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United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
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Research and Development
EPA/600/S2-85/061 Aug. 1985
<&ER& Project Summary
Enumeration and
Identification of Heterotrophic
Bacteria from Drinking Water
James T. Staley
Various spread-plating enumeration
media and procedures were tested to
determine the method of choice for
enumerating the highest numbers of
heterotrophic bacteria from chlorinated
drinking waters. Dilute media, includ-
ing a casemate peptone starch medi-
um, a dilute peptone medium, and R2A
medium provided greater recoveries
than the standard plate count medium
currently used. In addition, reduced
temperatures of 20°C and prolonged in-
cubation periods of 14 to 28 days re-,
suited in the highest recoveries of het-
erotrophic bacteria from the waters of
the two chlorinated distribution sys-
tems examined.
In the Seattle, Washington, water
treatment and distribution system,
unchlorinated source water had higher
diversities of heterotrophic bacteria
than did chlorinated drinking water
samples. The predominant types re-
covered from chlorinated drinking
waters of that system were Gram-
negative pigmented bacteria. Most of
these could not be readily identified to
known genera. Some of these could be
placed in the poorly described genus
Flavobacterium, but the great variety
of types of pigmented Gram-negative
bacteria included many that could not
be readily classified in this group with-
out further studies including DNA hy-
bridization.
A number of representatives of
known genera were encountered
among the isolates from the chlori-
nated drinking waters. Included were
members of the genera Caulobacter,
Hyphomonas, Aquaspirillum, Vibrio,
Gluconobacter, Azomonas, and
Aeromonas.
This Project Summary was devel-
oped by EPA's Water Engineering Re-
search Laboratory, Cincinnati, OH, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at,
back).
Introduction
Water is universally consumed in
large quantities by the public. Thus
from a public health perspective, it
would be desirable to know the types
and numbers of bacteria that are in-
gested by drinking chlorinated water.
Yet numerous investigations have
shown that the standard method for
enumerating heterotrophic bacteria
from drinking waters (i.e., the standard
plate count or SPC procedure) fre-
quently provides lower counts of bacte-
ria from drinking waters than other pro-
cedures. Furthermore, no adequate
procedure currently exists for character-
izing and identifying heterotrophic bac-
teria from drinking waters.
The objectives of this investigation
were twofold: a) to evaluate alternative
procedures for enumerating het-
erotrophic bacteria from chlorinated
water supplies and b) to develop a pro-
cedure that could be used to identify the
bacteria that commonly occur in drink-
ing waters.
The City of Seattle water distribution
system was used as a test system for
the study of enumeration procedures.
Various media and methods were com-
pared with the SPC procedure to pro-
vide an optimum count of the viable
heterotrophic bacteria.
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About 100 bacterial strains (50 iso-
lates each from the Cincinnati, Ohio,
and Seattle, Washington, water sys-
tems) were studied to develop a schema
for use in characterizing and identifying
the predominant bacteria from treated
distribution water. Morphologic and
biochemical tests were used to charac-
terize the isolates.
Materials and Methods
Drinking water samples from the
Seattle water system were collected in
sterile sample bags or bottles contain-
ing sodium thiosulfate to inactivate
residual chlorine, as recommended by
Standard Methods for the Examination
of Water and Wastewater (15th Ed.,
American Public Health Association,
1980). Samples were iced until pro-
cessed, and all samples were analyzed
within 8 hr of collection.
Enumeration of heterotrophic bacte-
ria was done by the spread plate proce-
dure using several media, including
Standard Methods agar (SMA), dilute
peptone agar (DP), caseinate peptone
starch agar (CPS), and R2A medium.
The pour plate procedure was also used
for some SMA plates.
Strain diversity in water samples was
determined based on colony type. The
Shannon index was calculated as an es-
timate of strain diversity from the for-
mula:
H' =
where
3.3 log10ni/N
H' = diversity index value
N =total number of colonies
counted
ni = number of colonies of one
type (i.e., ith strain)
s = number of strains
Direct counts were made microscopi-
cally using acridine orange (AO) stain-
ing and an epifluorescence microscope..
Bacterial strains used for the charac-
terization and identification studies
were isolated from winter and summer
water samples from both the Seattle
and Cincinnati distribution systems.
Seattle isolates were obtained from DP
plates, and Cincinnati isolates were se-
lected from R2A plates. Characterization
tests included Gram-stain, colonial and
cellular morphology, anaerobic growth,
oxidase test, growth on mineral media
with carbon sources, oxidation/
fermentation reaction, polyhydroxybu-
tarate incorporation, spore formation,
growth at pH 4.5, acid production from
ethanol, nitrification, cellulose degrada-
tion, pigmentation or fluorescence,
growth temperature, and other special
media tests as required. Media were in-
cubated at 20°C unless otherwise speci-
fied. Known baterial strains were ob-
tained for comparison from other
investigators and from the American
Type Culture Collection.
Experimental Results
Enumeration of Viable
Heterotrophic Bacteria from
Chlorinated Drinking Water
Water samples were collected and ex-
amined from 22 sites in the Seattle
water distribution system, representing
both pre- and post-chlorination loca-
tions.
All four media were used to examine
samples from three surveys. The results
showed that the higest counts were ob-
tained after 14 to 28 days of incubation,
depending on the sample. However,
counts from SMA plates incubated at
35°C for 48 hr were never as high as
those obtained by other means, and
20°C SMA counts were almost always
inferior to those obtained with other
media used in the surveys. Overall, the
best medium was DP, followed by CPS
and R2A. DP and CPS consistently pro-
vided the highest counts for chlorinated
water samples, but DP was less satisfac-
tory than CPS for enumerating bacteria
in water samples collected before chlo-
rine treatment. Because of the superior
performance of DP and CPS, these
media were used for subsequent viable
plate count analyses.
The length and temperature of incu-
bation strongly affected viable plate
counts. After 14 days of incubation,
most chlorinated samples showed vi-
able counts at 20°C that were 50% to
100% of the counts obtained after 28 to
30 days at 20°C. Incubation beyond 28 to
30 days at 20°C infrequently showed
significant increases in plate counts.
Bacterial counts on DP and CPS media
were compared after plates were incu-
bated at 20°, 30°, and 35°, and at a dual
temperature—20° for 48 hr, then at 30°C
for up to 30 days (20° -> 30°C). Ranked
from highest to lowest counts, the re-
sults were 20° > (20° -H> 30°) > 30°C.
Total direct counts by the AO method
were much higher than viable counts,
as expected. For unchlorinated water
samples, only about 1% of the total di-
rect count bacteria were cultivable; and
for chlorinated samples, the percentage
of viable organism recovery was even
lower. In some reservoirs, the ratio of
viable to AO counts exceeded 10%
(range 11.8% to 42.4%). Such high ratios
indicate that the water was eutrophic
rather than oligotrophic.
Diversity indices showed that chlori-
nation of the water caused a dramatic
change in bacterial diversity (Table 1).
Whereas the unchlorinated source
water contained a variety of colony
types, including many nonpigmented
ones, the chlorinated water contained
relatively few colony types and most
were pigmented. Since these results
were supported by those from an earlier
study in which diversity indices were
calculated for both chlorinated and
unchlorinated waters, it was concluded
that the disinfection and distribution
processes in the Seattle system com-
monly (and perhaps always) result in
selection of relatively few bacterial
species compared with the source
water.
Characterization and Identi-
fication of Bacteria from
Chlorinated Drinking Waters
The major purpose of this portion of
the study was to identify the predomi-
nant heterotrophic bacteria from two
different drinking water systems. The
process of identification assumes that
the organisms being investigated are
known organisms that have been previ-
ously characterized, described, and
named. We anticipated that most bacte-
ria found in chlorinated drinking waters
would be strains of known organisms
and that characterization of the isolates
and their comparison with known or-
ganisms would be rather straightfor-
ward. Such was not the case, however.
Because of the slow growth of the iso-
lates and their general nonreactivity in
biochemical characterization media,
they posed special problems for charac-
terization. Tests were developed to per-
mit phenotypic characterization and de-
scription of these bacteria, and a
dichotomous flow chart for characteri-
zation was prepared (Figure 1). Many of
the bacterial isolates were not identifi-
able to known species, but use of the
dichotomous key permitted grouping of
similar types of organisms.
Most of the bacterial isolates exam-
ined were Gram-negative rods, some of
which were morphologically distinctive.
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Table 1.
Diversity Indices of Source Water (CPRI) and Chlorinated Samples Collected
September 2, 1981, and Counted after 28 and 40 days
After 28 Days
After 40 Days
Sample
CPRI
13
K3
L3
M3
No. Bacteria/ml
3,600
210
1,700
2,900
150,000
Diversity Index*
4.14
1.85
2.81
1.23
1.08
No. Bacteria/mlt
-
570
-
3,070
160,000
Diversity Indext
-
2.45
-
1.45
1.55
*The diversity index is based on 55 colonies, except for sample M3, in which 40 colonies
were used.
tForty-day counts were determined from 28-day plates that had been refrigerated. In-
creased counts on plates were due to growth of Hyphomonas.
^Diversity index is based on Hyphomonas colonies. Total colonies counted for 13 were 81;
L3 were 58; and M3 were 44.
such as prosthecate bacteria. Prosthe-
cate bacteria identified included
Caulobacter and strains recognized as
belonging to the Hyphomonas—
Hyphomicrobium group. In some sam-
ples from the Seattle system, Hy-
phomonas constituted from 2.8% to
12.8% of the viable count. Other mor-
phologically distinct isolates included
a species of Vibrio, three spiral-
shaped organisms identified as
Aquaspirillum strains, and three large,
ovoid, motile cells identified as
Azomonas.
The majority of the isolates (44) were
Gram-negative, nonfermentative, oxi-
dase positive, aerobic rods, and 24 of
these were pigmented. Of the nonpig-
mented group, only one isolate was
identified as a typical Pseudomonas
Phase Morphology
Gram Stain (102)
Gram Negath
Anaerobi
-(•
Oxii
te Rods (72)
c Growth
17)
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strain, although several isolates resem-
bled Pseudomonas species in all re-
spects except for their inability to grow
on mineral medium with acetate as the
carbon source. About half of the pig-
mented Gram-negative rods (24) were
unable to grow at 37°C and 7 did not
grow at 30°C. Many did not grow suffi-
ciently well to permit thorough charac-
terization. All of the pigmented strains
were negative for cellulose digestion,
and none survived the heat survival
tests for sporeformers.
A few Gram-positive and some Gram-
variable isolates were obtained. Of the
five Gram-positive cocci, three were
identified as Staphylococcus, one was
Micrococcus luteus, and one was possi-
bly a strain of Planococcus. One of the
Gram-positive rods was identifiable to
genus; its characteristics were typical of
the genus Gluconobacter. The remain-
ing four Gram-positive rods and two
Gram-variable rods were not identifi-
able. All but one were pigmented
strains.
Because the pigmented Gram-
negative rods made up an especially
large group of the isolates and were
well represented in both distribution
systems, much effort was made to iden-
tify them. Because of the difficulty in
characterizing those isolates, however,
identification was not successful. Many
of these isolates could possibly be clas-
sified as members of the genus
Flavobacterium, which is a broadly de-
fined genus that encompasses virtually
all pigmented Gram-negative rods. So
broadly is it defined, however, that it is
known as a wastebasket genus. The pig-
mented isolates from this study were
not identified as Flavobacterium for
several reasons. First, there was no con-
vincing evidence that the pigmented
distribution system isolates were re-
lated to those that are currently placed
in the Flavobacterium genus. Second,
some nonpigmented strains closely re-
semble the pigmented strains except
for the pigmentation. Thus, until both
the pigmented and nonpigmented
strains are better characterized and
studied, it seemed inappropriate to
make an arbitrary assignment on the
basis of only one characteristic, pig-
mentation. The third reason was that
the group of pigmented isolates found
in this study is a very diverse group, and
because of the wide range of pheno-
typic characteristics, it is doubtful that
all pigmented isolates could be placed
in a single genus. Even the genus
Flavobacterium as currently defined
does not contain organisms that
demonstrate the diversity shown by the
pigmented bacterial isolates found in
this study.
Conclusions
A variety of media were tested to de-
termine which provided the highest vi-
able counts of heterotrophic bacteria in
chlorinated drinking waters from the
City of Seattle water distribution sys-
tem. The most dilute medium tested
consistently provided the highest recov-
eries of heterotrophic bacteria from the
chlorinated water samples examined
during this study.
The highest viable counts of het-
erotrophic bacteria from the chlorinated
samples were obtained when (1) plates
were incubated at 20°C rather than at
30° or 35°C, and (2) incubation time was
longer than 14 days. Use of dual-
temperature incubation (20°C for 48 hr
followed by 30°C for the remainder of
the incubation period) provided higher
counts than did incubation at 30°C but
lower counts than incubation at 20°C.
Twenty-eight days of incubation nor-
mally gave maximal recovery at 20°C,
but some bacteria did not appear until
after about 40 days.
Total direct microscopic count using
AO generally decreased following chlo-
rination. Viable count results indicate
that only about 1% of the total bacteria
can be cultivated on laboratory media.
The diversity of heterotrophic bacte-
ria on each enumeration medium was
determined using the Shannon diver-
sity index formula. Chlorination re-
sulted in a diversity index decrease
(1.08 to 2.81) compared with values be-
fore chlorination (>4.0).
A total of 102 bacterial isolates were
obtained from the two treated munici-
pal drinking water systems in Seattle
and Cincinnati. Of about 50 isolates
from each system, half were isolated
during the summer months and the
other half during the winter months.
The bacterial isolates were character-
ized and identified, where possible, to
known genera using phenotypic tests.
Seventeen of the isolates were morpho-
logically distinctive and included
Caulobacter (9 strains), Hyphomonas
(1 strain), Aquaspirillum (3 strains), a
black pigmented strain of Vibrio
(1 strain), and Azomonas (3 strains).
This is apparently the first report of
Caulobacter and Hyphomonas in chlori-
nated drinking water. Among a group
(14 cultures) of Gram-positive and
Gram-variable nonfermentative rods.
only one was identified, a strain of Glu-
conobacter.
Most of the isolates were Gram-
negative, obligately aerobic rods. Only
one was clearly identifiable as a strain
of Pseudomonas, although several
other isolates were motile and nonpig-
mented and shared many features of
the genus. Many of the Gram-negative
rods could be superficially classified in
the genus Flavobacterium because of
their pigmentation and other features.
However, these organisms showed
greater diversity than even that poorly
defined genus. Further study, including
DNA hybridization, is needed to deter-
mine whether any of the isolates resem-
ble known strains of the genus
Flavobacterium closely enough to be
classified as such.
Only a few of the Gram-negative rods
were facultative anaerobes, some of
which were identified as strains of
Aeromonas hydrophila.
The full report was submitted in fulfill-
ment of Cooperative Agreement No.
CR-807570 by the University of Wash-
ington under the sponsorship of the
U.S. Environmental Protection Agency.
US.QOVEHNMENT PRINTING OFFICE 1W5 559-111/20634
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James T. Staley is with University of Washington. Seattle. WA 98195.
Donald J. Reasoner is the EPA Project Officer (see below).
The complete report, entitled "Enumeration and Identification of Heterotrophic
Bacteria from Drinking Water," (Order No. PB 85-207 496/AS; Cost: $11.50.
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK RATE
POSTAGE & FEES PAI
EPA
PERMIT No. G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-85/061
0169064 HERL
60604
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