United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati, OH 45268
Research
,.l
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Mathematical Models
When the membrane filter (MF)
method is used to detect coliform
bacteria, the limit of detection is one
organism per 100 mL for an individual
grab sample. The results from one
sample do not, however, provide much
information about the density of coliform
bacteria in the body of water that was
sampled. If several samples are collected
from a relatively large amount of water
containing only a few coliform bacteria,
the limit of detection is approximate and
depends on the number of samples
examined and the probability of capturing
coliform bacteria in any sample. For any
body of water, there is a probability of
capturing one or more coliform bacteria
in any 100 mL sample, which can be
represented at P{ + /100 mL}. The
probability of capturing conforms with n
samples can be calculated from
P{detection} = 1-[1-P{ + /100 mL}]".
This formula is independent of the
frequency distribution of the coliform
counts and can be generalized for other
sample volumes by substituting P{ + /V
mL} as the probability of a positive result
in a sample of V mL. The manner in
which P{ + /V mL} changes with a change
in V can be calculated if the frequency
distribution is known. This information
can then be used to calculate the
difference in the probability of detecting
coliform bacteria by compositing a large
number of small volume samples or by
using a single large sample having the
same total volume.
The frequency distributions used for
the mathematical models were the
Poisson as a representation of a random
dispersion and the lognormal as a repre-
sentative of aggregated dispersion.
These frequency distributions have been
used to describe coliform data on large
numbers of samples from water distribu-
tion systems.
Closed Form Model
If coliform bacteria were ever random-
ly dispersed in the entire body of water
(i.e., the colony counts in unit volumes of
water fitting a Poisson distribution), the
probability of obtaining a count of x
coliform bacteria in a volume, V, would
be given by
P{x} = e-m(nv
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for estimating the sampled distribution.
For instance, a lognormal distribution with
GM = 0.00000112 and GSD - 100 has
an arithmetic mean density lower than
another lognormal distribution with GM
= 0.00112 and GSD = 20; however, it
can produce samples with densities of
one or more coliforms per sample
precisely because, with its greater
variance, it is more likely to produce
samples with a very high density. As an
example, for the GM - GSD pairs cited
above, the arithmetic mean and variance
and the probability of a positive sample
are shown in Table 1.
GSD values reported for water
distribution systems ranged from about
10 to 100. When the GSD is low,
composite sampling gives little advantage
for finding coliform bacteria. For example,
a system with a GM of 0.1 and a GSD of
10 has an arithmetic mean density of
1.42/100 ml, and composite sampling
will provide little advantage when
compared with grab sampling since the
probability of a positive 100 mL sample is
0.16. The more challenging case is the
system that has the same arithmetic
mean but much higher variability. A
system with a GM of 0.000025 and a
GSD of 100 also has an arithmetic mean
density of about 1 per 100 mL, but very
few 100 mL aliquots (about 1 in 100) will
have any coliforms at all. In this case,
composite sampling would be the
method of choice.
Summary on Mathematical
Models
It was shown by both closed form
models and computer simulations that
composite sampling from a lognormal
distribution is superior to grab sampling,
if the variance of the coliform count is
large. The larger the variance, the greater
the advantage of composite sampling for
capturing coliform bacteria. When, how-
ever, the value of the variance
approaches the mean density, composite
sampling does not provide any great
advantage for detecting coliform bacteria.
The agreement of the simulated results
with the predictions of the closed form
model provides some assurance of the
reliability of the conclusions.
Laboratory Study
The laboratory study included con-
trolled experiments for testing hypoth-
eses about the effectiveness of
composite sampling for capturing
bacteria entering or in a water distribution
system. Evidence of the efficacy and
fficiency of composite sampling as com-
pared with grab sampling was developed
by means of these experiments. The
laboratory results turned out to be the
primary verification of the results from the
mathematical models, since few coliform
bacteria were found in either grab or
composite samples during the field
study.
Methods
The criteria that governed selection of
a sampler were that it should (1) be easy
to clean, (2) be constructed so that the
parts coming in contact with the water
could be sterilized or sanitized, (3)
require only standard fittings and fixtures
to install, and (4) be portable and easy to
set up. The intent was to select a stock or
modified commercial sampler; however,
none of the suppliers queried had or
knew of such a stock sampler. The
Sigma Model 6301* (American Sigma PO
Box 300, Middleport, NY 14105-0300)
was selected largely because of its ability
to index through a series of sample
containers, which would provide
sequential composite samples. Additional
samplers were also constructed
consisting of variable rate peristaltic
pumps and polypropylene sample
containers (Nalgene 2319 series).
To produce large variances of the
coliform densities, a syringe pump driven
by a very accurate timer was selected to
deliver the inoculum. The composite
samples were collected by means of a
peristaltic pump operated at a rate
selected to provide the sample size
desired. Tubing and pump heads were
selected to provide residence times in
the experimental system consistent with
residence times expected or experienced
in field applications.
The laboratory composite sampling
systems were modified from time to time
as experimental requirements made
additional controls necessary. Composite
samples were collected with variable rate
peristaltic pumps set to collect the
desired volume. Volumes collected for
the composite samples ranged between
0.1 and 4.0 L/hr.
All composite samples were analyzed
in their entirety. Intermittent inoculation
was used to simulate the occurrence of
coliform bacteria in a real distribution
system; i.e., relatively high densities were
injected over relatively short periods of
time and relatively small portions of the
flow contained most of the bacteria. Both
"Mention of trade names or commercial
products does not constitute endorsement or
recommendation for use.
Escherichia coli (ATCC 8739) and
Enterobacter cloacae (ATCC 13047)
were used in the laboratory experiments,
and there were no differences in the
results obtained using the two different
organisms. The inoculum density was
changed by using different dilutions of
the initial culture; using different
inoculation rates; varying the flow rate;
and, to a much lesser extent, varying the
periodicity of inoculation.
Results
The laboratory experiments were
designed to show that (1) the recovery of
coliform bacteria by composite samples
from flows of water with known disper-
sions, in which very little of the water
under test contained all the coliforms,
were not significantly different from the
predicted frequency of occurrence, (2)
the threshold for coliform detection was
generally in the range predicted, and (3)
the mean coliform densities of the
samples were not significantly different
from those calculated from the inoculum
density. The control of the dispersions of
bacteria in the flow of water to mimic the
occurrence of coliform bacteria in actual
water systems was a central theme of
these experiments.
An agreement was obtained between
the theoretical frequency of occurrence of
various densities and the actual densities
obtained for all overnight (18 to 24 hr)
composite sampling runs. These results
indicated that the composite sample den-
sities were substantially in agreement
with the densities that were introduced
into the experimental system by the
syringe pump. The probability of captur-
ing a coliform was directly related to the
highest density in the flow of water
sampled. The 95% probability of captur-
ing a single coliform in the composite
was the 95% probability of a single
coliform occurring in the sample portion
given the sampled stream density.
The data showed that composite
sampling provided equal or superior
performance for times of both high and
low probability of capturing coliform
bacteria and hence, for both levels
coliform occurrence. All experiments
accepted for further analysis were from
systems tested for and found free of
coliform contamination. Bacterial
densities in composite samples were
consistent with stream densities
calculated from inoculum densities and
from the volume of flow for all
experiments. Experiments designed to
test the effect of variability on the
frequency of positive sample results
followed predictions of the closed form
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Table 1.
GS
0.00112
0.00000112
Effect of Variability of Conform Counts on Arithmetic Parameters
GSD
10
50
100
10
50
100
Arithmetic
Mean
0.0159
2.36
45.12
0.00002
0.00236
0.04512
Variance
0.0503
2.46 X TO7
3.305 x TO'2
5.03 x 10-8
24.6782
3.305 x 108
Probability of
Any Positive Result
0.0016
0.0412
0.0700
1 x 10'9
0.0002
0.0015
Result > 1
0.0015
0.0381
0.063
1 x 10'9
0.0002
0.0014
and simulation models; i.e., that, for any
given mean density, higher variability will
provide a greater frequency of positive
portions. Further, the frequency of
positive portions was directly related to
the maximum stream density for any
given size of aliquot. Finally, experiments
directly comparing composite and grab
sampling from the same stream conclu-
sively demonstrated the superiority of
composite sampling for capturing
coliform bacteria.
Field Studies
Field sampling results were obtained
from waters from two different distribution
systems, West Chester and Downing-
town. Both of these systems are in
Chester County, Pennsylvania, about 30
miles west of Philadelphia. Neither have a
record of violations of the microbiological
maximum contaminant level. The objec-
tives of the field sampling were to test the
use of the composite sampler in realistic
situations, to attempt to obtain further
verification of the mathematical model
and to compare grab samples with the
composite samples.
Methods
The composite sampling setups for
the field studies were identical to those
used in the laboratory. The West Chester
sites were both at system pressure,
approximately 160 to 180 psi at the plant
and 140 to 160 psi at the tank. The
Downingtown sampler was not used on a
pressurized line but drew from the top of
the filters and from the filter effluent.
Composite and grab samples were col-
lected over 18- to 24-hr periods and
protected from sunlight during transport
in insulated containers with artificial
coolant packs. All microbiological proce-
dures were completed within 6 hr of
collection.
Each sample, composite and grab,
was tested for total coliform bacteria and
heterotrophic plate count. Large aliquots
could be filtered by the MF procedure for
all the finished water samples and both
300 and 500 mL portions were routinely
filtered with no evident reduction in
filtrate flow due to occlusion or matting of
filter surfaces. In addition to the
microbiological analyses, each sample
source was tested each day for free and
total chlorine, temperature, and pH. The
turbidity of all except West Chester tank
samples was also determined each
sampling day.
West Chester Results
The East Branch of the Brandywine
Creek was the source of raw water during
the period of sampling. The raw water
had very high densities of coliform
bacteria from agricultural land runoff and
wastewater discharges. Treatment con-
sisted of prechlorination, flocculation,
settling, rapid sand filtration, and post
chlorination. Composite samples were
concurrently taken of the treated water
(the high pressure side of one of the high
service pumps) and the flow to the
storage tank. Overall, 36 sets of 2
composite samples and 5 individual
composite samples (77 total composite
samples) were obtained from this system
from June to November 1987. In addition,
grab samples of the raw, finished, and
distribution system water were collected.
The distribution system samples were
obtained from both private residences
and fire hydrants.
Only one of the composite samples,
which was obtained from the tank site,
had coliform bacteria. Four 500-mL
portions were positive, three with one
organism each and one with two. This
gave a sample density of 0.00098 per mL
for the approximately 2-gal composite,
thereby demonstrating the ability of
composite sampling to detect coliform
bacteria at low densities. Three of 104
grab samples of the finished water and 2
of 96 grab samples from private
residences had coliform bacteria. More
coliform bacteria were detected in the
grab samples than in the composite
samples. Overall coliform densities were
much lower than 1.0 per 100 mL, ai
these results were probably just happe
stance.
Dowingtown Results
Two water sources, Copeland Run ai
Beaver Creek, supplied about 30% ai
70% of the water treated, respective!
There were approximately 8 miles
mains, most 6 or 8 in., and 2,100 servii
connections. The system had three stc
age sites, a 4-MG open storage reserve
at the plant and two 2-MG steel grout
storage tanks on elevations north ar
south of the Borough proper.
A history of poor turbidity removal w;
one reason for selecting this plant f
sampling. There was no provision f
continuous sludge removal. Filter influe
turbidities were sometimes very high, ar
it was hoped that some coliform positi>
composite samples would result from th
sampling series despite the 2 mg/L fr«
chlorine residuals maintained through tt
process.
The composite samples of the influe
were analyzed in full. These data showe
that composite sampling can provide da
at least equivalent to even the stricte
grab sampling regimen. In these expei
ments, grab samples were taken alte
nately from the top of the filters (har
dipped) and the effluent sampling port
a rate of between 8 and 20 per hr f
each sampling location. Composite sar
piers were operated at a rate around 3(
mL/hr, which provided a 1.5 gal 24-I
composite sample.
The results of the field sampling d
not achieve the objectives of this part
the project. The occurrence of coliforn
in the plant effluent in these systems w<
not high enough to provide the da
needed. Unfortunately, the samples wi
coliform bacteria present came moi
often from the grab samples than fro
the composite sampler. This does n
prove that the theoretical model or tt
laboratory results are incorrect, only th
the systems selected for the fie
sampling did not provide the conditior
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needed for the composite sampler
performance to be demonstrated.
Conclusions
1. If coliform bacteria were ever
randomly dispersed in a body of water
(i.e., the variance of the count per unit
volume is equal to the mean density
per unit volume), the probability of
capturing coliform bacteria would be
proportional to the total volume of
water collected and would not depend
on the total number of samples or how
the samples were collected. A random
dispersion of bacteria in a body of
water can be achieved by complete
mixing of the water; this, of course, is
very unlikely to occur in a water
distribution system.
2. For water supply systems in which the
coliform bacteria are not randomly dis-
tributed but show an aggregated
arrangement (i.e., one in which the
variance of the counts per unit volume
is greater than the mean density),
composite sampling is more effective
than grab sampling in capturing
coliform bacteria in any volume of
water tested. This conclusion is based
on a closed form mathematical model,
computer simulation of sampling from
a lognormal distribution, and a lab-
oratory study of composite sampling.
3. The results of sampling municipal
water systems during this project did
not demonstrate the superiority of
composite sampling for collection of
coliforms under field conditions.
4. The commercially available composite
samplers could not be adapted for
truly continuous, aseptic sampling of a
stream treated water under pressure.
The best that could be achieved was
very frequent intermittent collection of
very small volumes of water.
5. For intermittent composite sampling of
a stream of water from a treatment
plant, the probability of capturing
coliform bacteria in any given sample
volume is a function of the volume of
sample tested, the size of the stream
of water sampled, and the frequency
of collecting the component aliquots.
The full report was submitted in
fulfillment of Cooperative Agreement No.
CR 813337 by Drexel University under
the sponsorship of the U.S. Environ-
mental Protection Agency.
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Wesley 0. Pipes and Harvey A. Minnigh are with Drexel University, Philadelphia,
PA 19104.
Donald J. fteasoner is the EPA Project Officer (see below).
The complete report, entitled "Composite Sampling for Detection of Coliform
Bacteria in Water Supply," (Order No. PB 90 192-7581 AS; Cost: $17.00,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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