Assessing Defined-Substrate Technology
for Meeting Monitoring Requirements
of the Total Coliform Rule
M. Michael Katamay
The Illinois Environmental Protection Agency is responsible for drinking water anaJyses for
approximately 2,200 public water utilities serving more than half the state's population. The
recently developed defined-substrate technology (DST, commercially known as Colilert),
which simultaneously enumerates both totaJ coliforms and Escherichia coli and does not
r~quire confirmatory tests, was compared in this geographic area with the membrane filter
(MF) procedure from Standard Methods. Overall, there were no differences between MF and
OST. Subcultures of positive DST tubes demonstrated this technology's specificity. Yellow
(total coliform-positive) tubes yielded species consistent with total coliforms, and fluorescent
(E. coli-positive) tubes contained E. coli. The DST system was easy to use and is compatible
with new drinking water regulations that will intrease monitoring and resampling
requirements, mandate a fecal indicator (either an E. coli or fecal coliform analysis), and use a
maximum contaminant level In the frequency-of-occurrence mode. DST also has severaJ
important advantages for state regulators, inducting its abiUty to help utilities comply with
new transportation and storage requirements.
ln 1987 an innovative bactcnological
method that could simultaneously detect
tot a I col iforms and E. coli was developed.
This method (known commercially as
the Colilert test) IS an example of
defined-substrate technology (DST).
whtch allows the specific identification
of target microbes. This new test for the
drinking water industry was a techno!·
ogy transfer from DST applied in the
field of clinical microbiology. The Amer·
ican Water Works Association Research
Foundation and the US Environmental
Protection Agency (USEPA) supported
the testing of this new method. In 1989
the USEPA approved it for total coliform
analysis. With the understanding that a
new coliform rule was under develop·
ment. the lllinois Environmental Protection Agency <IEPAl D1v. of Labora·
tories 1n Chicago conducted a study that
encompassed a wide range of water
sources m Illinois. It was believed un·
necessary to repeat the USEPA investi·
gat ion because there were favorable
reports in the literature. If the commer·
cia! test's performance characteristics
were comparable with those reported by
SEPTEMBER 1990
the USEPA, the !EPA could then deter·
mine whether the test wou.ld be com·
patible with actual utility use and the
new coliform rule.
The IEPA Division of Laboratories is
responsible for the testing of a statewide
network of approximately 2,200 public
water supplie::;. Routine water samples
are analyzed in the Chicago laboratory,
which serves the northern half of the
slate, or nearly 54 percent of the supplies
in Jllinois. Water from this region is
primarily obtained from three different
sources-deep aquifers, glacial drift
wells, and s urface waters. Of the total
number of water supplies in all regions.
22.5 percent are exempt from chlorine
disinfection; the balance of the public
water supplies are chlor inated. All of
these sources are routinely analyzed for
total coliforms by the membrane filler
(M F') procedure 2 MF can produce false·
positive and false-negative results. J·!·
These phenomena are well known in MF
testing, especially if large numbers of
heterotrophic bacteria are present with
total coliforms. 2 False-positive tests can
result for several reasons. Most impor·
tant, the genus Aero monas shares many
DST simulloneously identifies E. coli
and lola/ coliforms. a11d il requtres no
confirmatory tests.
M MICHAEL KATAMAY
83
characteristics with the species that target microbes, i.e.. total coliforms or
comprise the total coliform group and E. coli. Such an innovation would allow
can inflate total coliform denstties and utilities to obtain results within hours
give a false indication of water quality.z instead of days.
As many as 20 percent of total coliformSome of the major strengths of the
positive confirmed tests can be attributed DST test are:
to Aeromonas.6 False-negatives are com·
• sensitivity to total coliform and E.
monly caused by the suppression of total coli concentrations as low as 1 cfu/100
colifor ms by heterotrophic bacteriaP mL (equivalent to multiple-tube fermen·
Particulates from turbid waters also tation [MTF] and MF);
suppress coliforms on membranes.
• results in 4 to 24 h, depending on
Of critical importance to state regu· the coliform or E. coli concentration;
• simultaneous identification of both
Ia tors is the recent report that 61 percent
of membrane filters, as evaluated by the total coli forms and E. colt;
USEPA. are deemed unsatisfactory.~ Of
• no confirmatorv tests needed;
special public health significance is that
• specific E. coii identification to
up to 10 percent of E. coli are not species with no additional work;
• low cost (up to a third Jess than
detected by the fecal coliform test. 9
The new coliform rule, to become current methods);
effective]an. 1, 1991, will have profound
• no refrigeration of prepared formula
effects on water purveyors and water (long shelf-life);
quality laboratories. In addition to the
• configuration as either a P-A or
adoption of presence-absence (P-A) con· most·probable·numbcr (MPN) test; and
figuration, three repeat samples are
• equal utilization by small and large
required within 24 h for each total utilities.
coliform-po5itive sample, regardless of
DST is unique because it directs the
the overall percentage positive. A fecal metabolism of the target bacteria to
indicator analysis will be required for all specific indicator nutrients. In the
total coliform-posith·e samples. both commercial DST test. simple nutrients.
primary and repeats. The USEPA will salts, and solanium make up the rest of
allow either the elevated-temperature, the formula. The complete formula is a
fecal-coliform test or E. coli to be used as stable powder that can be added directly
the fecal indicator. Positive fecal indi· to the sample or vtce versa. For each
cators in conjunction with a total coli· target microbe, there is a substrate for a
form positive will result in an acute specific enzyme. There is one substrate
violation and possibly the need for public for total coliforms and another for E.
coli. The hydrolyzable substrates are o·
notification.
Also of concern to state agencies are nitrophenyl-.8-D-galactopyranoside (that
the new storage and transportation changes from clear to yellow if total
requirements. The transit period from coliforms are present) and 4·methylum·
the time of sample collectton until the belliferyi-.8-D-glucuronide (that produces
total coliform analysis is performed a distinct Cluorescence if E. coli is
cannot exceed 24 h. ln addition. the present). This directed metabolism oc·
holding temperature must not be higher curs because only the target microbe can
chemically detect the substrate. Thus,
than l0°C.
In anticipation of the pending coliform under such restrictive conditions, hetero·
rule and with the introduction and trophic bacteria cannot replicate and
USEPA approval of a rapid and specific interfere in the test. The commercial
method* for the simultaneous detection DST test contatns all that is necessary
of total coli forms and E. coli from drink· to establish the identity of the target
ing water, the IEPA compared this new microbe Thus, there is no need for
technique in a field e\·aluation against additional tests after a color change
the MF method. The field-testing of because the change is spectfic.
To use a commercial DST test. water
public water supply samples was mod·
eled after the USEPA'sMethods Equiva- is added to the powder (or vice versa) and
lency Program for Drinking Water the mixture is incubated. Bacterial
Samples.ll1 It was also enluated con· metabolism begins immediately, and the
cerning Hs compatibility with the new test is positive from the water sample
regulations and utility usefulness.
within 24 h. The larger the number of
bacteria, the sooner the test is positive.
The new method can be performed as a
DST- development and function
quantitative MPN test or a qualitative
The baste goal was to ref me DST ust>d P-A analysis.
in eli nicallaboratories to enumerate and
Materials and methods
identify microbes from urine and to
Water sampling procedure. The purpose
overcome the problems faced in the
analvsis of water. This refinement re· of the field evaluation was to verify the
suited in a test in which a water sample accuracy and specificity of the new
was added to a vessel containtng the method in Illinois. The method of com·
powdered, stable formula. A simple color parisonwas based on that of the USEPA's
change indicated the presence of the Environmental Monitoring and Support
84 RESEARCH AND TECHNOLOGY
Laboratory (EMSL). Sensitivity was
determined by comparing the number of
positive and negative tubes on a statis·
tical basi:;.'0 Specificity was determined
by subculturing positive tubes (com mer·
cial DST test) and sheen colonies (MF)
and identifying them to species. Water
samples typically were collected from
the source, the effluent, and a distribu·
tion point from the same system. In
addition. sites experiencing particular
problems were more intensively sampled.
The water sources for the study were
representative of a variety of geograph·
ically and hydrologically diverse condi·
tions. Transportation and storage were
carried ·out according to USEPA guide·
lines.2.11 On each of the 44 samples. a
total of 4 repeats was performed. There·
fore, for each sample there were 40 DST
tubes and 4 membrane fi ltrations. This
extensive number permitted a complete
statistical analysis of the data.
Heterotrophic plate count. A hetero·
trophic plate count (HPC) was deter
mined for each water sample according
to Standard 1\,fethods. R2A agar was used
and incubated at 35°C for 72 h.
Total coliform MF. Coliform bacteria
were enumerated using the single-step
MF test and interpreted as a single 100·
mL sample.1.2 Colonies consistent with
total coli forms from the membrane were
confirmed in brilliant green bile broth as
outlined in Standard Methods. 1
DST commercl1l system. Tests were
performed in accordance with the test
manufacturer':; instructions. Develop·
ment of a yellow color after incubatton
indicated the presence of total coli forms
in the test tube. Each total coliformpositive test tube was exposed to a hand·
held fluorescent <366 nm) light.t Fluores·
cence in the test 1u be noted the presence
of E. coli. A small amount of liquid from
all positive tubes was subcultured onto a
Lev me EMB plate on the same workiog
day. Lactose-fermenting colonies were
identified to spec1es. Bacteria I identi·
fications were performed by pri\'ate
laboratories.t
Results
Overall comparison. The number of
samples tested is well above the min·
imum number required by the EMSL.
and the data were analyzed statistically.
A total of 440 DST lO·mL tubes were
compared with 176MFtests. There was
an overall mean. for all samples com·
bined. of 11.9 total coliforms/100 mL
confirmed by Standard .vtetlzods and
12.2. 100 ml. by the DST system (Table
ll. MF had a confirmation rate of 78
percent. E. coli was isolated from 11
percent of samples by MF and 18 percent
·coliltrt
~nttm, ,,«~'~>'
,,nahuat Sy•tt!n$, Rranlord,
Conn
Rlak R"Y· S;~n Cabr>rl. Cahf
tEnH•rotuht II nnd O•yiFrrm Tu~. Roche Dtagno•toc
Syotrm•. D11· Hollman 1-<l Rocht. Nutley, N,J ,
t
JOURNAL AWW A
total coliform group. More than 80 percent of sheen colonies from MF that did
not confirm belonged to the genus
Aeromonas.
25.0
, • 0,17 )( -o.ot~ ~
o.n
Discussion
20.0
rf.S
15.0
12.5
10..0
rs
s.o
l.S
5.0
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
OsTTnl
Fipre 1. RegressiOn analysis of the DST
system compared with the MF procedure
(numbers on each axis represmf the total
coliforms per 100 mi/lililres from Mch
method: the r 1 valuco/0.93 indicates a t•cry
strong correialron bt>lwel'll the two proudures)
TABLE 1
C()mparison of MF and DST recoveries
Parameter
Total <"umulauv~ conftrmed
total coliforms
Total unconfirmed positive TC
Iota! c:oliforms
Total E roli
MP
DST"
524
78
46
0
55
•Number of c:olnntl'S or MPN ~'Quivalents
by DST (Table 2). The yellow colors
produced by total coliform-positive tests
and the nuorescent colors produced by
E. coli were easy to read. [n a small
number (approximately 5 percent) of
tubes, a light yellow or nuorescence was
noted. As per the manufacturer's package
insert, these samples were mcubated an
additional4 h. In all cases, the reactions
became more pronounced.
Statistical analyses. There were no
differences between the methods by chisquare analysis, including the Kendall
and Spearman tests (Kendall correlation
= 0. 751, Spearman correlation = 0 2 592.5,
p = 0.98, Z = of 7.266, Pearson p > 0.05,
Mantei-Hanzel P"> 0.05). The /-tests also
showed no differences between the two
methods. The results of the F-statistic
showed that the DST system had equal
precisiOn with MF (F-test = 724.8). The
likelihood ratio test. used to compare
result s from different methods. also
showed equivalence. 12
The data were analyzed by simple and
multiple regression analysis with DST
taken as the x variable and MF taken as
the y variable. Although most of the
SEPTEMBER 1990
values fell in the zero or "greater than"
range. regressiOn analysis showed that
the two analytical methods resulted in
equivalent results with an r2 of 0.93
(Figure L).10.1l.11
Therefore, statistical analysis of the
two methods from split samples showed
that DST and MF were m agreement.
Accordingly, the data base generated
from one method could be used to superimpose the data base for the other.
Therefore, changing from DST to MF
testing, or vice versa, should result in
the same overall data base.
Effect of llOIICOiifonn hetemroplas. There
was no effect of noncoliform heterotrophs (HPC) on the ability of the commercial DST test to enumerate total
coliforms and E coli.
Baderial identifications. Table 2 shows
the results of the bacterial identifications
from DST and MF. A species commonly
cons1dered part of the total coliform
group was isolated from DST tubes that
became yellow. Likewise. an E. coli was
isolated from tubes that fluoresced at
366 nm. MF showed 78 percent of sheen
colonies confirmed as a member of the
MF and DST compuiSOII. The advantage
of the DST system is that it can specifically detect and confirm total colirorms
and E. coli in 100 mL of water sample
within 24 h in the same container. DST
eliminates any potential heterotrophic
bacterial interference. lt has previously
been ~ompared with MPN 15 and P-A 16
procedures from Standard Methods and
found to produce equivalent data. It has
been approved by the USEPA. 10 DST is
also highly compatible with new regulations for the testing of drinking water,
wh1ch will require analysis for both
total coliforms and E. coli.'~
Field personnel of a certification
agency may find that audit sampling of
participating water utilities is more convenient using a DST system because the
integrity of the sample is enhanced.
There are fewer steps and less probability of contamination occurring during
sample collection. A DST system, requiring only the addition of water to the
tubes for MPN or vessel for P-A, requires considerably less subjectivity
than MF.
Confirmation, which means corroboration or verification. is not necessary
with DST but is essential for the MPN
and MF methods. which are screening
methods: i.e .. they are not specific and
result in an inordinate number of falsepositives unless confirmed. Because the
commercial DST test is coliform· and E.
coli-specific, confirmation is unnecessary. This lack of need for confirmatory
tests is a major advantage for both water
utilities and state regulators. Neither
mdividual tests nor some percentage of
positives should be confirmed-either
would create unnecessary costs. Quality
control becomes much easier. requiring
only a positive and negative control. In
add1tion, because the ingredients come
in a ready-to·use powdered form. preparation errors would be minimal. The
commercial DST test could also be used
w1th water samplE's that are difficult to
filter.
Accurate and dependable results are
available within 24 h with th1s new
method. (This is important in the case of
public notification or issuance of a boilwater order.) DST provides definitive
measures of both total coli forms and E.
coli within 24 h. The Lime to achieve
positive results also decreases as the
density of indicator bacteria increases,
compared with other procedures in
Standard M~lhods that require two to six
days for a completed Lest. Because of the
Significantly reduced time of analysis
using DST. public health authorities
can quickly respond to incidents of bacM. MICHAEL KATAMAY 85
TABLE2
Species isolated from DST and M F
Species
DST
MF
CitrobaclerJ"u11dii
Entuobocter atrogenes
Enleroborler cloacae
29
27
Escherichia coli
Klrbsiella /JIIeumoniae
Strratia marcesctns
Strratia species
18
10
teriological pollution. Increased monitoring and other measures can be taken
rapidly. The USEPA has calculated the
overall cost of the new system to be a
third less than current methods. 9
There are limitations of DST that
should be noted. First, it is a direct water
test. It s hould not be used as a confirmatory test. Next, if Aeromonas hydrophila are present in concentrations
greater than 20,000/mL(2x 106/100 mL)
a false-positive may result.•s Also, DST
should not be incubated longer than 28
h. 15 If extended incubation occurs and
the test is negative, it is valid; if the test
s hows positive results. however, it
should be voided.
It s hould also be noted that approximately 5 percent of E. coli will be
fluorescence·negative. 17 However, be·
cause feces contai n many strains of E.
coli, there would always be significant
numbers of fluorescent-positive bacteria
present in a contamination event associated with drinking waterY By contrast, th1s is much better than results
obtained with the fecal coliform test.
With that test, approximately 10 percent
of E. coli will be missed because they are
anaerogenic. Furthermore, there will be
a significant false-positive rate because
approximately 15 percent of Klebsiella
are thermotolerant. The specificity and
sensitivity of the DST test for E. coli,
therefore, make it much easier for a
state regulator to deal with truly positive
samples and take appropriate action with
greater confidence.
DST altd tlte new coliform nale. State
agencies will be significantly affected by
the new coliform rule. They need to
begin planning how to modify existing
bacteriological monitoring programs in
order to manage their constituencies.
The following issues pertaining to the
new coliform rule were addressed by the
author when considering the use of DST:
Transit time. Meeting the 24 h max·
imum transit time criterion will be difficult for smaller utilities and rural water
producers that mail or send water samples to laboratories for analysis. Although it is possible to use overnight
delivery, the cost of the shipment would
exceed the cost of the analysis. lnocu-
86 RESEARCH AND TECHNOLOGY
18
12
~
9
21
14
11
11
6
10
lating a DST test at the utility and
mailing it to a laboratory are attractive
means of dealing with the 24-h transit
time requirement. Some state agencies
are already considering this option. Even
if a DST test requires more than 24 h to
reach a laboratory and is positive, the
laboratory can still confirm or further
analyze the bacteria present in order to
establish specificity. rt the test does not
become positive, then the analysis is
complete.
Maximum storage temperature.
The requirement of a maximum storage
temperature of l0°C will severely hamper
small utilities and rural water providers,
especially during summer months. Maintaining a temperature below 10°C will be
an impossible burden on many utilities,
even using cumbersome temperaturecontrol systems. Inoculation of the DST
system in the field is a possible solution
for this limitation.
Increased testing requirements.
The new regulations wlll require significantly increased testing, especially for
smaller utilities. First, the testing base
for smaller utilities has been increased.
Second, there is the requirement to perform repeat total coliform tests from
each locale that has shown a total
coliform-positive analysis. It has been
estimated that this increased testing
will amount to approximately a 33 percent increase over current monitoring
levels.'8
Il is unlikely that utilities and state
laboratories will see an expansion of
their personnel to deal with this increased testing. Many state laboratories
have determined that a DST system is
their best answer to increased monitoring requirements. Furthermore, it is
expected that microbial analysis will be
extended beyond the total coliform-fecal
coliform-E. coli realm to Giardia, Cryptosporidium, and, perhaps, viruses. The
ease of performmg a DST test will allow
laboratories to perform many additional
tests with thesamenumberofpersonnel.
Repeat testing requirements. For
each total coliform-positive sample,
regardless of the overall number positive,
new samples must be collected within
the next24 h. One of these three samples
must be from the same tap showing the
total coliform. one may be within five
connections upstream, and one within
five connections downstream. There are
no variances or exceptions to this rule.
Obviously, utilities will face a treadmill
of repeat and continued analyses extend·
ing through weekends.
Of great concern to utilities and state
agencies is the implicit requirement for
significantly increased weekend work.
Weekend work will not be limited to
Saturdays because repeat samples must
be collected and analysis started within
24 h. Few utilities or state agencies are
geared to performing large-scale weekend
testing.
DST can significantly alleviate the
repeat testing problem in a number of
ways. Because of its ease of use, an
operator can be trained to inoculate and
read a DST test. Because DST requires
no verification steps, a quick determina·
tion of yellow or fluorescence can be
made on the weekend and would conclude
the analysis. With MTF or MF, the
analyst would not only have to closely
examine the plates or tubes but would
then have to transfer any positives to
both confirm atory total coliform tests
(e.g., BGLB) and confirmatory fecal coli·
form tests (EC at 44.5°C). Then someone
would have to return to the laboratory
the next day to read these confirmatory
tests. It has been estimated that the
virtual elimination of weekend work,
along with its attendant overtime and
personnel disruption, would more than
pay for the cost of the commercially
purchased DST tests.
Fecal coliform or E. coli tesli11g.
Under the new coliform rule, each total
coliform-positive sample, regardless of
the percentage of total coliform positive
in the system, must be further analyzed
for either fecal coliform or E. coli. This
means that each sheen colony that
confirms as a total coliform must then be
tested to determine whether it is a fecal
coliform or an E. coli.
Fecal-coliform testing is an expensive,
complex series of steps. The fecal -coliform media must be prepared by trained
individuals, and incubators must be
rigidly controlled at 44.5°C ± 0.2. ft has
been shown that decreases as little as
0.2°C below 44°C will permit a much
higher percentage of the nonfecal Kleb·
siella to yield a positive lest and that
temperatures as little as 0.2°C above
45°C will inhibit the growth of many
strains of E. coli. It has also been found
that overinoculation of fecal coliform
tests. which ts very difficult to control,
will significantly increase the number of
fecal coliforms. 1''
The fecal-coliform procedure is not
only exacting; it also provides a considerably less sensitive and specific assess
ment of recent fecal pollution than does
the analysis of E. coli. Approximately 10
JOURNALAWWA
percent of E. coli are anaerogenic, and
gas variability has been reported. 2u It
has been well-established that approx·
imately 15 percent of Klebsiella pneu·
mo,iae, many of which can be found in
pristine sites and are not of fecal origin.
are thermotolerant and produce a posi·
tive fecal-coliform test. State regulators
will have to cope with a number of fecal
positive samples from sites that appear
to be m good order. Even well-managed
utilities could expect to see several fecalcoliform positives per year. On the other
hand, E. coli is almost unheard of m a
distribution system without overt evi·
dence of fecal contamination. State regu·
lators would be in a much better position
to judge the risk to the public's health
with an E. coli indicator. and the public
would be better served. E. coli is the
health risk indicator that public health
officials can utilize when determining
whether a boil-water order should be
issued. a sanitary survey performed. or
other measures taken.
P-A testing. The greatest overall
reason not to use coliform-density measurements is that after extensive study
the USEPA has found there is no assoCiation bet ween the numbers of total
coliforms per millilitrc and disease outbreaks.21 The enumeration of coliform
bactena, therefore. ts not as importam
as measuring the frequency of occurrence
(P-A).u Although a OST system can be
used as either a quantitative MPN or
P-A procedure, a particular strength for
utilil!es tn meetmg the new regulattons
is its compatibility m the P-A format. A
OST formula can etthE'r be added to a
water sample ora water sample added to
it. The alternatiH~ ways of determining
P-A are much more cumbersome. For
the ~tTF procedure. a 10-tube analysis
(or 5-tubeanalysis, 20 ml. per tube) must
be done or one 100 mL broth must be
made. For the MF procedure, the presence or absence of one total coliform per
100 mL must b<· established, which
means that every possible sheen colony
on a plate must be tt>stcd to determine
whether it is a total coliform or not.
Furthermore, this same colony must
tht>n he retested to determine whether it
is also a fecal coliform.
By using a DSTsystem, stateagencics
can better control and ensure the testing
analysis of all water supplies (including
small rural systems) than is now possible. State regulators can have better
control over their peripheral operations.
Another possibility is that OST can be
used by those operators who now perform
such direct tests as chlorine and turbid·
ity. This would best protect the public's
health by bringing the most important
tests for the cleanliness of water as close
to the time of production as possible.
Therefore. if a problem t>xists. it can be
expediently addressed. The state regu·
lator could be instrumental in bringing
SEPTEMBER 1990
about thb advance in public health
protection.
Therefore, the author concurs with
the USEPA in its assessment of OST:
"In summary, the AC test [Colilert] can
detect and enumerate total coliforms
and E. coli from a water sample within
24 h with no additional confirmatory
tests. The AC test is very easy to use,
only requiring the addition of sample to
the tubes, incubation for 24 h. and inter·
pretation of the reactions." 23 It offers
state regulators better information con·
cerning the public health significance of
bacterial analysis and provides utilities
with a cost-effective means of protecting
the public's health.
Acknowledgment
The author thanks Michael Alan for
his help in the statistical analysis of the
data and their tabular organization.
References
I. Standard Methods/or the Examination of
2.
3.
4.
5.
6.
7.
Water and IVastewattr. APHA. AWWA,
and WPCF. Washington, D.C. (16th ed..
1985).
Microbiological Methods for Monitoring
the Environment-Waterand Wastes(R
Bordner and j. Winter. editors). EPA60018-78·017, Cincmnat1, Ohio(l978).
Lt:CliFVAu.rER, M.w.: c.~~1ERos. s.c.: &
MCFF'TE!t~. G.A. New Medium for lm
proved Recovery of Coliform Bacteria
From Drinking Water. App/. Etwir.
Microbwl.. 45:484 Cl983)
SCHIH, LJ.; MORRISO~. S.M.: & MAYEI'X,
J V Synergistic False-Positive Coliform
Reaction on m-endo MF Medium. Appl.
Micrublol, 20:778 (1970}.
SlltP~. E.L. ]R. & CAMI'R0:-1, G.M. A
Comparison oft he Membrane Filter With
the Most Probable·Number Method for
Coliform Determinations From Several
Waters. rlppf. .1/icrobiol.• 2:85 (1954l.
LECt£Kc, H. ETAL. Microbiological Muni·
toring-A New Test for Fecal Contam
mat10n Baclerialllldicalors!Health Haz
ard:: Association With Water (A. W.
Hoadley and B.J. Dutka. editors). Amcr
Soc. for Testmg and ,\1atcrials. Philadel ·
phia, l'a. ( 1977).
Gfl.llRI'lCII, E. E. Handbook for Evaluating
Water Bacteriological Laboratories
EPA 670/ 9-75-006, C1ncinnat1, Ohio
tion m Drinking Water. four. ,'\-licrobiol.
1.,.lethods, 4:67 (1985).
13. BISHOP, Y.M., FtENBERG, W.E.: & HOL·
LAND, P. W. Discrete Multivariate A11alysis:
Theory and Practice. MIT Press, Cambridge, Mass. (1975).
14. F'LEISS. j L. Statistical Mttlrods for Rates
a11d Proporlrons. john Wiley & Sons Inc..
New York (2nd ed .. 1981).
15. EDBERG, S.C. ET At .. National Field Eval·
uation of a Defined-Substrate Method for
the Simultaneous Enumeration of Total
Coliforms and Esclrerich1a coli From
Drinking Water: Comparison With the
Standard Multiple-Tube Fermentation
Me\hod. Appl. Envir. Microbial.. 54:6:
1595 (1988).
16. EDBERG, S.C. ET AL. National Field Eval·
uation of a Defined-Substrate Method for
the Simultaneous Detection of Total
Coliforms and Escherichia coli From
Drinking Water: Companwn With Pres·
ence-Absence Techniques. Appl. Eflvir
Microbial.. 55 (4):1003 (1989).
17. ADAMS, M.R. ET AL. Colonmetric Enu·
meratton of Escherichia coli Based on {3·
Glucuronidase Activity. App/ Em•ir
.~!icrobiol.. 56 (7):2021 (1990).
18. Commonwealth of Virgima, Rept. of the
Dept. of Health. The Impact of the Safe
Drinking Water Act Amend men ts of 1986
on the Commonwealth of Virginia. House
Document 30 (1990).
19 GELDREI<..H, E.E.: ALLEN, MJ.: & TAYLOR,
R.H. Interferences to Coliform Detection
in Potable Water Supplies. Evaluation of
the Microbiology Standards for Drinkmg
Water (C. W. Hendricks, editor). USEPA,
Washington. D.C. (197H).
20. MEADOW!>, P.S. ET Al. Variability in Gas
Producuon by Escherichia coli in Enrich·
ment Media and Its Relat10nsh1p to pH
Appl. Em·ir. Micrabiol.. 40:309 (1980}.
21. PtPES, W.O. El AL. Companson of Clark's
Presence-Absence Test and the Mem·
brane Filler Method for Coliform Delee·
lion in Potable Water Samples. App/
E~~t·ir. Mirrobiol.. 54:-139 11986).
22. CLARK. j.A. A Presence-Absence CP-A)
Test Prondmg Sensitive and Inexpensive
Detection of Coliforms. Fecal Coliforms
and F<>cal Streptococci in Municipal
Drinking Water Supplies. Can. jour.
!t-ficrobiol.. 14: 13 (1968).
23. Con.H1. T.C. ET M .. Evaluation of the
Autoanalvsi~ Colilert Test for Detection
and Enumrration of Total Cohforms.
Appl. Enr•i1 . Microbiul.. 55CIOI ( 1989).
(1975)
8. BKE:->:->EK, K.P. & RAI'Kts. C.C. New
Scrt.!eningTest to Determme the Accept ·
ab1lit~· of 0.45-pm Membrane Filters for
Analy~is of Water. .4ppl. Em·ir. Mirro bial.. 56(1):54 {1990).
9. USEPA. National Primar>' Drinking Wa
ter Regulations: Total Cohforms(lnclud
ing Fecal Coliforms and E. coil). Fed.
Reg.. 54:27544-30002.
10. ( 0\'F.RT. T.C. US Environmental Protec·
tion Agf'ncy's MNhod!: Equivalency Program for Drinking Water Samples.
US EPA. Cmcinnat i. Ohio (1985).
I I. l'SEPA. Nationallntenm Primary Drink·
mg Water Regulations. Frd Reg.. ·10:
59566 (1 975).
12.
A.: BLOCK. j.C.: & ELSHAAR~\\'1,
A.H Statisucal Approach for Companson
Between Methods of Bacterial Enumera·
M At: l.,
About the author:
M. ,'t,fichnel Katamav
is microbiology mm;.
ager, Illinois Envi
romtzental Protectiotz
Agency,2121 W Tay-
lor St .. Chicago. !L
60612. Agraduateof
Roosevelt Unive1·sify
(Chicago, Jll.) wtlh a BSr, of the Keller
Graduatp Srhool of Management (Chtcago,
Ill.) with an MBA, at1d of Columbin
PaCific l/nll'(mity (San Rafael, Calif)
u•ilh a PhD, Kafamay has 34 years' experience in lht• field of industrial microbiology, food microbiology. and sanitary
microbiology
M. ?\11CHAF.I. KATAMAY 87
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JOURt\AI. AM! RICMI WAll R \\Oitlo.S ASS()(
\hi H~. Nn 41, \\:f'll,·tnhc:l 11~1
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