Validation of Colilert-18/Quanti-Tray

for the Enumeration of E. coli and

Coliform Bacteria from Water

January 2008

One IDEXX Drive Westbrook, Maine 04092 USA

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2011 IDEXX Laboratories, Inc. All rights reserved. 7537-01
All /TM marks are owned by IDEXX Laboratories, Inc.
or its affiliates in the United States and/or other countries.

Validation of Colilert-18/Quanti-Tray for the Enumeration

of E. coli and Coliform Bacteria from Water
1 Introduction
The Colilert-18/Quanti-Tray method is a test specifically designed for the MPN
enumeration of E. coli and coliform bacteria from drinking waters and other similar
treated and untreated waters.
Colilert-18 simultaneously detects total coliforms and E. coli in water. It is based on
Defined Substrate Technology (DST). The DST reagent is mixed with 100 ml of
sample and incubated either as an presence/absence (PA) test or a most probable
number (MPN) test. When coliform bacteria metabolize the nutrient-indicator, ONPG, the
sample turns yellow. When E. coli metabolize a second nutrient-indicator, MUG, the
sample fluoresces under UV illumination. Colilert-18 allows simultaneous detection of
these bacteria at 1 cfu/100 ml within 18 hours in the presence of heterotrophic bacteria
in numbers as high as 2 x 106 per 100 ml sample.
The Quanti-Tray is designed to produce quantitative bacterial counts from 100 ml
samples using DST reagents. The reagent/sample mixture is added to a Quanti-Tray
pouch which is then sealed in a Quanti-Tray Sealer prior to incubation. The pouch is
designed so that after sealing there are 51 wells containing reagent/sample mixture. The
Sealer is a motor-driven, heated roller instrument designed to seal a Quanti-Tray. The
number of positive wells is counted and from an appropriate table the MPN of coliform
bacteria and/or E. coli is determined.
2 Scope of application of Colilert-18/Quanti-Tray
Colilert-18/Quanti-Tray is primarily designed for the analysis of drinking water and
similar waters. As such it has wide approval in North America (USA, Canada and
Mexico), South America (Brazil, Argentina, Chile and Columbia), Europe (Denmark,
Germany, Hungary, Iceland, Ireland, Italy, Norway, Spain and UK) and the Far East
(Japan and Korea). It is also approved for the analysis of bottled waters by the
International Bottled Water Association and of pharmaceutical purified water by the US
Pharmacopeial Convention. In USA it has also been approved by the USEPA for the
analysis of source waters and groundwaters for coliform bacteria and E. coli and of
ambient and recreational waters and wastewaters for the enumeration of E. coli.
3 Target organisms identification (ISO/TR 13843 sections 10.2.1 and 9.2)
In the Colilert-18/Quanti-Tray method coliform bacteria are those bacteria which
produce a yellow colouration through the action of -galactosidase on ortho-nitrophenyl-D-galactopyranoside (ONPG) and E. coli are those coliform bacteria that also produce
blue fluorescence under UV illumination through the action of -glucuronidase on 4methyl-umbelliferyl--D-glucuronide (MUG).

3.1 Pure culture challenge (ISO/TR 13843 section 10.2.1)

Definitions of reactions by target and non-target bacteria were confirmed by challenging
Colilert-18/Quanti-Tray with pure cultures of reference strains of E. coli, coliform
bacteria and non-coliform Gram-negative bacteria sourced from the American Type
Culture Collection. Typical reactions of these reference strains are listed in Table 1.
These strains are used by IDEXX for routine quality control of Colilert-18/Quanti-Tray.
Table 1

Reference strains of E. coli, coliform bacteria and non-target Gramnegative bacteria used for confirming typical positive and negative
reactions in Colilert-18/Quanti-Tray

Bacterium

Reaction in Colilert-18
Strong yellow colouration
Strong blue fluorescence under UV
Strong yellow colouration
No fluorescence under UV
Strong yellow colouration
No fluorescence under UV
Strong yellow colouration
No fluorescence under UV
No growth
No yellow colouration
No fluorescence under UV
No growth
No yellow colouration
No fluorescence under UV

ATCC* No.

Escherichia coli

25922

Citrobacter freundii

8090

Klebsiella pneumoniae

31488

Enterobacter aerogenes

13048

Pseudomonas aeruginosa

10145

Aeromonas hydrophila

35654

* American Type Culture Collection

3.2 Sensitivity, specificity and selectivity studies (ISO/TR 13843 section 9.2)
In terms of microbiological methods these characteristics are defined by ISO/TR 13843
as:
Sensitivity - the fraction of the total positives correctly assigned in the presumptive
count;
Specificity - the fraction of the total negatives correctly assigned in the presumptive
count;
Selectivity - the ratio of the number of target colonies to the total number of colonies in
the sample volume.
For Colilert-18/Quanti-Tray, these characteristics relate to the number of positive
wells for coliforms or E. coli that actually contained target bacteria and the number of
negative wells that are truly negative for the target bacteria. Assessment of the
sensitivity, specificity and selectivity of Colilert-18/Quanti-Tray was achieved through the
identification of -galactosidase and -glucuronidase positive isolates from positive wells
generated using natural samples derived by spiking dechlorinated drinking water with
river waters. Three sources of surface water (two rivers and one surface water reservoir)

were used to derive 1 litre spiked dechlorinated drinking water samples. After incubation
100 yellow but non-fluorescing wells were selected from the 30 samples (3 5 from
each sample). These were selected in a manner so to ensure that the range of yellow
colour intensities encountered were included. Similarly, 100 yellow and fluorescing wells
were selected to cover the range of fluorescence intensity encountered. From the 30
samples only 54 negative wells were available, of which two showed cloudy growth. Galactosidase and -glucuronidase positive and/or negative reactions were confirmed
and the isolates identified using BBL Crystal E/NF miniaturised identification panels
(Becton, Dickinson and Company, Sparks, MD, USA).
3.2.1 Calculation of sensitivity, specificity, selectivity, false positive rate and
false negative rates
For each parameter the identification data were divided into four categories:a = number of positive wells found to contain coliforms or E. coli (true positives);
b = number of negative wells found to contain coliforms or E. coli (false negatives);
c = number of positive wells found not to contain coliforms or E. coli (false positives);
d = number of negative wells showing growth and found not to contain coliforms or E.
coli (true negatives).
The sensitivity, specificity, selectivity, false positive rate and false negative rates for
coliforms and E. coli from the data were calculated as follows:Sensitivity = a / (a+b)
Specificity = d / (c+d)
Selectivity = log10 [(a+c) / (a+b+c+d)]
False positive rate = c / (a+c)
False negative rate = b / (b+d)
A further parameter, efficiency (E), which gives the fraction of wells correctly assigned,
was calculated as E = (a+d) / (a+b+c+d).
3.2.2 Non-E. coli coliforms
-Galactosidase isolates were obtained from all 100 yellow but non-fluorescing wells
subcultured. No -glucuronidase reactions were observed on these plates. The oxidase
and indole reactions, BBL Crystal E/NF profiles and identifications of these isolates are
presented in Appendix A1. All were identified as -galactosidase positive and glucuronidase negative members of the Enterobacteriaceae other than E. coli. In all, 23
species of coliforms were recovered (Table 2).
3.2.3 E. coli
-Glucuronidase isolates were obtained from all 100 yellow and fluorescing wells
subcultured. The oxidase and indole reactions, BBL Crystal E/NF profiles and
identifications of these isolates are presented in Appendix A2. Ninety-nine of the isolates

were identified as E. coli. The remaining isolate was identified as Klebsiella oxytoca.
This isolate had a -glucuronidase positive reaction in its Crystal E/NF panel.
3.2.4 Negative wells
Fifty-four negative wells were subcultured, of which only two had shown growth. These
two yielded -galactosidase and -glucuronidase negative colonies that were oxidase
positive and indole negative (Appendix A3). Both were identified as Shewanella
putrefaciens by the BBL Crystal E/NF system. No growth was obtained from the
remaining 52 wells.
Table 2

Identification of coliform bacteria (number of isolates) recovered from

non-E. coli coliform positive wells of Colilert-18/Quanti-Tray
Cedecea lapegei (1)
Citrobacter amalonaticus (2)
Citrobacter freundii (10)
Enterobacter aerogenes (1)
Enterobacter asburiae (1)
Enterobacter cancerogenus (1)
Enterobacter cloacae (25)
Enterobacter sakazakii (7)
Escherichia vulneris (4)
Hafnia alvei (1)
Klebsiella oxytoca (4)
Klebsiella pneumoniae (6)

Kluyvera ascorbata (12)

Kluyvera cryocrescens (3)
Leclercia adecarboxylata (1)
Pantoea agglomerans (9)
Serratia fonticola (1)
Serratia liquefaciens (3)
Serratia marcescens (1)
Serratia odorifera (1)
Serratia plymuthica (4)
Serratia rubidea (1)
Serratia spp (1)

3.3 Determination of characteristics related to sensitivity and specificity

3.3.1 Coliform Bacteria
In the context of the Colilert-18/Quanti-Tray test coliforms are organisms that produce a
yellow colouration with or without fluorescence and the outcomes for the calculated
parameters are:
Sensitivity = a / (a+b) = 200 / (200+0) = 1
Specificity = d / (c+d) = 2 / (0+2) = 1
Selectivity = log10 [(a+c) / (a+b+c+d)] = log10 [(200+0) / (200+0+0+2)] = -0.004
False positive rate = c / (a+c) = 0 / (200+0) = 0
False negative rate = b / (b+d) = 0 / (0+2) = 0
Efficiency (E) = (a+d) / (a+b+c+d) = (200+2) / (200+0+0+2) = 1

3.3.2 E. coli
In the context of the Colilert-18/Quanti-Tray test E. coli are organisms that produce a
yellow colouration and fluorescence and the outcomes for the calculated parameters
are:
Sensitivity = a / (a+b) = 99 / (99+0) = 1
Specificity = d / (c+d) = 102 / (1+102) = 0.99
Selectivity = log10 [(a+c) / (a+b+c+d)] = log10 [(99+1) / (99+0+1+102)] = -0.305
False positive rate = c / (a+c) = 1 / (99+1) = 0.01
False negative rate = b / (b+d) = 0 / (0+102) = 0
Efficiency (E) = (a+d) / (a+b+c+d) = (99+102) / (99+0+1+102) = 0.995
The outcomes of the analyses of the data show that for coliform bacteria the Colilert18/Quanti-Tray method is highly sensitive and specific, with zero false-positive and falsenegative rates. The method is also very selective with a value of
-004, which is much better than the guidance value of -1 suggested by ISO/TR 13843 for
colony count methods. The method can be said to be highly efficient for coliform bacteria
with an efficiency value of 1. The identification data shows that Colilert-18/Quanti-Tray
recovers a wide range of coliform bacteria.
For E. coli Colilert-18/Quanti-Tray is also highly sensitive and specific. The selectivity (0.305) is less than that for coliform bacteria, but this is to be expected from a dual test
system where E. coli is a subset of the coliform bacteria group. The value is still better
than the guidance value of ISO/TR 13843. The method is still highly efficient (E = 0.995)
for the detection of E. coli.
4 Counting uncertainty (ISO 13843 section 10.2.1 and Annex B)
Repeatability and reproducibility are two estimates of the reliability that can be achieved
with an analytical method. These can be assessments of the whole method using
appropriate natural samples, or of the counting uncertainty associated with reading the
results of a method. The result produced by any method will be dependent upon the
ease with which a count of colonies or positive MPN reactions can be made by analysts.
This will be affected by the distinctiveness of colonial morphology of target and nontarget organisms on an enumeration agar, or the clarity of positive and negative
reactions in broth-based MPN tests. Assessments of counting uncertainty can, therefore,
provide an indication of any potential problems that could occur with wide adoption of a
method.
Repeatability (r) and reproducibility (R) are defined as follows:
Repeatability

closeness of the agreement between the results of successive

measurements of the same measurand carried out under the same
conditions of measurement

Reproducibility

closeness of the agreement between the results of measurements on

the same measurand carried out under changed conditions of
measurement

When applied to counting of microbiological colonies on an agar plate or positive

reactions in an MPN test, the same or changed condition is the counting analyst. Thus,
in this context, repeatability is the agreement in counts obtained by repeated counting by
one analyst, and reproducibility is the agreement in counts obtained by repeated
counting by two or more analysts. The assessment of reproducibility is generally more
informative than the assessment of repeatability.
ISO/TR 13843 Annex B provides guidance on the assessment of counting repeatability
and reproducibility using relative standard deviations (RSD) of repeated counts. It also
recommends that, when using pure cultures, RSDs should ideally be < 0.02 (i.e. not
more that 2 % deviation). Counting uncertainty studies were conducted by IDEXX
analysts and the data is presented in Appendix B. The counting was done so that each
analyst were not aware of other analysts counts and the resulting MPNs recorded as
whole integers. The calculated RSDs for MPNs from Colilert-18/Quanti-Tray
inoculated with a typical coliform (Klebsiella pneumoniae ATCC 33186) and E. coli
(ATCC 25922) were:
Klebsiella pneumoniae

Repeatability
Reproducibility

=
=

0.022
0.020

Escherichia coli

Repeatability
Reproducibility

=
=

0.000
0.007

The values for the typical coliform are of the order of the guidance value of < 0.02
(ISO/TR 13843 section B1), whilst those for E. coli are well below the guidance value,
indicating that reliable MPN counting for Klebsiella pneumoniae and E. coli can be
achieved with Colilert-18/Quanti-Tray.
5 Robustness time sensitivity (ISO/TR 13843 sections 10.2.2 and B.4)
Two aspects of robustness are relevant to Colilert-18/Quanti-Tray. These are the
recommended incubation period of 18-22 hours and the shelf life of up to 15 months with
storage at 4-25 C for the Colilert-18 medium. Replicate counts of analyses of
suspensions of low numbers of reference cultures of E. coli, Klebsiella pneumoniae and
Citrobacter freundii after 18 hours and 22 hours incubation for the same batch of
Colilert-18 stored for a period up to 541 days at 25 C are presented in Appendix C.
5.1 Robustness of incubation time
There were 48 paired results for counts after 18 hours and 22 hours incubation. For E.
coli all 48 paired counts were the same after the two incubation time, while for the
Klebsiella pneumoniae 47 (98 %) of the counts were the same and the remaining paired
count showed an increase of only one positive well at 22 hours incubation. However, for
the Citrobacter freundii there were increased counts after 22 hours incubation compared
to 18 hours incubation from 13 (27 %) of the samples. All except one of these higher
counts were increases of only one or two positive wells. This strain of Citrobacter
appears to show a relatively slower growth rate or weaker enzyme kinetics compared to
the other bacteria tested.
The data shows that for E. coli and robust coliform bacteria such as Klebsiella
pneumoniae the recommended incubation period of 18-22 hours is robust, but that for

some slower growing coliform bacteria or weak enzyme producers there can be an
increase in counts at 22 hours incubation compared to that at 18 hours incubation.
Increases in counts over an prescribed incubation period (e.g. 18-24 hours for typical
membrane filtration methods such as ISO 9308-1) is a well recognised phenomenon,
particularly with environmental coliforms. However, provided that increases in counts
after extended incubation are not excessive, for practical and operational considerations
counts after 18 hours incubation are considered acceptable.
5.3 Robustness of storage of Colilert-18
The Colilert-18 medium has a stated shelf of 12 months with storage at 4-25 C. The
data for all three bacteria tested with the same batch of medium stored at 25 C shows
no difference in performance with storage up to 541 days, well in excess of the one year
stipulated. The higher storage temperature was chosen as this would present a greater
challenge to the stability of the Colilert-18 medium than storage at refrigeration
temperatures.
6 Upper working limit (ISO/TR 13843 sections 10.2.4 and 6.3.3)
The Colilert-18/Quanti-Tray test is an MPN method with an arbitrary counting range of
0 to 201, set by the number of wells in the Quanti-Tray pouch. According to ISO/TR
13843 section 6.3.3 no upper limit can be set for MPN methods for practical reasons and
for statistical reasons because precision does not depend in a simple way on the
number of particles introduced in the detection set.
7 Precision (ISO/TR 13843 sections 10.2.5 and 9.5.5)
The precision of an MPN method is described by the 95 % confidence intervals
calculated for each MPN value (ISO/TR 13843 section 9.5.5). The confidence intervals
for counts from Colilert-18/Quanti-Tray, calculated using the program available from
the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM)
online (http://www.cfsan.fda.gov/~ebam/bam-a2.html#excl), are given in Appendix D.
A theoretical repeatability for each MPN can be derived from the MPN and its confidence
intervals. Relative uncertainty values for each MPN (log10SD, calculated from the MPN
95 % confidence interval values) and repeatability (relative standard uncertainty) as
coefficients of variation (CV%) are also presented in Appendix D. These Poisson values
can be used as minimum precision values to which any appropriate data a laboratory
may have (e.g. on variation in decanting the test volume or counting uncertainty) can be
combined to derive an overall estimate of repeatability for each MPN.
8 References
8.1 ISO standards
ISO 9308-1:2000 Water Quality Detection and enumeration of Escherichia coli and
coliform bacteria Part 1: Membrane filtration method. Geneva: International
Organisation for Standardization.
ISO/TR 13843:2000(E) Water Quality Guidance on validation of microbiological
methods. Geneva: International Organization for Standardization.

Appendix A
3.2)
Appendix A1

Data from sensitivity, specificity and selectivity studies (see Section

Identification of isolates from yellow but non-fluorescing wells

from Colilert-18/Quanti-Tray

Isolate Ref Oxidase

Y1
Y2
Y3
Y4
Y5
Y6
Y7
Y8
Y9
Y10
Y11
Y12
Y13
Y14
Y15
Y16
Y17
Y18
Y19
Y20
Y21
Y22
Y23
Y24
Y25
Y26
Y27
Y28
Y29
Y30
Y31
Y32
Y33
Y34
Y35
Y36
Y37
Y38
Y39
-

Indole
+
+
+
+
+
+
+
+
+
+
+
+

Crystal E/NF profile

5765637153
4764417153
4724477151
7764477153
4764477151
4724467153
5764475555
4760427053
5764637051
5766477152
7424676151
7764672153
7764476157
5764637151
5765677151
4764627151
7734457051
5765477157
7766276157
5764476153
3764217153
5764427151
7764637157
5764627452
7764627555
0764437152
7765275557
4760056153
5765276157
5764617151
5764677557
7564677151
4560477153
5464454151
5464654155
5764427151
5664456055
4764475555
5760467555

Identification
Enterobacter sakazakii
Pantoea agglomerans
Kluyvera cryocrescens
Enterobacter cloacae
Kluyvera cryocrescens
Pantoea agglomerans
Klebsiella oxytoca
Pantoea agglomerans
Kluyvera ascorbata
Enterobacter cloacae
Citrobacter freundii
Enterobacter cloacae
Enterobacter sakazakii
Kluyvera ascorbata
Kluyvera ascorbata
Escherichia vulneris
Pantoea agglomerans
Klebsiella oxytoca
Serratia liquefaciens
Enterobacter cloacae
Cedecea lapagei
Pantoea agglomerans
Enterobacter sakazakii
Leclercia adecarboxylata
Klebsiella pneumoniae
Pantoea agglomerans
Serratia rubidaea
Enterobacter asburiae
Serratia liquefaciens
Kluyvera ascorbata
Klebsiella oxytoca
Enterobacter cloacae
Enterobacter cloacae
Citrobacter freundii
Citrobacter freundii
Pantoea agglomerans
Citrobacter freundii
Klebsiella pneumoniae
Klebsiella pneumoniae

Confidence
0.5861
0.7217
0.9829
0.9962
0.6287
0.7127
0.9825
0.9481
0.9264
0.9886
0.9335
0.8268
0.6102
0.9925
0.8702
0.5764
0.9932
0.8524
0.5712
0.8943
0.9835
0.7613
0.9734
0.999
0.9154
0.8776
0.9942
0.9504
0.5545
0.982
0.9555
0.9987
0.9961
0.9998
0.9994
0.7613
0.9973
0.9241
0.9413

Y40
Y41
Y42
Y43
Y44
Y45
Y46
Y47
Y48
Y49
Y50
Y51
Y52
Y53
Y54
Y55
Y56
Y57
Y58
Y59
Y60
Y61
Y62
Y63
Y64
Y65
Y66
Y67
Y68
Y69
Y70
Y71
Y72
Y73
Y74
Y75
Y76
Y77
Y78
Y79
Y80
Y81
Y82
Y83
Y84
Y85
Y86

+
+
+
+
+
+
+
+
+
+
-

4764627051
5764677150
4760473151
5764637151
5766476156
7765627157
5774677153
7775677555
7765627157
5464477553
7775637153
4774437151
5764677557
5464457051
7764272153
5766477152
5764476152
5764256057
5764676557
5420677151
5766476152
5470473153
5774637151
5764676153
4764476057
5766476153
7765671151
7764477153
5764276057
4675477153
5765276057
5624657151
5725654447
5766477152
5764477555
5724627052
5766476153
5420677151
7767276157
5420444051
7764276557
5766477153
5423614153
5764627151
5765677557
5765476553
5767476152

Escherichia vulneris
Enterobacter cloacae
Enterobacter cloacae
Kluyvera ascorbata
Enterobacter sakazakii
Enterobacter sakazakii
Kluyvera ascorbata
Klebsiella pneumoniae
Enterobacter sakazakii
Enterobacter cloacae
Enterobacter cancerogenus
Kluyvera cryocrescens
Enterobacter aerogenes
Citrobacter freundii
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae
Serratia plymuthica
Serratia odorifera
Citrobacter amalonaticus
Enterobacter cloacae
Enterobacter cloacae
Kluyvera ascorbata
Kluyvera ascorbata
Enterobacter sakazakii
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae
Serratia plymuthica
Enterobacter cloacae
Serratia plymuthica
Citrobacter freundii
Serratia fonticola
Enterobacter cloacae
Klebsiella pneumoniae
Escherichia vulneris
Enterobacter cloacae
Citrobacter amalonaticus
Serratia liquefaciens
Citrobacter freundii
Serratia marcescens
Enterobacter cloacae
Hafnia alvei
Kluyvera ascorbata
Klebsiella oxytoca
Enterobacter cloacae
Enterobacter cloacae

0.9475
0.8043
0.9673
0.9925
0.8111
0.9416
0.9439
0.9853
0.9416
0.9999
0.5033
0.8422
0.8747
0.9962
0.8189
0.9886
0.9048
0.8677
0.7963
0.8735
0.6927
0.9996
0.9864
0.9308
0.7089
0.6618
0.937
0.9962
0.8302
0.9921
0.6877
0.996
0.9933
0.9886
0.9919
0.9987
0.6618
0.8735
0.6819
0.9799
0.9547
0.9872
0.9847
0.9942
0.9715
0.9226
0.7434

Y87
Y88
Y89
Y90
Y91
Y92
Y93
Y94
Y95
Y96
Y97
Y98
Y99
Y100

+
+
+
+
-

5764637051
5764276057
5664677151
4464654141
5764627051
5764455555
5765647151
7765256157
5765477153
5464654151
5765637151
5764427151
5764077153
5420656050

10

Kluyvera ascorbata
Serratia plymuthica
Kluyvera ascorbata
Citrobacter freundii
Escherichia vulneris
Klebsiella pneumoniae
Pantoea agglomerans
Serratia spp.
Enterobacter cloacae
Citrobacter freundii
Kluyvera ascorbata
Pantoea agglomerans
Enterobacter cloacae
Citrobacter freundii

0.9876
0.8302
0.9349
0.999
0.6454
0.9504
0.6856
0.9846
0.9957
0.9771
0.8306
0.7613
0.9948
0.994

Appendix A2

Identification of isolates from yellow and fluorescing wells from

Colilert-18/Quanti-Tray

Isolate Ref Oxidase

F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
F13
F14
F15
F16
F17
F18
F19
F20
F21
F22
F23
F24
F25
F26
F27
F28
F29
F30
F31
F32
F33
F34
F35
F36
F37
F38
F39
F40
F41
F42
F43
-

Indole
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Crystal E/NF profile

5464444171
5461444171
5464444171
4464444171
5465464171
5464444171
5464444171
5464444171
5464444171
5464444171
5424444171
4464444171
5424444171
5464444171
5464444171
5425444571
5464444171
5464444171
5464444171
5464444171
5424444171
5464444171
5424444171
5465444161
5465444161
5464044171
5465446071
5464444171
5464444171
5464444171
5464444171
5425444571
5464444171
5464444171
5424444171
5464444171
5464044171
5464444171
5464444171
5465464171
5464444171
5464444171
5464444171

11

Identification
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli

Confidence
0.9998
0.9737
0.9998
0.9813
0.991
0.9998
0.9998
0.9998
0.9998
0.9998
0.9997
0.9813
0.9997
0.9998
0.9998
0.9679
0.9998
0.9998
0.9998
0.9998
0.9997
0.9998
0.9997
0.9976
0.9976
0.999
0.9945
0.9998
0.9998
0.9998
0.9998
0.9679
0.9998
0.9998
0.9997
0.9998
0.999
0.9998
0.9998
0.991
0.9998
0.9998
0.9998

F44
F45
F46
F47
F48
F49
F50
F51
F52
F53
F54
F55
F56
F57
F58
F59
F60
F61
F62
F63
F64
F65
F66
F67
F68
F69
F70
F71
F72
F73
F74
F75
F76
F77
F78
F79
F80
F81
F82
F83
F84
F85
F86
F87
F88
F89
F90

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

5464444171
5424444171
5464444171
5665444171
5464444171
5465404171
5464444171
5465464171
5464444171
5464444171
5464444171
5424444171
5464444171
5464444171
5464444171
5464444171
5464444171
5464444171
5464444171
5464444171
5464444171
5424444171
5665444171
5464444171
5464444171
5435444171
5464444171
5464444171
4464444171
5464444171
5724444571
5465464171
5424444171
5424444171
5464444171
5464444171
5464444171
5424444061
5464044171
5465446071
5464444171
5464444171
5564044171
5464444171
5464444171
5464444171
5424444171

12

Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli

0.9998
0.9997
0.9998
0.9962
0.9998
0.9314
0.9998
0.991
0.9998
0.9998
0.9998
0.9997
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9998
0.9997
0.9962
0.9998
0.9998
0.9803
0.9998
0.9998
0.9813
0.9998
0.9999
0.991
0.9997
0.9997
0.9998
0.9998
0.9998
0.8035
0.999
0.9945
0.9998
0.9998
0.9911
0.9998
0.9998
0.9998
0.9997

F91
F92
F93
F94
F95
F96
F97
F98
F99
F100

+
+
+
+
+
+
+
+
+
+

5464444171
5464444171
5424444171
5420444571
5425444571
5464444171
5464444171
5464444171
5465464171
5765475577

13

Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Klebsiella oxytoca

0.9998
0.9998
0.9997
0.9983
0.9679
0.9998
0.9998
0.9998
0.991
0.7486

Appendix A3

Isolate
Ref
N1
N2
N3
N4
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
N16
N17
N18
N19
N20
N21
N22
N23
N24
N25
N26
N27
N28
N29
N30
N31
N32
N33
N34
N35
N36
N37
N38
N39
N40
N41
N41

-Gal

Identification of isolates from negative wells from Colilert18/Quanti-Tray

-Gluc Oxidase Indole

Crystal E/NF
profile

3103310212

3103310212

14

Identification
NO GROWTH
NO GROWTH
NO GROWTH
Shewanella putrefaciens
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
Shewanella putrefaciens
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH

Confidence

0.9994

0.9994

N43
N44
N45
N46
N47
N48
N49
N50
N51
N52
N53
N54

NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH
NO GROWTH

15

Appendix B

Data from counting uncertainty studies (see Section 4)

Appendix B1

Counting uncertainty for MPNs* of yellow non-fluorescing wells

from Colilert-18/Quanti-Tray inoculated with Klebsiella
pneumoniae ATCC 33186
* Most Probable Number recorded as whole integers

Analyst Analyst Analyst Analyst

A
A
B
C
Repeatability
Reproducibility
Read 1 Read 2 Read
Read
Sample MPN
MPN
MPN
MPN
Mean St Dev RSD sq Mean St Dev RSD sq
1
2
2
2
2
2.00 0.0000 0.0000 2.00 0.0000 0.0000
2
6
6
6
6
6.00 0.0000 0.0000 6.00 0.0000 0.0000
3
19
19
19
19
19.00 0.0000 0.0000 19.00 0.0000 0.0000
4
18
18
18
18
18.00 0.0000 0.0000 18.00 0.0000 0.0000
5
36
36
36
36
36.00 0.0000 0.0000 36.00 0.0000 0.0000
6
34
34
34
36
34.00 0.0000 0.0000 34.67 1.1547 0.0011
7
31
31
31
31
31.00 0.0000 0.0000 31.00 0.0000 0.0000
8
27
27
27
27
27.00 0.0000 0.0000 27.00 0.0000 0.0000
9
56
56
56
56
56.00 0.0000 0.0000 56.00 0.0000 0.0000
10
50
50
50
50
50.00 0.0000 0.0000 50.00 0.0000 0.0000
11
41
41
41
41
41.00 0.0000 0.0000 41.00 0.0000 0.0000
12
48
48
48
48
48.00 0.0000 0.0000 48.00 0.0000 0.0000
13
56
56
56
56
56.00 0.0000 0.0000 56.00 0.0000 0.0000
14
89
89
89
89
89.00 0.0000 0.0000 89.00 0.0000 0.0000
15
78
78
78
78
78.00 0.0000 0.0000 78.00 0.0000 0.0000
16
66
70
70
70
68.00 2.8284 0.0017 68.67 2.3094 0.0011
17
109
109
109
109
109.00 0.0000 0.0000 109.00 0.0000 0.0000
18
78
89
89
89
83.50 7.7782 0.0087 85.33 6.3509 0.0055
19
95
95
95
95
95.00 0.0000 0.0000 95.00 0.0000 0.0000
20
95
95
95
95
95.00 0.0000 0.0000 95.00 0.0000 0.0000
21
95
101
101
101
98.00 4.2426 0.0019 99.00 3.4641 0.0012
22
83
89
89
89
86.00 4.2426 0.0024 87.00 3.4641 0.0016
23
109
109
109
109
109.00 0.0000 0.0000 109.00 0.0000 0.0000
24
101
101
109
109
101.00 0.0000 0.0000 106.33 4.6188 0.0019
25
145
145
145
145
145.00 0.0000 0.0000 145.00 0.0000 0.0000
26
145
145
145
145
145.00 0.0000 0.0000 145.00 0.0000 0.0000
27
201
201
201
201
201.00 0.0000 0.0000 201.00 0.0000 0.0000
28
165
165
165
165
165.00 0.0000 0.0000 165.00 0.0000 0.0000
29
165
165
165
165
165.00 0.0000 0.0000 165.00 0.0000 0.0000
30

201

201

201

201

201.00 0.0000 0.0000 201.00 0.0000 0.0000

Sum RSD
RSDc

16

0.0147
0.0221

Sum RSD
RSDc

0.0125
0.0204

Appendix B2

Counting uncertainty for MPNS* of yellow fluorescing wells from

Colilert-18/Quanti-Tray inoculated with E. coli ATCC 25922
* Most Probable Number recorded as whole integers

Analyst Analyst Analyst Analyst

A
A
B
C
Repeatability
Reproducibility
Read 1 Read 2 Read
Read
Sample MPN
MPN
MPN
MPN
Mean St Dev RSD sq Mean St Dev RSD sq
1
6
6
6
6
6.00 0.0000 0.0000 6.00 0.0000 0.0000
2
11
11
11
11
11.00 0.0000 0.0000 11.00 0.0000 0.0000
3
9
9
9
9
9.00 0.0000 0.0000 9.00 0.0000 0.0000
4
29
29
29
29
29.00 0.0000 0.0000 29.00 0.0000 0.0000
5
32
32
32
32
32.00 0.0000 0.0000 32.00 0.0000 0.0000
6
21
21
21
21
21.00 0.0000 0.0000 21.00 0.0000 0.0000
7
36
36
36
36
36.00 0.0000 0.0000 36.00 0.0000 0.0000
8
32
32
32
32
32.00 0.0000 0.0000 32.00 0.0000 0.0000
9
56
56
56
56
56.00 0.0000 0.0000 56.00 0.0000 0.0000
10
50
50
50
50
50.00 0.0000 0.0000 50.00 0.0000 0.0000
11
48
48
48
48
48.00 0.0000 0.0000 48.00 0.0000 0.0000
12
45
45
45
48
45.00 0.0000 0.0000 46.00 1.7321 0.0014
13
78
78
78
78
78.00 0.0000 0.0000 78.00 0.0000 0.0000
14
70
70
70
70
70.00 0.0000 0.0000 70.00 0.0000 0.0000
15
66
66
66
66
66.00 0.0000 0.0000 66.00 0.0000 0.0000
16
78
78
78
78
78.00 0.0000 0.0000 78.00 0.0000 0.0000
17
95
95
95
95
95.00 0.0000 0.0000 95.00 0.0000 0.0000
18
109
109
109
109
109.00 0.0000 0.0000 109.00 0.0000 0.0000
19
101
101
101
101
101.00 0.0000 0.0000 101.00 0.0000 0.0000
20
89
89
89
89
89.00 0.0000 0.0000 89.00 0.0000 0.0000
21
130
130
130
130
130.00 0.0000 0.0000 130.00 0.0000 0.0000
22
95
95
95
95
95.00 0.0000 0.0000 95.00 0.0000 0.0000
23
70
70
70
70
70.00 0.0000 0.0000 70.00 0.0000 0.0000
24
74
74
74
74
74.00 0.0000 0.0000 74.00 0.0000 0.0000
25
109
109
109
109
109.00 0.0000 0.0000 109.00 0.0000 0.0000
26
101
101
101
101
101.00 0.0000 0.0000 101.00 0.0000 0.0000
27
145
145
145
145
145.00 0.0000 0.0000 145.00 0.0000 0.0000
28
165
165
165
165
165.00 0.0000 0.0000 165.00 0.0000 0.0000
29
118
118
118
118
118.00 0.0000 0.0000 118.00 0.0000 0.0000
30

130

130

130

130

130.00 0.0000 0.0000 130.00 0.0000 0.0000

Sum RSD
RSDc

17

0.0000
0.0000

Sum RSD
RSDc

0.0014
0.0069

Appendix C

Replicate counts from suspensions of E. coli, Klebsiella

pneumoniae and Citrobacter freundii from Colilert-18/QuantiTray after 18 hours and 22 hours incubation using Colilert-18
stored at 25 C for up to 541 days (see Section 5)

Day post
manufacture

Escherichia coli
%
18 h
22 h
change
2
2
0
4
4
0
7
7
0
9
9
0
16
16
0
8
8
0
14
14
0
9
9
0
10
10
0
13
13
0
17
17
0
11
11
0
8
8
0
13
13
0
14
14
0
8
8
0
5
5
0
9
9
0
13
13
0
4
4
0
4
4
0
10
10
0
8
8
0
8
8
0
9
9
0
5
5
0
21
21
0
3
3
0
2
2
0
9
9
0
7
7
0
11
11
0
12
12
0
10
10
0
8
8
0
14
14
0
8
8
0
10
10
0
10
10
0
13
13
0
18
18
0

8
15
24
30
36
44
56
84
111
138
175
205
269
319

Number of positive wells

Klebsiella pneumoniae
%
18 h
22 h
change
12
12
0
11
11
0
15
15
0
9
9
0
19
19
0
13
13
0
14
14
0
12
12
0
16
16
0
18
18
0
16
16
0
13
13
0
18
18
0
13
13
0
17
17
0
24
24
0
14
14
0
19
19
0
10
10
0
16
16
0
14
14
0
14
14
0
16
16
0
19
19
0
20
20
0
12
12
0
12
12
0
14
14
0
15
15
0
12
12
0
15
16
+7%
20
20
0
22
22
0
17
17
0
20
20
0
18
18
0
13
13
0
26
26
0
19
19
0
15
15
0
17
17
0

18

Citrobacter freundii
%
18 h
22 h
change
11
11
0
14
14
0
16
16
0
2
3
+ 50 %
5
5
0
7
7
0
14
14
0
16
16
0
18
18
0
18
18
0
18
18
0
8
8
0
16
16
0
15
15
0
16
16
0
11
12
+9%
16
18
+ 13 %
10
11
+ 10 %
6
7
+ 17 %
7
14
+ 100 %
11
11
0
11
11
0
8
8
0
12
12
0
20
20
0
17
17
0
17
17
0
8
9
+ 13 %
10
10
0
13
13
0
16
16
0
20
21
+5%
30
30
0
18
18
0
18
18
0
22
22
0
14
14
0
17
17
0
14
14
0
4
5
+ 25 %
13
15
+ 15 %

367
541

13
2
11
10
7
10
9

13
2
11
10
7
10
9

0
0
0
0
0
0
0

11
12
21
20
13
17
16

19

11
12
21
20
13
17
16

0
0
0
0
0
0
0

13
17
20
21
11
11
8

14
17
20
21
11
12
9

+8%
0
0
0
0
+9%
+ 13 %

Appendix D

Ninety-five percent confidence intervals and theoretical

repeatabilities for each MPN value for the 51 well Colilert18/Quanti-Tray (see Section 7)

95% Confidence
Intervals

No. of wells
giving
positive
reaction

MPN per 100

ml sample

0
1

Theoretical repeatability

Lower

Upper

<1.0

0.0

3.7

Repeatability
standard
deviation
Log10SD
-

Relative
standard
uncertainty
CV%
-

1.0

0.3

5.6

0.4343

100.0
70.7

2.0

0.6

7.3

0.3071

3.1

1.1

9.0

0.2508

57.7

4.2

1.7

10.7

0.2172

50.0

5.3

2.3

12.3

0.1943

44.7
40.9

6.4

3.0

13.9

0.1774

7.5

3.7

15.5

0.1643

37.8

8.7

4.5

17.1

0.1537

35.4

9.9

5.3

18.8

0.1450

33.4
31.7

10

11.1

6.1

20.5

0.1376

11

12.4

7.0

22.1

0.1313

30.2

12

13.7

7.9

23.9

0.1257

29.0

13

15.0

8.8

25.7

0.1209

27.8
26.8

14

16.4

9.8

27.5

0.1166

15

17.8

10.8

29.4

0.1127

26.0

16

19.2

11.9

31.3

0.1092

25.1

17

20.7

13.0

33.3

0.1061

24.4

18

22.2

14.1

35.2

0.1032

23.8
23.1

19

23.8

15.3

37.3

0.1005

20

25.4

16.5

39.4

0.0981

22.6

21

27.1

17.7

41.6

0.0959

22.1

22

28.8

19.0

43.9

0.0938

21.6
21.2

23

30.6

20.4

46.3

0.0919

24

32.4

21.8

48.7

0.0902

20.8

25

34.4

23.3

51.2

0.0885

20.4

26

36.4

24.7

53.9

0.0870

20.0
19.7

27

38.4

26.4

56.6

0.0856

28

40.6

28.0

59.5

0.0843

19.4

29

42.9

29.7

62.5

0.0830

19.1

30

45.3

31.5

65.6

0.0819

18.9
18.6
18.4

31

47.8

33.4

69.0

0.0809

32

50.4

35.4

72.5

0.0799

20

33

53.1

37.5

76.2

0.0791

18.2

34

56.0

39.7

80.1

0.0783

18.0

35

59.1

42.0

84.4

0.0776

17.9
17.7

36

62.4

44.6

88.8

0.0770

37

65.9

47.2

93.7

0.0765

17.6

38

69.7

50.0

99.0

0.0761

17.5

39

73.8

53.1

104.8

0.0758

17.4
17.4

40

78.2

56.4

111.2

0.0756

41

83.1

59.9

118.3

0.0756

17.4

42

88.5

63.9

126.2

0.0757

17.4

43

94.5

68.2

135.4

0.0761

17.5
17.7

44

101.3

73.1

146.0

0.0768

45

109.1

78.6

158.7

0.0778

17.9

46

118.4

85.0

174.5

0.0794

18.3

47

129.8

92.7

195.0

0.0819

18.9

48

144.5

102.3

224.1

0.0859

19.8
21.4

49

165.2

115.2

272.2

0.0929

50

200.5

135.8

387.6

0.1094

25.2

51

> 200.5

146.1

infinite

21