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EFFECT OF HOLDING TEMPERATURE AND TIME ON
TAL COL IF ORM DENSITY IN SEWAGE EFFLUENT SAMPLES
by
Charlotte V. Davenport
Ronald C. Gordon
AERS, WORKING PAPER NO. 31
CERL 041
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
ARCTIC ENVIRONMENTAL RESEARCH STATION
COLLEGE, ALASKA
April 1978
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DISCLAIMER
This working paper has been reviewed by the Corvallis Environ-
mental Research Laboratory, U. 5. Environmental Protection Agen-
cy, and approved for distribution. Mention of trade names or
commercial products does not constitute endorsement or recommen-
dation for use. A Working Paper presents results of investiga-
tions which are to some extent limited or incomplete.
Therefore, conclusions or recommendations--ex pressed or
implied--are tentative.
i i
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INTRODUCTION
Sample integrity has always been of prime importance to
sanitary microbiologists. Over the years, a variety of tempera-
ture and storage conditions have been recommended as a means of
maintaining sample integrity when immediate coliform analysis
was not possible. These recommendations were developed, in
large part, from analysis of drinking water samples which
usually have a low coliform and nutrient content. However, in
river water samples with high (i.e. 100,000/100 ml) initial
coliform densities, Henson(l) showed that the population tended
to increase, at both refrigeration (2°-6°C) and room tempera-
tures (23°-27°C), when analyzed after 6, 12, and 30 hours of
storage. He found the coliform population increases in the room
temperature samples to be more than double those observed for
the refrigerated samples. Hendricks(2) showed enteric bacteria
were capable of growth in waters just slightly above 10°C when
provided with nutrients from a nearby sewage outfall, and a
similar nutrient effect may be involved in bacterial density
changes in samples. Cohen and Shuval(3) observed an order of
magnitude increase in total and fecal coliform counts when un-
treated sewage effluent samples were stored overnight. However,
this did not occur if the samples were diluted before storage.
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These studies on river water(l) and untreated sewage effluent(3)
suggest that samples with high coliform densities display
greater population variability during storage than do drinking
water samples. However, the same time and temperature condi-
tions are often used for storage of both pristine and polluted
water samples. The current recommendation for preserving stream
pollution samples is a maximum holding temperature of 10°C and
coliform analysis within 8 hours after sampling(4). This study
was designed to examine the effects of temperature and length
of storage on total coliform densities in unchlorin a ted primary
effluent samples.
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METHODOLOGY
Unchlorinated primary effluent sample volumes of 19 liters
were obtained from a local treatment plant having a 9.5°-10°C
in-plant effluent temperature. Within 90 minutes of sampling,
the initial sample processing in the laboratory was completed.
In general, processing was as follows: 200 ml sample volumes
were placed in 240 ml (8 oz.) containers (Falcon Plastics);
sample temperatures in the containers were determined; and con-
tainers were placed in 11-liter water baths which had been
equilibrated to maintain the desired holding temperature.
Measurements of pH were also made at this time and again after
8 hours holding time.
Two methods were used to bring the effluent to the desired
holding temperature. In the siow-cool(SC) method, 200 ml
volumes were taken from the 19-liter container and placed
directly in the appropriate water bath. In the rapid-cool (RC)
method, a 2-liter reagent bottle containing about 1800 ml of ef-
fluent from the 19-liter container was placed in a -1°C ethylene
glycol bath. The effluent was stirred continuously and the tem-
perature monitored during cooling. When the desired temperature
was reached, a 200 ml volume of effluent was withdrawn and
placed in the appropriate water bath.
~3~ LIBRARY "
U.S. Environmental Protection A(j*nCT
Corvallis Environmental iUiifiac*. 1 I
200 S.W 3Sth ;
Cocvalks, OrwynrN 9?"?
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The membrane filter technique was used for coliform
analysis(4). An aliquot of effluent obtained directly from the
19-liter container was used for zero time SC coliform counts.
After cooling the effluent to the appropriate temperature, an
aliquot was taken from the 2-liter reagent bottle for zero time
RC coliform counts. All sample dilutions were filtered in
triplicate through 0.45 micron filters (Millipore, type
HAWG 047 SO). The filters were incubated at 35°C for 24 hours
on m-Coliform broth(BBL). After incubation, the total coliform
colonies were counted and 15 typical sheen colonies were ran-
domly picked from each of the three replicate filters for
verification as co 1 iforms(5).
In this study, five temperature conditions were compared:
<1° C-5C , <10 C-RC , 5°C-5C, 5°C-RC, and 10°C. There were no ex-
periments done using the 10°C-RC test condition because the ini-
tial plant effluent temperatures were always very close to 10°C.
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RESULTS
The results of three 8-hour experiments examining the ef-
fects of holding time and temperature are presented here. Table
1 shows the temperature and pH measurements for each experiment.
In the SC test conditions, the desired sample temperatures were
reached within the first 2 hours and remained stable for the
rest of the test period. The pH was stable during each of the
three experiments except for the 5°C-RC sample in experiment 3,
which had an aunormally low initial pH.
The mean membrane filter-total coliform (m-TC) count/100 ml
and percent verification for each time interval and test condi-
tion are shown in Tables 2, 3, and 4. The mean and standard
deviation of the count/100 ml and percent verification for each
test condition over the 8-hour holding time are also presented.
In experiment 1 (Table 2), <1°C-SC, <1°C-RC, and 10°C holding
conditions were examined. The counts showed the <1°C-SC
coliform population declining slightly after 6 hours, but over
the 8 hours it was relatively stable as was the <1°C-RC popula-
tion. The verification percentages for the <1°C samples were
very similar, with the <1°C-SC sample being slightly lower in
mean percent verification than the <1°C-RC sample. During the
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8-hour storage period at 10UC, the m-TC counts showed a con-
tinual increase, while the percent verification showed a con-
tinual decrease .
In experiment 2 (Table 3), an intermediate holding tempera-
ture, 5°C-5C was added. There was no significant difference
between the <1°C-5C and <1°C-RC counts although the zero time
count for <1°C-RC samples was relatively low. The 5°C-SC sample
has a low count at 2 hours, but was not significantly different
from the <1°C samples for the remainder of the test period. The
10°C sample showed a slight increase after 4 hours. Comparing
the mean percent verifications, the <1°C samples were higher
than either 5°L SC or 10°C samples. But in general, the mag-
nitude of the population density differences under the various
test conditions in this experiment were not as great as those
in experiment 1.
In experiment 3 (Table 4), a 5°C-RC test was added so that
<1°C-SC, <1°C-RC, 5°C-SC, 5°C-RC, and 10°C test conditions could
be compared. The coliform densities for the <1°C and 5°C
samples showed about the same degree of variability. However,
for both RC samples, the zero time counts were lower than the
SC zero time counts. This may be an effect of rapid-cooling.
During the remainder of the test period, the 5°C densities were
slightly higher than the <1°C samples. Relatively stable counts
were found at <1°C and 5°C as compared to the 10°C sample in
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which the population doubled over the 8-hour test period. The
stability of the counts at <1°C and at 5°C was confirmed by the
percent verification and of the five test conditions, the sample
at 10°C had the lowest mean percent verification and the highest
standard deviation for verification of any of the samples. In
general, the magnitude of the differences in the 10°C counts
relative to the other test conditions was greater than in ex-
periment 2, but not as great as in experiment 1.
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DISCUSSION AND CONCLUSIONS
This study was designed to examine the effects of various
temperatures and storage times on coliform density. Time was
not a factor except that the coliform density differences at the
various holding temperatures became more accentuated with in-
creasing storage time. There was insufficient testing to deter-
mine the relative effect on the coliform population density, of
either the rapid-cool or slow-cool treatment. However, the
results do sugy^st that storage temperature is a critical factor
in maintaining sample integrity when immediate coliform analysis
is not possible. In each of the three experiments, the coliform
population in the 10°C samples showed density increases over the
8-hour storage period. These 10°C samples also exhibited the
lowest mean percent verification compared to the other holding
temperatures. The <1°C samples, while usually having the
highest mean percent verification, sometimes showed a density
decrease after 6 hours. The samples at 5°C showed little change
over the 8-hour test period and the SC samples had a percent
verification generally as high as the <1°C samples.
These results suggest that an adequate assessment of the
coliform density in the sample source can be provided if the
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sample temperature is reduced to 5°C or less with analysis com-
pleted in 8 hours. However, these observations may only be ap-
plicable to samples containing high coliform densities as
analyzed by the membrane filter technique. It should be noted
that since the Most-Probable-Number (MPN) confidence limits en-
compass a wide density range, the differences seen in the mem-
brane filter counts for the three experiments could be masked
if the MPN technique were used.
Coliform isolates were verified to determine if false-
positive colonies were being counted on the membrane filter.
The study showed that there were a higher number of false-
positive orgarusms at 10°C than at the lower temperatures, but
there was a great variability, in percentage of the population
verified, from one plant effluent sample to the next. This
fluctuation suggests that verification is necessary for accurate
membrane filter count interpretation, and should include the ex-
amination of negative as well as positive colonies on the
filter. Very little work has been done on the false-positive
and false-negative reactions which occur during membrane filter
coliform analysis. One group of investigators has found that
false-positive reactions can result from synergistic interac-
tions among microorganisms(6) . However, the why of false-
negative reactions has not been examined.
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The current recommendations for sample preservation^) do
not consider the wide variety of sample sources that can be en-
countered in pollution surveys. This study has shown that main-
tenance of bacteriological sample integrity during transport and
storage requires consideration of sample temperature during
storage, nutrient content and coliform density. Additionally,
interpreting results from analyses of stored samples may require
further evaluation of the accuracy and reliability of coliform
analytical methods. Development of preservation guidelines
should include studies examining a wide variety of sample
sources and using several analytical techniques. Such studies
may result in preservation guidelines which suggest using a
variety of s to l age and analytical methods depending on sample
source.
In the meantime, sample integrity remains a problem for
microbiologists. But as we continue to learn more about en-
vironmental factors affecting coliform populations in samples
and to understand more fully the limitations of the analytical
techniques, we will be better able to provide accurate assess-
ment of the bacteriological quality of an environment.
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REFERENCES
1. Henson, E. B. 1971. Investigations on storage and preserva-
tion of water samples for microbiological examination.
Master of Science Thesis, Dept. of Civil and
Environmental Engineering, Univ. of Cincinnati,
Cincinnati, Ohio.
2. Hendricks, C. W., and S. M. Morrison. 1967. Multiplication
and growth of selected enteric bacteria in clear mountain
stream water. Water Research. 1^:567-576.
3. Cohen, 3., and H. I. Shuval. 1973. Coliforms, fecal
coliforms, and fecal streptoccocci as indicators of water
pollution. Water, Air and Soil Pollut. 2:85-95.
4. American Public Health Association. 1975. Standard methods
for the examination of water and wastewater, 14th ed.
American Public Health Association, Inc., New York.
5. U. S. Environmental Protection Agency. 1974. Current prac-
tices in water microbiology. EPA-430/1-74-009. U. S.
Environmental Protection Agency, Washington, D. C.
6. Schiff, L. J.,S. M. Morrison, and 3. V. Mayeux. 1970.
Synergistic false-positive coliform reaction on m-endo
MF medium. Appl. Micro. 20;778-781 .
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Table 1. Sample temperatures and pH readings recorded during the eight-hour storage period.
Experiment
no.
Time
(hr)
<1°C-SC
<1°C-RC
5°C-SC
5°C-RC
10°C
Temp. pH
Temp. pK
Temp. pH
Temp. pH
Temp pH
1
0
2
4
6
8
10.9 7.2
0.2 —
0.2
0.6 —
0.7 7.4
1.8 7.2
0.1
0.2
0.6 ---
0.8 7.4
— —
— —
12.7 7.2
10.2
9.7 —
9.8 —
9.8 7.2
2
0
2
4
6
8
9.3 7.3
0.7
0.6 ---
0.5
0.4 7.4
1.6 7.3
0.6 ---
0.6 ---
0.5
0.4 7.3
9.6 7.3
5.5
5.3
5.2 —
5.1 7.3
— —
10.3 7.3
10.0
10.0 —
9.8
9.8 7.2
3
0
2
4
6
8
10.8 7.3
0.7
0.5 ---
0.5
0.5 7.4
1.8 7.2
0.7 ---
0.5
0.5 ---
0.6 7.2
11.4 7.3
5.6 ---
5.4
5.2 ---
5.6 7.3
7.4 6.0
5.6 —
5.4 —
5.4 —
5.6 7.5
72.1 7.3
9.9 —
9.7
9.6 ---
9.7 7.3
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Table 2. Mean m-TC counts and percent verified for experiment 1.
Time
<1°C-SC
<1°C-RC
10°C
(hr)
m-TC Percent
count/100 ml verified
m-TC Percent
count/100 ml verified
Percent
count/100 ml verified
0
2
4
6
8
2.6 x 107 62.3
2.5 x 107 75.6
2.8 x 107 79.7
2.0 x 107 64.3
1.6 x 107 71.1
4.2 x 107 62.3
2.0 x 107 78.0
2.4 x 107 73.7
2.2 x 107 75.7
2.9 x 107 75.3
2.6 x 107 62.3
5.0 x 107 53.0
6.3 x 107 46.7
7.8 x 107 44.3
1.4 x 108 26.3
Mean
Standard
Deviation
2.3 x 107 70.6
±0.5 x 107 ±7.4
2.7 x 107 73.0
±0.9 x 107 ±6.2
7.1 x 107 46.5
±4.3 x 107 ±13.3
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Table 3. Mean m-TC counts and percent verified for experiment 2.
Time
(hr)
<1°C-
sc
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Table 4. Mean m-TC counts and percent verified for experiment 3.
Time
A
0
o
1
¦sc
A
0
o
1
¦RC
5°
c-sc
5°C-
RC
10
°c
(hr)
M-TC
count/
100 ml
Percent
verified
m-TC
count/
100 ml
Percent
verified
m-TC
count/
100 ml
Percent
verified
m-TC
count/
100 ml
Percent
veri fi ed
m-TC
count/
100 ml
Percent
verified
0
1.4
X
107
78.0
8.0
X
106*
80.0*
1.4
X
107
78.0
1.2
X
107
85.0
1.4
X
107
78.0
2
9.5
X
106
89.0
1.2
X
107
89.0
9.9
X
106
80.0
1.3
X
107
71.0
1.4
X
107
60.0
4
I. T
X
107
80.0
7.2
X
TO7
84.0
7.4
X
107
80.0
1.3
X
107
78.0
2.7
X
107
47.0
6
1.7
X
107
67.0
1.8
X
107
89.0
1.3
X
107
89.0
2.1
X
107
62.0
5.7
X
107
49.0
8
1.2
X
107
84.0
1.1
X
107
80.0
1.7
X
107
76.0
1.7
X
107
69.0
2.8
X
107
73.0
Mean
1.3
X
107
79.6
1.2
X
107
84.4
1.4
X
107
80.6
1.5
X
107
73.0
2.8
X
107
61.4
Std.
Dev.
±0.3
X
107
±8.2
±0.4
X
107
±4.5
±0.3
X
107
±5.0
±0.4
X
107
±8.8
±1.8
X
107
±13.9
* Mean of two instead of three replicates because of laboratory error.
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