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Validation of Colilert-18/Quanti-Tray

for the Enumeration of E. coliand

Coliform Bacteria from Water

JANUARY 2008

One IDEXX Drive Westbrook, Maine 04092 USA


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Validation of Colilert-18/Quanti-Trayfor the Enumerationof E. coliand Coliform Bacteria from Water

1 Introduction

The Colilert-18/Quanti-Tray method is a test specifically designed for the MPNenumeration 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, thesample turns yellow. When E. coli metabolize a second nutrient-indicator, MUG, thesample 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 bacteriain numbers as high as 2 x 106per 100 ml sample.

The Quanti-Tray is designed to produce quantitative bacterial counts from 100 mlsamples 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. Thenumber of positive wells is counted and from an appropriate table the MPN of coliformbacteria and/or E. coliis 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 andMexico), 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 theInternational Bottled Water Association and of pharmaceutical purified water by the USPharmacopeial 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 ofambient 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. coliare those coliform bacteria that also produce

blue fluorescence under UV illumination through the action of !-glucuronidase on 4-methyl-umbelliferyl-!-D-glucuronide (MUG).


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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 TypeCulture 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 Gram-negative bacteria used for confirming typical positive and negativereactions in Colilert-18/Quanti-Tray

Bacterium ATCC* No. Reaction in Colilert-18

Escherichia coli 25922Strong yellow colouration

Strong blue fluorescence under UV

Citrobacter freundii 8090Strong yellow colourationNo fluorescence under UV

Klebsiella pneumoniae 31488

Strong yellow colouration

No fluorescence under UV

Enterobacter aerogenes 13048Strong yellow colourationNo fluorescence under UV

Pseudomonas aeruginosa 10145No growthNo yellow colouration


Aeromonas hydrophila 35654No growthNo yellow colouration


* 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 presumptivecount;

Specificity - the fraction of the total negatives correctly assigned in the presumptivecount;

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 ofnegative wells that are truly negative for the target bacteria. Assessment of the

sensitivity, specificity and selectivity of Colilert-18/Quanti-Tray was achieved through theidentification of !-galactosidase and !-glucuronidase positive isolates from positive wells

generated using natural samples derived by spiking dechlorinated drinking water withriver waters. Three sources of surface water (two rivers and one surface water reservoir)


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were used to derive 1 litre spiked dechlorinated drinking water samples. After incubation100 yellow but non-fluorescing wells were selected from the 30 samples (3 5 fromeach 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 30samples only 54 negative wells were available, of which two showed cloudy growth. !-

Galactosidase and !-glucuronidase positive and/or negative reactions were confirmedand 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. colifrom 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 arepresented in Appendix A1. All were identified as !-galactosidase positive and !-

glucuronidase negative members of the Enterobacteriaceae other than E. coli. In all, 23species 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


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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 oxidasepositive 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. colicoliform positive wells of Colilert-18/Quanti-Tray

Cedecea lapegei (1) Kluyvera ascorbata (12)

Citrobacter amalonaticus (2) Kluyvera cryocrescens(3)

Citrobacter freundii (10) Leclercia adecarboxylata(1)

Enterobacter aerogenes (1) Pantoea agglomerans(9)

Enterobacter asburiae (1) Serratia fonticola(1)

Enterobacter cancerogenus (1) Serratia liquefaciens(3)

Enterobacter cloacae(25) Serratia marcescens(1)

Enterobacter sakazakii(7) Serratia odorifera(1)

Escherichia vulneris(4) Serratia plymuthica(4)

Hafnia alvei(1) Serratia rubidea(1)

Klebsiella oxytoca(4) Serratia spp (1)

Klebsiella pneumoniae(6)

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 ayellow colouration with or without fluorescence and the outcomes for the calculatedparameters are:

Sensitivity = a / (a+b) = 200 / (200+0) = 1Specificity = d / (c+d) = 2 / (0+2) = 1Selectivity = 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) = 0Efficiency (E) = (a+d) / (a+b+c+d) = (200+2) / (200+0+0+2) = 1


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3.3.2 E. coli

In the context of the Colilert-18/Quanti-Traytest E. coliare organisms that produce a

yellow colouration and fluorescence and the outcomes for the calculated parameters

are:

Sensitivity = a / (a+b) = 99 / (99+0) = 1Specificity = d / (c+d) = 102 / (1+102) = 0.99

Selectivity = log10[(a+c) / (a+b+c+d)] = log10[(99+1) / (99+0+1+102)] = -0.305False positive rate = c / (a+c) = 1 / (99+1) = 0.01

False negative rate = b / (b+d) = 0 / (0+102) = 0Efficiency (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 Colilert-

18/Quanti-Tray method is highly sensitive and specific, with zero false-positive and false-

negative 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-Trayrecovers a wide range of coliform bacteria.

For E. coliColilert-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. coliis 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 usingappropriate 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 theease 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 non-

target organisms on an enumeration agar, or the clarity of positive and negativereactions 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 successivemeasurements of the same measurand carried out under the same

conditions of measurement

Reproducibility closeness of the agreement between the results of measurements onthe same measurand carried out under changed conditions of

measurement


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When applied to counting of microbiological colonies on an agar plate or positivereactions 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 moreinformative 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 alsorecommends that, when using pure cultures, RSDs should ideally be < 0.02 (i.e. not

more that 2 % deviation). Counting uncertainty studies were conducted by IDEXXanalysts and the data is presented in Appendix B. The counting was done so that eachanalyst 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 = 0.022

Reproducibility = 0.020

Escherichia coli Repeatability = 0.000Reproducibility = 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. coliare well below the guidance value,

indicating that reliable MPN counting for Klebsiella pneumoniae and E. coli can beachieved 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 therecommended 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 ofsuspensions 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 pneumoniae47 (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, forthe Citrobacter freundiithere 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 Citrobacterappears to show a relatively slower growth rate or weaker enzyme kinetics compared tothe 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


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some slower growing coliform bacteria or weak enzyme producers there can be anincrease 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 countsafter 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-18medium has a stated shelf of 12 months with storage at 4-25 C. Thedata for all three bacteria tested with the same batch of medium stored at 25 C showsno 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-Traytest 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/TR13843 section 6.3.3 no upper limit can be set for MPN methods for practical reasons andfor 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 intervalsfor 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) ascoefficients of variation (CV%) are also presented in Appendix D. These Poisson valuescan 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 becombined 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 coliandcoliform bacteria Part 1: Membrane filtration method. Geneva: InternationalOrganisation for Standardization.

ISO/TR 13843:2000(E) Water Quality Guidance on validation of microbiological

methods. Geneva: International Organization for Standardization.


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Appendix A Data from sensitivity, specificity and selectivity studies (see Section

3.2)

Appendix A1 Identification of isolates from yellow but non-fluorescing wellsfrom Colilert-18/Quanti-Tray

Isolate Ref Oxidase Indole Crystal E/NF profile Identification Confidence

Y1 - + 5765637153 Enterobacter sakazakii 0.5861

Y2 - - 4764417153 Pantoea agglomerans 0.7217

Y3 - + 4724477151 Kluyvera cryocrescens 0.9829

Y4 - - 7764477153 Enterobacter cloacae 0.9962



Y7 - + 5764475555 Klebsiella oxytoca 0.9825


Y9 - - 5764637051 Kluyvera ascorbata 0.9264Y10 - - 5766477152 Enterobacter cloacae 0.9886

Y11 - - 7424676151 Citrobacter freundii 0.9335


Y13 - - 7764476157 Enterobacter sakazakii 0.6102

Y14 - + 5764637151 Kluyvera ascorbata 0.9925


Y16 - - 4764627151 Escherichia vulneris 0.5764



Y19 - - 7766276157 Serratia liquefaciens 0.5712


Y21 - - 3764217153 Cedecea lapagei 0.9835


Y23 - + 7764637157 Enterobacter sakazakii 0.9734

Y24 - + 5764627452 Leclercia adecarboxylata 0.999

Y25 - - 7764627555 Klebsiella pneumoniae 0.9154


Y27 - - 7765275557 Serratia rubidaea 0.9942

Y28 - - 4760056153 Enterobacter asburiae 0.9504











Y39 - + 5760467555 Klebsiella pneumoniae 0.9413


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Y45 - - 7765627157 Enterobacter sakazakii 0.9416Y46 - + 5774677153 Kluyvera ascorbata 0.9439




Y50 - - 7775637153 Enterobacter cancerogenus 0.5033


Y52 - - 5764677557 Enterobacter aerogenes 0.8747





Y57 - - 5764256057 Serratia plymuthica 0.8677

Y58 - + 5764676557 Serratia odorifera 0.7963

Y59 - + 5420677151 Citrobacter amalonaticus 0.8735









Y68 - - 5764276057 Serratia plymuthica 0.8302Y69 - - 4675477153 Enterobacter cloacae 0.9921



Y72 - - 5725654447 Serratia fonticola 0.9933





Y77 - + 5420677151 Citrobacter amalonaticus 0.8735


Y79 - - 5420444051 Citrobacter freundii 0.9799Y80 - - 7764276557 Serratia marcescens 0.9547


Y82 - - 5423614153 Hafnia alvei 0.9847






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Y94 - - 7765256157 Serratia spp. 0.9846


Y96 - + 5464654151 Citrobacter freundii 0.9771






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Appendix A2 Identification of isolates from yellow and fluorescing wells from

Colilert-18/Quanti-Tray

Isolate Ref Oxidase Indole Crystal E/NF profile Identification Confidence

F1 - + 5464444171 Escherichia coli 0.9998












F13 - + 5424444171 Escherichia coli 0.9997F14 - + 5464444171 Escherichia coli 0.9998






























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F80 - - 5464444171 Escherichia coli 0.9998











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F100 - + 5765475577 Klebsiella oxytoca 0.7486


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Appendix A3 Identification of isolates from negative wells from Colilert-

18/Quanti-Tray

IsolateRef

!-Gal !-Gluc Oxidase IndoleCrystal E/NF

profileIdentification Confidence

N1 NO GROWTHN2 NO GROWTH

N3 NO GROWTH

N4 - - + - 3103310212 Shewanella putrefaciens 0.9994

N5 NO GROWTH

N6 NO GROWTH

N7 NO GROWTH

N8 NO GROWTH

N9 NO GROWTH

N10 NO GROWTH

N11 NO GROWTH


N14 NO GROWTH

N15 NO GROWTH

N16 NO GROWTH

N17 - - + - 3103310212 Shewanella putrefaciens 0.9994

N18 NO GROWTH

N19 NO GROWTH

N20 NO GROWTH

N21 NO GROWTH

N22 NO GROWTH

N23 NO GROWTH

N24 NO GROWTH

N25 NO GROWTH

N26 NO GROWTH

N27 NO GROWTH

N28 NO GROWTH

N29 NO GROWTH

N30 NO GROWTH

N31 NO GROWTH

N32 NO GROWTH

N33 NO GROWTH

N34 NO GROWTH

N35 NO GROWTH

N36 NO GROWTH

N37 NO GROWTH

N38 NO GROWTH

N39 NO GROWTH

N40 NO GROWTH

N41 NO GROWTH

N41 NO GROWTH


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N43 NO GROWTH

N44 NO GROWTH

N45 NO GROWTH

N46 NO GROWTH

N47 NO GROWTH


N50 NO GROWTH

N51 NO GROWTH

N52 NO GROWTH

N53 NO GROWTH

N54 NO GROWTH


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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-Trayinoculated with KlebsiellapneumoniaeATCC 33186

* Most Probable Number recorded as whole integers

AnalystA

Read 1

AnalystA

Read 2

AnalystB

Read

AnalystC

ReadRepeatability Reproducibility

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 0.0147 Sum RSD 0.0125

RSDc 0.0221 RSDc 0.0204


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Appendix C Replicate counts from suspensions of E. coli, Klebsiella

pneumoniaeand Citrobacter freundiifrom Colilert-18/Quanti-Trayafter 18 hours and 22 hours incubation using Colilert-18

stored at 25 C for up to 541 days (see Section 5)

Number of positive wells

Escherichia coli Klebsiella pneumoniae Citrobacter freundiiDay postmanufacture

18 h 22 h%

change18 h 22 h

%change

18 h 22 h%

change

8 247

247

000

121115

121115

000

111416

111416

000

15 9168

9168

000

91913

91913

000

257

357

+ 50 %00

24 14910

14910

000

141216

141216

000

141618

141618

000

30 1317

11

1317

11

00

0

1816

13

1816

13

00

0

1818

8

1818

8

00

0

36 813

14

813

14

00

0

1813

17

1813

17

00

0

1615

16

1615

16

00

0

44 85

9

85

9

00

0

2414

19

2414

19

00

0

1116

10

1218

11

+ 9 %+ 13 %

+ 10 %

56 134

4

134

4

00

0

1016

14

1016

14

00

0

67

11

714

11

+ 17 %+ 100 %

0

84 10

88

10

88

0

00

14

1619

14

1619

0

00

11

812

11

812

0

00

111 9

5

21

9

5

21

0

0

0

20

12

12

20

12

12

0

0

0

20

17

17

20

17

17

0

0

0

138 3

2

9

3

2

9

0

0

0

14

15

12

14

15

12

0

0

0

8

10

13

9

10

13

+ 13 %

0

0

175 711

12

711

12

00

0

1520

22

1620

22

+ 7 %0

0

1620

30

1621

30

0+ 5 %

0

205 108

14

108

14

00

0

1720

18

1720

18

00

0

1818

22

1818

22

00

0

269 8

1010

8

1010

0

00

13

2619

13

2619

0

00

14

1714

14

1714

0

00

319 13

18

13

18

0

0

15

17

15

17

0

0

4

13

5

15

+ 25 %

+ 15 %


20/22

19

13 13 0 11 11 0 13 14 + 8 %

367 211

10

211

10

00

0

1221

20

1221

20

00

0

1720

21

1720

21

00

0

541 7

10

9

7

10

9

0

0

0

13

17

16

13

17

16

0

0

0

11

11

8

11

12

9

0

+ 9 %

+ 13 %


21/22

20

Appendix D Ninety-five percent confidence intervals and theoretical

repeatabilities for each MPN value for the 51 well Colilert-18/Quanti-Tray(see Section 7)

95% ConfidenceIntervals Theoretical repeatabilityNo. of wellsgiving

positivereaction

MPN per 100ml sample

Lower Upper

RepeatabilitystandarddeviationLog10SD

Relativestandard

uncertaintyCV%

0


22/22

21

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

36 62.4 44.6 88.8 0.0770 17.7

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

40 78.2 56.4 111.2 0.0756 17.4

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

44 101.3 73.1 146.0 0.0768 17.7

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 19.849 165.2 115.2 272.2 0.0929 21.4

50 200.5 135.8 387.6 0.1094 25.2

51 > 200.5 146.1 infinite - -

colilert raport

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