. H., and B. J. Carroll. Introduction and Summary. In:
Proceedings: Seminar on the Significance of Fecal Coliforms in Indus-
trial Wastes, R. H. Bordner and B. J. Carroll, eds. U.S. Environmental
Protection Agency, Denver, Colo., 1972. p. vii.

Geldreich, E. E., H. F. Clark, C. B. Huff, and L. C. Best. Fecal-Coli-
form-Organism Medium for the Membrane Filter Technique. J. Amer. Water
Works Assoc,, 57:208-214, 1965.

Dutka, B. J., and S. E. Tobin. Study on the Efficiency of Four Proced-
ures for Enumerating Coliforms in Water. Can. J. Microbiol. 22:630-635,

1976.

Dutka, B. J. Coliforms Are an Inadequate Index of Water Quality. J.
Environ. Health, 36:39-46, 1973.

12


TABLE 2. IDENTIFICATION OF VERIFIED TOTAL COLIFORM ISOLATES FROM EACH SAMPLE STATION*

: Reaction Number of isolates from each sample station
Genus and species in EC Total

broth at isolates T-700 1-600 1-500 1-400 1-300 T-200 T-100
44.5°C verified

Encherichia coli positive 40 9 1 6 6 5 10 3
negative 3 1 0 0 0 0 2 0
Klebsiella pnewnoniae positive 61 5 14 10 12 7 5 8
negative 36 7 5 4 4 7 3 6
Enterobacter cloacae negative 19 1 3 0) 2 4 1 8
Enterobacter agglomerans positive 12 3 2 3 0 2 2 0
negative 8 1 1 2 1 1 2 0
Citrobacter freundii negative 6 0 0 1 2 0 2 1
Serratia liquefactens positive 1 0 0 0 0 0 0 1
negative 3 0 0 1 0 1 1 0
Proteus morganit negative 1 0 am!) 0 oO am!) J a
Total isolates verified
as TC 190 27 26 27 27 27 29 27
Total isolates verified
as FC 114 WV W7 19 18 14 W7 12

* 210 colonies subcultured from membrane filters: 30 from each sample station, 10 from each replicate
aliquot.


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

ii


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)

1. REPORT NO, 2. |3. RECIPIENT'S ACCESSION NO.
EPA-600/3-79-073

4, TITLE AND SUBTITLE 5. REPORT DATE

Identification of Fecal Indicator Bacteria Isolates July 1979 issuing date

from an Ice-Covered River 6. PERFORMING ORGANIZATION CODE

7. AUTHOR(S) . PERFORMING ORGANIZATION REPORT NO.

Elena B. Sparrow, Charlotte V. Davenport, and Ronald C. P

Gordon

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10, PROGRAM ELEMENT NO.

U. S. Environmental Protection Agency

Arctic Environmental Research Station 71 CONTRACT/GHANT NO.

College, Alaska 99701

12, SPONSORING AGENCY NAME AND ADDRESS. 13. TYPE OF REPORT AND PERIOD COVERED

U. S. Environmental Protection Agency inhouse

Corvallis Environmental Research Laboratory 14. SPONSORING AGENCY CODE

200 S. W. 35th Street

Corvallis, Oregon 97330 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 Klebsiella pneumoniae (46.2%), Escherichia coki (20.5%), Entero-
bacter sp. (18.6%), 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. pnewnon-
Aae (53.5%) and E. coli (35.1%). In contrast, the 210 m-FC cultures were predominantly
E. coli (77.6%), with K. pneumoniae (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 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.

17. KEY WORDS AND DOCUMENT ANALYSIS

a. DESCRIPTORS Ib. IDENTIFIERS/OPEN ENDED TERMS jc. COSATI Field/Group
Coliform Bacteria, Enterobacteriaceae, Enterobacter, Entero- 06/F, M
Escherichia coli, Klebsiella, icoccus, Fecal Coliform,

Microorganisms, Stream Pollution, Fecal Indicator Bacteria,| 08/H, L
Streptococcus Fecal Streptococcus, Low

Temperature Survival,
Membrane Filter Tech-
nique, Total Coliform

Hg, DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report] SINO-OF PAGES
lUnclassified 19
Release to public 20. SECURITY CLASS (This page) 22. PRICE
Iinclassified

EPA Form 2220~1 (Rev. 4~77) PREVIOUS EDITION IS OBSOLETE

13 GPO 699.320



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 Z. 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 X. 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. pnewnoniae 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 X. pnewnontae 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. pnewnoniae 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


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.

The coliforms isolated from the river were predominantly fecal coliform-
positive (from warm-blooded animal feces) with Escherichia colt and
Klebstella pneumoniae as the most frequent isolates, while the principal
fecal streptococcus was Streptococcus faecalis which is generally preva-~
lent in domestic wastes.

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.

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.

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.

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.

New or modified techniques for enumerating fecal coliforms should mini-
mize the problem of underestimating the fecal coliform population.


Foreword

Abstract

CONTENTS

Page

Fc Vii
a rs cre 7
a vi
Introduction. ..... eee re 1
Conclusions and Recommendations ......... wee eee ee 2
Materials and Methods... 1 1 ee ee ee ee ee ee ett 3
Results... 2. eee eee eee ecw ee eee we ee ee 4
Discussion. ©... ee ee ee eh ee ee ee ee ee 9
OSL ee ee ee ee |


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 Klebstella pneumoniae (46.2%), Escherichia coli (20.5%), Entero-
baeter 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 x. pnewnoniae (53.5%) and EB. coli (35.1%). In
contrast, the 210 m-FC cultures were predominantly £. colz (77.6%), with x.
pneumoniae (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 fabstract n83].

iv


10.

11.

REFERENCES

Bagley, S. T., and R. J. Seidler. Significance of Fecal Coliform-Posi-
tive Klebsiella. Appl. Environ. Microbiol., 33:1141-1148, 1977.

Brown, C., and R. J. Seidler. Potential Pathogens in the Environment:

Klebstella pneumoniae, a Taxonomic and Ecological Enigma. Appl. Micro-
biol., 25:900-904, 1973.

Dufour, A. P., and V. J. Cabelli. Membrane Filter Procedure for Enumer-
ating the Component Genera of the Coliform Group in Seawater. App].
Microbiol., 29:826-833, 1975.

Dufour, A. P., and V. J. Cabelli. Characteristics of Klebsiella From
Textile Finishing Plant Effluent. J. Water Pollut. Contr. Fed., 48:872-
879, 1976.

Huntley, B. E., A. C. Jones, and V. J. Cabelli. XZebstieZZa Densities in
Waters Receiving Wood Pulp Effluents. J. Water Pollut. Contr. Fed.,
48:1766-1771, 1976.

McFeters, G. A., G. K. Bissonnette, J. J. Jezeski, C. A. Thomson, and D.
G. Stuart. Comparative Survival of Indicator Bacteria and Enteric
Pathogens in Well Water. Appl. Microbiol., 27:823-829, 1974.

Vasconcelos, G. J., and R. G. Swartz. Survival of Bacteria in Seawater
Using a Diffusion Chamber Apparatus In Situ. Appl. Environ. Microbiol.,
31:913-920, 1976.

Geldreich, E. E. Sanitary Significance of Fecal Coliforms in the En-
vironment. Publication No. WP-20-3, Federal Water Pollution Control
Administration, Cincinnati, Ohio, 1966. 122 pp.

Geldreich, E. E., R. H. Bordner, C. B. Huff, H. F. Clark, and P. W.
Kabler. Type Distribution of Coliform Bacteria in the Feces of Warm-
Blooded Animals. J. Water Pollut. Contr. Fed., 34:295-301, 1962.

Brezenski, F. T. Fecal Streptococci. In: Proceedings of the First .
Microbiology Seminar on Standardization of Methods, San Francisco, Cali-
fornia. EPA-R4-73-022, U.S. Environmental Protection Agency, Washing-
ton, DC, 1973. pp. 47-53.

Geldreich, E. E. The Use and Abuse of Fecal Streptococci in Water Qual-
ity Measurements. In: Proceedings of the First Microbiology Seminar on
Standardization of Methods, San Francisco, California. EPA-R4-73-022,
U.S. Environmental Protection Agency, Washington, DC, 1973. p. 54-68.

WW


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
Tevel 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 al. (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.


United States Environmental Research EPA-600 -3-79-073
Environmental Protection Laboratory July 1979
Agency Corvallis OR 97330

Research and Development

Identification of
Fecal Indicator
Bacteria

Isolates from an
Ice-Covered River

>> Hy


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 Enterobactertaceae
were identified (Table 2). KlebsteZla pnewnontae (46.2%) was the predominant
total coliform isolated and was found, along with Escherichia colt (20.5%) and
Enterobacter 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, X. pnewnontae
(53.5%) and #. eol¢ (35.1%) were found at all sample stations with Entero-
baeter 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 £. colt (77.6%) was the predominant isolate and
was the only m-FC isolate found at all stations. x. pnewnoniae (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
faecium (Table 4). S. faecalis 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. faecalts and S.
faecium 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. ,
aymogenes 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).


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 m-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, NJ) 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.


TABLES

Number Page
1. Membrane Filter Densities and Percent Verification of Indicator
Bacteria From Each Sample Station. ..........-. eee ee ee 5
2. Identification of Verified Total Coliform Isolates From Each Sample
Station, 2. ee ee 6
3. Identification of Verified Fecal Coliform Isolates From Each Sample
Station, ©. ee we ee 7
4. Identification of the Enterococcus Group Isolates From Each Sample
Station

vi


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.

ii


TABLE 3. IDENTIFICATION OF VERIFIED FECAL COLIFORM ISOLATES FROM EACH SAMPLE STATION*

Total Number of isolates from each sample station
Genus and species isolates

verified T-700 T-600 T-500 T-400 T-300 T-200 T-100
Escherichia coli 163 27 24 19 22 26 24 21
Klebsiella pneumoniae 21 0 3 7 3 2 1 5
Klebsiella ozaenae 1 0 1 0 0 0 0 0
Enterobacter eloacae 1 0 0 0 0 0 0 1
Enterobacter agglomerans 5 3 0 1 0 0 0 1
Serratia liquefaciens 1 am) 0 m!) J m') 0 a)
Total isolates verified 192 30 28 27 26 28 25 28

* 210 colonies subcultured from membrane filters: 30 from each sample station, 10 from each replicate
aliquot.


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. colt was the most frequently isolated coliform, followed by K. pneumoniae,
and Enterobacter 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, S. faecalis 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 aZ. (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 tail K. pnewnoniae) 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 XK. pnewnoniae) and E. colt (35%). The EC-positive total coli-
form cultures in the current study also proved to be mostly X. pnewnoniae
(54%) and £. coli (35%).

Fecal coliforms are considered to be a heterogeneous group with £. colt
and Klebsiella as principal components (3). However, Geldreich (18) cited a
report (L. A. Vinogradova, Hyg. Sanit. 36:157, 1971) in which E. colt isolates


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
Corvallis Environmental Research Laboratory
College, Alaska 99701

CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
US. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330


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
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

Environmental Health Effects Research

Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)
Interagency Energy-Environment Research and Development
“Special” Reports

Miscellaneous Reports

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-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.

DWONAHARwWNH =

This document is available to the public through the Nationa! Technical Informa-
tion Service, Springfield, Virginia 22161.


TABLE 1. RIVER FLOW TIMES BETWEEN STATIONS, AND MEMBRANE FILTER DENSITIES OF
INDICATOR BACTERIA AT EACH STATION*

Indicator bacteria densities per

Days mean 100 ml of river water sample**
Sample flow time
station between Total Fecal Fecal
stations coliforms coliforms streptococci
T-700 6,200 2,200 78
1.9
T-600 1,800 430 65
1.0
T-500 700 100 VW
1.3
T-400 500 200 29
1.1
T-300 230 100 17
1.0
T-200 180 68 13
0.8
T-100 200 90 14

* Samples collected on day five of the survival study (12).
** Mean of three replicate aliquots.


TABLE 4. IDENTIFICATION OF THE ENTEROCOCCUS GROUP ISOLATES FROM EACH SAMPLE STATION*

. Total Number of isolates from each sample station
Genus and species isolates
verified T-700 T-600 T-500 T-400 T-300 T-200 T-100

Streptococcus faecalis 125 23 19 20 17 15 9 22

subsp. faecalis

subsp. zymogenes 7 0 1 1 1 0 2 2

subsp. Ziquefaciens 3 1 0 0 0 0 1 1

other* 29 2 5 7 3 5 7 0
Atypical Streptococcus 1 0 1 0 0 0 0 0

faecalis**
Streptococcus faecium _2 a 0 0 a) 0 0 J
Total isolates verified 167 27 26 28 21 20 19 26

* Litmus milk peptonized, gelatin not hydrolyzed, not beta hemolytic.
** Starch hydrolyzed.