Journal of Microbiological Methods 99 (2014) 66–70

Contents lists available at ScienceDirect

Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

Evaluation of the compartment bag test for the detection of

Escherichia coli in water
Christine Stauber ⁎, Candace Miller, Brittany Cantrell, Kate Kroell
School of Public Health, Georgia State University, P.O. Box 3995, Atlanta, GA 30302, USA

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Annually, more than 2 million diarrheal disease deaths can be attributed to the lack of access to water, san-
Received 3 December 2013 itation and hygiene. These deaths occur mostly in developing countries where water quality testing resources are
Received in revised form 13 February 2014 limited. Several tests are currently used to detect and quantify Escherichia coli and other fecal bacteria in drinking
Accepted 13 February 2014 water; however they can be expensive, complex, and technically demanding. There is a need for a simple, reli-
Available online 21 February 2014
able, low-cost water quality test that can be used in resource limited settings. Therefore, the purpose of this re-
search was to perform a rigorous evaluation of the recently developed compartment bag test for detection and
Keywords:
Environmental health
quantification of E. coli against the standard method of membrane filtration.
Drinking water quality Methods and results: A total of 270 water samples were collected from forty-five various naturally contaminated
Microbial water testing water sources around metro-Atlanta from August 2011 through April 2012. Samples were processed using the
Fecal indicator compartment bag test and membrane filtration with mI agar. Concentrations of E. coli were significantly correlat-
E. coli ed with a correlation coefficient of 0.904 (95% CI 0.859–0.950). Sensitivity and specificity were 94.9% and 96.6%,
Safe water respectively.
Conclusions: These results suggest that the compartment bag test produces results consistent with those pro-
duced by membrane filtration on mI agar. Based upon its performance, the compartment bag test has the poten-
tial to be used as a reliable, affordable drinking water quality test where other microbial water quality testing
resources are not readily available.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Globally, roughly 783 million people lack access to improved drinking
water, and over two billion have no basic sanitation (WHO/GLAAS, 2012).
There are approximately two billion cases of diarrheal disease globally
every year, making it one of the leading causes of preventable deaths
worldwide and the second leading cause of mortality and morbidity in
children under the age of 5 years (Liu et al., 2012). One of the major
causes of the more than 2 million deaths annually due to diarrheal disease
is the consumption of contaminated water (WHO/UNICEF, 2009).
The microbial quality of water has a large impact on health in de-
veloping countries where access to safe drinking water is limited
(Fewtrell and Bartram, 2001). Contaminated drinking water may
contain unsafe levels of microorganisms that pose a risk to human
health (JMP, 2010). Fecal (also called thermotolerant) coliforms,
particularly Escherichia coli (E. coli), have been used as indicators
of fecal contamination of drinking water (Horan, 2003). The World
⁎ Corresponding author. Tel.: +1 404 413 1128.
E-mail address: cstauber@gsu.edu (C. Stauber).
Health Organization has specified that zero E. coli per 100 ml of
water is the goal for all water supplies (WHO, 2006). There are sev-
eral current tests used to detect and quantify E. coli and other fecal
bacteria in drinking water; however they can be expensive, complex,
and time consuming (Boubetra et al., 2011). Most of these tests require
trained laboratory personnel and a laboratory setting that may not be
available in remote areas or those with limited resources (NRC, 2004).
In particular, the lack of access to microbial water analysis kits or
laboratories is an issue for many communities in the developing
world (Sundram et al., 2000).
There is a need for simple, reliable, low-cost microbial tests that will
be readily available to people and institutions in developing countries
with limited access to water, sanitation, and hygiene in order to provide
data that facilitate the prevention of waterborne diarrheal disease
(McMahan et al., 2011). To meet this testing need, the compartment
bag test, developed by researchers at the University of North Carolina
at Chapel Hill, is a self-contained, portable, less expensive and simple
to use test to detect and quantify the presence of E. coli in drinking
water based upon the most probable number principle of quantification.
This test can be used without extensive laboratory equipment that is
normally needed for standard tests for drinking water quality. The

http://dx.doi.org/10.1016/j.mimet.2014.02.008
0167-7012/© 2014 Elsevier B.V. All rights reserved.
 C. Stauber et al. / Journal of Microbiological Methods 99 (2014) 66–70 67

purpose of this study was to evaluate the compartment bag test for the
detection and quantification of E. coli in water against a standard meth-
od, membrane filtration using mI agar, for various natural water sources
in Atlanta, GA.

2. Materials and methods

Water samples were collected from forty-five various naturally con-

taminated water sources around metro-Atlanta. The samples were col-
10 30 56 3 1
lected from August 2011 through April 2012. Each sample was labeled
with the location of the water source and date of collection, stored at
2–8 °C, and processed within 24 h of collection. A total of 270 samples
were processed and tested in the School of Public Health Laboratory at
Georgia State University using the compartment bag test and the con-
ventional membrane filtration method.

2.1. Membrane filtration method

Water sample volumes of 10, 50, or 100 ml were processed in dupli-

cates using membrane filtration and selective medium (mI agar, Becton
Dickinson, Sparks, MD) containing chromogenic and fluorogenic β-
glucuronide and β-galactoside substrates for the detection and enumer-
ation of E. coli and coliforms, respectively following standard method
1604 (EPA, 2002). After applying the membranes of filtered water to
the agar medium, the plates were inverted and incubated for 18–24 h
at 35 °C. E. coli colonies were quantified and reported as colony-
forming units (CFU) per 100 ml (EPA, 2002).
2.2. Compartment bag test method
A total volume of 10, 50, or 100 ml of each water sample was mixed
with a chromogenic E. coli broth culture medium (HiMedia Laboratories,
Mumbai, India) in the form of a medium bud in a sterile 120 ml vessel.
Water samples of b 100 ml were brought to a 100 ml volume with sterile
reagent water prior to adding the medium. The water was allowed to
mix with the reagent medium bud for 20 min for medium dissolution
prior to pouring into the compartment bag. The compartment bag
consisted of a clear plastic bag with five internal compartments
with individual volumes of 1, 3, 10, 30, and 56 ml each (Nasco, Salida,
California). After 20 min, the water sample was poured into the com-
partment bag (typically into the largest compartment first). Then the
water sample was manually distributed into each compartment by
gently squeezing the bag exterior to ensure that each sample volume
was filled to the set mark (Fig. 1). Each bag was then sealed using a
two-piece plastic clip to isolate each compartment, and incubated for
18–24 h at 35 °C. The clip was placed across the bag above the sample
levels so that each compartment was isolated from one another. Addi-
tional instructions regarding the performance of the compartment bag
test can be found on the company's website (Aquagenx, 2013) (http://
www.aquagenx.com/wp-content/uploads/2013/12/Aquagenx-CBT-
Instructions-v3.pdf).
After incubation, each compartment of the bag was scored as posi-
tive or negative for the presence of E. coli. Concentrations of E. coli
were determined using the observed positive and negative compart-
ment bag results which correspond to the specified most probable num-
ber (MPN) values. Positive compartments of the bag were identified as
those which turned a blue-green color, indicating the presence of E. coli
due to the hydrolysis of the β-glucuronide substrate (McMahan et al.).
This outcome is based upon the principle that most E. coli strains pro-
duce β-glucuronidase (Watkins et al., 1988). Indeterminate results
were noted and re-evaluated.
2.3. Isolation and purification of presumptive E. coli positives
Presumptive E. coli isolates from positive compartments of the com-
partment bag test and isolates from the mI agar plates were streaked for
Fig. 1. Compartment bag test with sample prior to incubation. Numbers are compartment
volumes in milliliters.
isolation successively to Bio-Rad RAPID'E.coli 2™ agar and then
trypticase soy agar (TSA) to obtain pure colony isolates for subsequent
organism identification of isolates from 123 of the water samples. For
each compartment bag test with at least one positive compartment,
the smallest volume compartment that turned positive was first
streaked for isolation onto Bio-Rad RAPID'E.coli 2™ agar and incubated
for 18–24 h at 44.5 °C. A colony with the typical appearance of E. coli
from each of these plates was then re-streaked onto another plate of
Bio-Rad RAPID'E.coli 2™ agar and incubated for 18–24 h at 44.5 °C to en-
sure a pure culture. Colonies with the typical appearance of E. coli were
then streaked onto TSA and incubated for 18–24 h at 35 °C. Pure isolat-
ed colonies from the TSA plates were added to individual 1 ml aliquots
of TSB with 20% glycerol and stored at −80 °C for future use. Frozen iso-
lates were thawed and streaked onto TSA for further identification. Or-
ganism identification was confirmed with the pure isolates using the
BBLTM EnterotubeTM II multiple biochemical test system for the identifi-
cation of Enterobacteriaceae.
2.4. Data analysis
Data for each water sample were recorded and entered into
Microsoft Excel and copied into Stata 10.0 (StataCorp, College Station,
Texas, USA) for further analysis. The water quality testing results from
membrane filtration and the compartment bag test were characterized
using descriptive statistics. These included geometric and arithmetic
means with 95% confidence intervals, variance, and standard deviation
using categorical and continuous data in both log10 transformed and
non-log10 transformed format. Correlation analysis was used to deter-
mine how the two methods compared with each other for the same
water sample based on the presence and concentration of E. coli. The
analysis included correlations between estimates for E. coli within the
decimal categories commonly used to indicate ranges of fecal contami-
nation. Spearman's rank correlation coefficient was used to measure
how closely the E. coli concentrations for the two methods compared.
The compartment bag test and the membrane filtration method were
also compared on the basis of sensitivity [(true positives) / (true posi-
tives + false negatives)] and specificity [(true negatives) / (true nega-
tives + false positives)] for the presence of E. coli.
Parametric and non-parametric statistical tests were used to
compare results where data were and were not normally distributed,
68 C. Stauber et al. / Journal of Microbiological Methods 99 (2014) 66–70

respectively. Using continuous and log10 transformed data, comparisons isolates from both the compartment bag test and membrane filtration
were made with mean comparison tests. A Wilcoxon rank sum test was not presumed to be E. coli based on colony color were also biochemically
also performed to compare the two methods. tested. Only 1 of these isolates was identified as E. coli. The biochemical
identities of these isolates included Enterobacter cloacae, Enterobacter
aerogenes, Serratia liquefaciens, Klebsiella pneumoniae, Enterobacter
3. Results amnigenus, Citrobacter freundii, and Entrobacter agglomerans. Based on
the biochemical analysis of presumptive E. coli isolates, 99% were iden-
A total of 270 naturally contaminated water samples from various tified as E. coli with one isolate (1%) from membrane filtration identified
water sources were tested and grouped by the type of water source as Enterobacter aerogenes.
(Table 1). The geometric and arithmetic mean E. coli counts were 1.52
(95% CI 1.39–1.64) MPN/100 ml and 212.4 (95% CI 170.5–254.2) MPN/
4. Discussion
100 ml, respectively for the compartment bag test, versus 1.63 (95% CI
1.51–1.76) CFU/100 ml and 232.5 (95% CI 190.7–274.3) CFU/100 ml for
In this study, the compartment bag test, a self-contained, compact,
membrane filtration. The Wilcoxon rank sum test for paired samples field portable method to detect and quantify E. coli in water, performed
revealed that the arithmetic median concentrations of E. coli were signif-
just as effectively as membrane filtration by the US EPA Method 1604 in
icantly different between methods (p = 0.0002). a variety of different water samples collected seasonally in metropolitan
As shown in Table 2, E. coli concentrations for the two methods were
Atlanta, GA. Documenting the reliability of this test is an important step
significantly correlated with a Spearman's rank correlation coefficient of to provide evidence that this simple, low cost water quality test is not
0.904 (95% CI 0.859–0.950). Furthermore, when compared on the basis
only reliable but also practical for use in limited resource settings.
of categorical E. coli concentrations, there was relatively high agree- This evaluation indicated that the compartment bag test had high
ment, especially at higher concentrations as shown in Table 3. These
specificity (96.6%), positive predictive value (N97%), and sensitivity
categories [b 1 CFU/100 ml (safe or low risk), 1–10 CFU/100 ml (inter- (94.9%), but the negative predictive value was somewhat lower
mediate risk), 11–100 CFU/100 ml (high risk), and N100 CFU/100 ml
(~ 70%). When tested against membrane filtration with mI agar, the
(very high risk)] are used to indicate fecal contamination and the possi- compartment bag test was able to correctly detect 94.9% of all E. coli
ble association with level of waterborne disease risk. As shown in positive results, and 96.6% of all E. coli negative results. As a result of pro-
Table 3, the extent of agreement in categorical concentrations of E. coli ducing few false positives (0.45%), a positive result observed with the
between the two methods was 70%, 71.4%, 73.5% and 92.8% for the con- compartment bag test is a good predictor of the presence of E. coli
centration categories of b1, 1–10, 11–100 and N100/100 ml, (PPV = 99.6%). However, the compartment bag test produced a moder-
respectively. ate number of false negatives (30%), suggesting that a negative result
Out of 263 samples tested with both methods, 12 were negative for does not always assure the absence of E. coli (NPV = 70%). These results
E. coli (b1 MPN/100 ml) when tested with the compartment bag test, were comparable to those found by McMahan et al. with sensitivity,
whereas the same samples were positive for E. coli (N1 CFU/100 ml) specificity, NPV, and PPV determined to be 73%, 100%, 68%, and 100%,
when tested with membrane filtration. These were considered to be respectively for the compartment bag test when applied to the detec-
false negatives. One (0.45%) sample was considered to be a false positive tion of hydrogen sulfide producing bacteria in surface water. In studies
as it was positive for E. coli when tested using the compartment bag test, of the performance of some other E. coli methods, results for these pa-
but negative for E. coli when tested using membrane filtration. For the
rameters have been similar. Brenner et al. (1993) found that the sensi-
samples analyzed, the compartment bag test produced a sensitivity of tivity and specificity for mI agar when used to detect E. coli was 69.6%
94.9% and a specificity of 96.6% when compared with membrane filtra-
and 95.7%, respectively.
tion on mI agar (Tables 4 and 5). When compared using a two sample t-test and Wilcoxon rank sum
By EnterotubeTM II biochemical test results it was found that 100%
test for paired samples, we found statistically significant differences in
(43 out of 43) of the presumptive E. coli isolates obtained from the com- concentrations for the compartment bag test compared to membrane
partment bag test were identified as E. coli (Table 6). Out of a total of 109
filtration with mI agar. The statistically significant difference in concen-
presumptive E. coli isolates from the compartment bag test (43 pre- tration of E. coli between the two methods is likely due to the difference
sumptive E. coli isolates) and membrane filtration (66 presumptive
in media formulation as well as in how each method enumerates E. coli.
E. coli isolates), a total of 108 isolates were identified as E. coli. Fourteen Similar to how IDEXX Colilert Quantitray works, the compartment bag
test uses a broth culture medium to promote the growth of E. coli. Due
Table 1
Descriptive characteristics of water samples collected between August 2011 and April
to differences in the format as well as media formulation, it will likely
2012. not detect metabolically the exact same organisms as membrane filtra-
tion. Differences in concentrations may also be due to the relatively limit-
Type of source Number of samples Season Total
ed most probable number combinations available in a five compartment
Creek 18 Summer 84 bag test compared to a wide range of colony count outcomes (0 to 100
22 Fall
colonies per test) conforming to the Poisson distribution in membrane fil-
24 Winter
20 Spring tration analysis. In their comparison of seven different E. coli enumeration
Lake 22 Summer 86 methods, Hörman and Hänninen (2006) documented similar results.
27 Fall They found that the correlation coefficient suggested high correlation be-
17 Winter tween the MPN method (Colilert) and membrane filtration but mean
20 Spring
Pond 4 Winter 9
concentrations were significantly different between the two methods
5 Spring (Horman and Hanninen, 2006).
Stream 1 Summer 24 Statistically significant correlation of E. coli concentrations (by
3 Fall Spearman's rank correlation) by the two methods illustrates that quan-
8 Winter
tification of E. coli was comparable for both. Hence, the compartment
12 Spring
Rainwater 6 Summer 64 bag test was generally comparable to membrane filtration with mI
42 Fall agar for the quantification of E. coli in source waters of Atlanta, GA. Fur-
8 Winter thermore, based upon the decimal categories of E. coli concentrations,
8 Spring the compartment bag test and membrane filtration methods were in
Other 3 Winter 3
general agreement for matched results in each category. Agreement
 C. Stauber et al. / Journal of Microbiological Methods 99 (2014) 66–70 69

Table 2
Results from Spearman's rank correlation comparing E. coli concentrations of the compartment bag test and membrane filtration.

Membrane filtration (CFU/100 ml) Coefficient Standard error t P 95% CI Spearman's rho

CBT (MPN/100 ml) 0.904 0.0233 38.89 b0.001 0.8588–0.9504 0.9278

for the two methods was 70%, 71.4%, 73.5%, and 92.8% for E. coli concen-
tration categories of b1, 1–10,11–100, andN100 MPN/100 ml or CFU/
100 ml, respectively. Hence, the compartment bag test is able to effec-
tively indicate categorical concentrations of fecal contamination in
water samples, possibly predicting the level of waterborne disease risk.
Current methods for detection and quantification of fecal bacteria in
water can be complex and technically demanding (Boubetra et al.,
2011). They require extraneous laboratory equipment, and are difficult
to perform in developing countries and during natural disasters. In addi-
tion, other resources including a cold chain, electricity, and incubators
are too expensive and impractical for use in resource limited settings
(Brown et al., 2011). These requirements present tremendous obstacles
for microbial water quality testing in developing countries (Chuang
et al., 2011). The compartment bag test is self-contained, compact, light-
weight and field portable, which is beneficial in situations where access
to laboratory equipment and supporting materials like pipets, graduat-
ed cylinders, and membrane filtration apparatus is not feasible. It can be
used in settings where electricity may not readily be available with the
utilization of ambient temperature incubation. Despite the fact that the
water samples were processed in a laboratory setting for this analysis,
our results demonstrate that, the compartment bag test is likely to be
capable of performing efficiently in remote settings where resources
are limited, such as in developing countries. Furthermore, the compart-
ment bag test is similar in cost to the other methods available. The cost
per test is between $5 and $10, with the cost per test depending on how
many are purchased. When purchased in progressively larger quanti-
ties, the price incrementally decreases (personal communication, Dr.
Mark Sobsey). Estimates for membrane filtration and Colilert MPN
methods range from $0.50 to $6.00 per sample (Bain et al., 2012).
While the compartment bag test is at the higher end of this range, it
does not require additional equipment for processing, which can cost
between $2000 and $4000 depending on the method (Bain et al.,
2012), and the test includes its own decontamination system whereas
the others do not.
There are possible sources of analyst variation in reading the results
of this test, which include color detection and interpretation of the var-
iation in color production by hydrolysis of the chromogenic E. coli sub-
strate in the bacteriological medium. There may be a small or light
production of color change that may only be visible with close examina-
of most probable number values are possible within the range of b 1 to
nearly 100 MPN/100 ml (McMahan et al.).
Although some differences in E. coli presence were observed with
samples collected from rainwater sources when tested with the com-
partment bag test and membrane filtration, they were not statistically
significant (data not shown). However, membrane filtration on mI
agar gave higher concentrations of E. coli than did the compartment
bag test, and in some cases there were wide differences in the concen-
trations of E. coli detected by the two methods. These findings suggest
the need for further investigation into why these differences were ob-
served. It is noteworthy that in a study of the compartment bag test
compared to the IDEXX Quanti-Tray method for E. coli detection in har-
vested rainwater samples collected in rural households in Thailand,
there was not a significant difference in E. coli detection (unpublished
results presented at the 2011 Annual General meeting of the American
Society for Microbiology). Reasons for differences found in rainwater
samples in our study versus the study performed in Thailand may be
due to difference in the estimation format (membrane filtration enu-
meration of colonies and defined substrate technology quantified by
Quantitray MPN) (Hornan and Hinnanen, 2006).
The main limitations of the compartment bag test are the upper de-
tection limit of 100/100 ml and potential for variation in reading the re-
sults of this test, which include color detection and interpretation of the
variation in color production by hydrolysis of the chromogenic E. coli
substrate in the bacteriological medium. Since we performed these ex-
periments, the method of color interpretation has been somewhat
more standardized as the test is being sold commercially and detailed
instructions on interpretation are now provided (see http://www.
aquagenx.com/how-to-use-the-cbt/).
One additional problem with the test was that the compartment
bags occasionally leaked which resulted in the need to repeat or the
loss of the sample. This happened in very few instances (b2%) but
with production and distribution of the test increasing, additional mea-
sures of quality assurance and control have been implemented to re-
duce this likelihood, according to the test developers and producers. In
Table 4
Comparison of results from the compartment bag test and membrane filtration based on
sample positivity for E. coli.

tion, and observers may interpret the presence of the diagnostic blue or Membrane filtration
blue-green color differently. With only 5 sample volumes in the com-
CBT Negative Positive Total
partmentalized bag, there are a small number of likely positive bag com-
Negative 28 12 40
partment combinations for the test, and as a result only a small number
70% 30% 100%
Positive 1 222 223
0.45% 99.6% 100%
Total 29 234 263
Table 3 11% 89% 100%
Comparison of categorical concentrations of E. coli from the compartment bag test (CBT)
and membrane filtration.

Membrane filtration (CFU/100 ml)

CBT (MPN/100 ml) b1 1–10 11–100 N100 Total Table 5

Performance characteristics of the compartment bag test for
b1 28 8 4 0 40 the detection of E. coli compared to membrane filtration for
70.0% 20.0% 10.0% 0% 100% water samples from Atlanta.
1–10 1 30 9 2 42
2.38% 71.4% 21.4% 4.76% 100% Sensitivity 94.9%
11–100 0 7 50 11 68 Specificity 96.6%
0% 10.3% 73.5% 16.2% 100% PPV 99.6%
N100 0 0 8 103 111 NPV 70.0%
0% 0% 7.21% 92.8% 100% False positive rate 3.4%
Total 29 45 71 116 261 False negative rate 5.1%
11.1% 17.2% 27.2% 44.4% 100% Accuracy 95%
70 C. Stauber et al. / Journal of Microbiological Methods 99 (2014) 66–70

Table 6 Conflict of interest

Enterotube™ II biochemical test results for presumptive E. coli isolates from the compart-
ment bag test and membrane filtration analysis.
No conflict of interest declared.
Type of source Number of isolates Total number of isolates
identified as E. coli identified as E. coli

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This work was supported in part by a Research Initiation grant from challenge of extending and sustaining services. World Health Organization, Geneva,
the Georgia State University. Supplies for the compartment bag test Switzerland.
were provided by Dr. Mark Sobsey from the University of North WHO/UNICEF (United Nations Children's Fund), 2009. Diarrhea: why children are still
dying and what can be done. World Health Organization, Geneva, Switzerland.
Carolina.