Validation of Colilert®-18/Quanti-Tray®

for the Enumeration of E. coli andColiform Bacteria from Water

January 2008

One IDEXX Drive • Westbrook, Maine 04092 USAidexx.com

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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 β-galactosidase on ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and E. coli are those coliform bacteria that also produce blue fluorescence under UV illumination through the action of β-glucuronidase on 4-methyl-umbelliferyl-β-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 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 Gram-

negative bacteria used for confirming typical positive and negative reactions in Colilert®-18/Quanti-Tray®

Bacterium ATCC* No. Reaction in Colilert®-18

Escherichia coli 25922 Strong yellow colouration Strong blue fluorescence under UV

Citrobacter freundii 8090 Strong yellow colouration No fluorescence under UV

Klebsiella pneumoniae 31488 Strong yellow colouration No fluorescence under UV

Enterobacter aerogenes 13048 Strong yellow colouration No fluorescence under UV

Pseudomonas aeruginosa 10145 No growth No yellow colouration No fluorescence under UV

Aeromonas hydrophila 35654 No growth No yellow colouration No fluorescence under UV

* 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 β-galactosidase and β-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)

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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. β-Galactosidase and β-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 β-Galactosidase isolates were obtained from all 100 yellow but non-fluorescing wells subcultured. No β-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 β-galactosidase positive and β-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 β-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 β-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 β-galactosidase and β-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) 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 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

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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 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-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 non-target 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

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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 = 0.022 Reproducibility = 0.020

Escherichia coli Repeatability = 0.000 Reproducibility = 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

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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.

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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 wells

from 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 Y5 - + 4764477151 Kluyvera cryocrescens 0.6287 Y6 - - 4724467153 Pantoea agglomerans 0.7127 Y7 - + 5764475555 Klebsiella oxytoca 0.9825 Y8 - - 4760427053 Pantoea agglomerans 0.9481 Y9 - - 5764637051 Kluyvera ascorbata 0.9264

Y10 - - 5766477152 Enterobacter cloacae 0.9886 Y11 - - 7424676151 Citrobacter freundii 0.9335 Y12 - - 7764672153 Enterobacter cloacae 0.8268 Y13 - - 7764476157 Enterobacter sakazakii 0.6102 Y14 - + 5764637151 Kluyvera ascorbata 0.9925 Y15 - + 5765677151 Kluyvera ascorbata 0.8702 Y16 - - 4764627151 Escherichia vulneris 0.5764 Y17 - - 7734457051 Pantoea agglomerans 0.9932 Y18 - + 5765477157 Klebsiella oxytoca 0.8524 Y19 - - 7766276157 Serratia liquefaciens 0.5712 Y20 - - 5764476153 Enterobacter cloacae 0.8943 Y21 - - 3764217153 Cedecea lapagei 0.9835 Y22 - - 5764427151 Pantoea agglomerans 0.7613 Y23 - + 7764637157 Enterobacter sakazakii 0.9734 Y24 - + 5764627452 Leclercia adecarboxylata 0.999 Y25 - - 7764627555 Klebsiella pneumoniae 0.9154 Y26 - - 0764437152 Pantoea agglomerans 0.8776 Y27 - - 7765275557 Serratia rubidaea 0.9942 Y28 - - 4760056153 Enterobacter asburiae 0.9504 Y29 - - 5765276157 Serratia liquefaciens 0.5545 Y30 - + 5764617151 Kluyvera ascorbata 0.982 Y31 - + 5764677557 Klebsiella oxytoca 0.9555 Y32 - - 7564677151 Enterobacter cloacae 0.9987 Y33 - - 4560477153 Enterobacter cloacae 0.9961 Y34 - - 5464454151 Citrobacter freundii 0.9998 Y35 - - 5464654155 Citrobacter freundii 0.9994 Y36 - - 5764427151 Pantoea agglomerans 0.7613 Y37 - - 5664456055 Citrobacter freundii 0.9973 Y38 - - 4764475555 Klebsiella pneumoniae 0.9241 Y39 - + 5760467555 Klebsiella pneumoniae 0.9413

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Y40 - - 4764627051 Escherichia vulneris 0.9475 Y41 - - 5764677150 Enterobacter cloacae 0.8043 Y42 - - 4760473151 Enterobacter cloacae 0.9673 Y43 - + 5764637151 Kluyvera ascorbata 0.9925 Y44 - - 5766476156 Enterobacter sakazakii 0.8111 Y45 - - 7765627157 Enterobacter sakazakii 0.9416 Y46 - + 5774677153 Kluyvera ascorbata 0.9439 Y47 - - 7775677555 Klebsiella pneumoniae 0.9853 Y48 - - 7765627157 Enterobacter sakazakii 0.9416 Y49 - - 5464477553 Enterobacter cloacae 0.9999 Y50 - - 7775637153 Enterobacter cancerogenus 0.5033 Y51 - + 4774437151 Kluyvera cryocrescens 0.8422 Y52 - - 5764677557 Enterobacter aerogenes 0.8747 Y53 - - 5464457051 Citrobacter freundii 0.9962 Y54 - - 7764272153 Enterobacter cloacae 0.8189 Y55 - - 5766477152 Enterobacter cloacae 0.9886 Y56 - - 5764476152 Enterobacter cloacae 0.9048 Y57 - - 5764256057 Serratia plymuthica 0.8677 Y58 - + 5764676557 Serratia odorifera 0.7963 Y59 - + 5420677151 Citrobacter amalonaticus 0.8735 Y60 - - 5766476152 Enterobacter cloacae 0.6927 Y61 - - 5470473153 Enterobacter cloacae 0.9996 Y62 - + 5774637151 Kluyvera ascorbata 0.9864 Y63 - + 5764676153 Kluyvera ascorbata 0.9308 Y64 - - 4764476057 Enterobacter sakazakii 0.7089 Y65 - - 5766476153 Enterobacter cloacae 0.6618 Y66 - - 7765671151 Enterobacter cloacae 0.937 Y67 - - 7764477153 Enterobacter cloacae 0.9962 Y68 - - 5764276057 Serratia plymuthica 0.8302 Y69 - - 4675477153 Enterobacter cloacae 0.9921 Y70 - - 5765276057 Serratia plymuthica 0.6877 Y71 - - 5624657151 Citrobacter freundii 0.996 Y72 - - 5725654447 Serratia fonticola 0.9933 Y73 - - 5766477152 Enterobacter cloacae 0.9886 Y74 - - 5764477555 Klebsiella pneumoniae 0.9919 Y75 - - 5724627052 Escherichia vulneris 0.9987 Y76 - - 5766476153 Enterobacter cloacae 0.6618 Y77 - + 5420677151 Citrobacter amalonaticus 0.8735 Y78 - - 7767276157 Serratia liquefaciens 0.6819 Y79 - - 5420444051 Citrobacter freundii 0.9799 Y80 - - 7764276557 Serratia marcescens 0.9547 Y81 - - 5766477153 Enterobacter cloacae 0.9872 Y82 - - 5423614153 Hafnia alvei 0.9847 Y83 - + 5764627151 Kluyvera ascorbata 0.9942 Y84 - + 5765677557 Klebsiella oxytoca 0.9715 Y85 - - 5765476553 Enterobacter cloacae 0.9226 Y86 - - 5767476152 Enterobacter cloacae 0.7434

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Y87 - + 5764637051 Kluyvera ascorbata 0.9876 Y88 - - 5764276057 Serratia plymuthica 0.8302 Y89 - + 5664677151 Kluyvera ascorbata 0.9349 Y90 - - 4464654141 Citrobacter freundii 0.999 Y91 - - 5764627051 Escherichia vulneris 0.6454 Y92 - - 5764455555 Klebsiella pneumoniae 0.9504 Y93 - - 5765647151 Pantoea agglomerans 0.6856 Y94 - - 7765256157 Serratia spp. 0.9846 Y95 - - 5765477153 Enterobacter cloacae 0.9957 Y96 - + 5464654151 Citrobacter freundii 0.9771 Y97 - + 5765637151 Kluyvera ascorbata 0.8306 Y98 - - 5764427151 Pantoea agglomerans 0.7613 Y99 - - 5764077153 Enterobacter cloacae 0.9948

Y100 - - 5420656050 Citrobacter freundii 0.994

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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 F2 - + 5461444171 Escherichia coli 0.9737 F3 - + 5464444171 Escherichia coli 0.9998 F4 - + 4464444171 Escherichia coli 0.9813 F5 - + 5465464171 Escherichia coli 0.991 F6 - + 5464444171 Escherichia coli 0.9998 F7 - + 5464444171 Escherichia coli 0.9998 F8 - + 5464444171 Escherichia coli 0.9998 F9 - + 5464444171 Escherichia coli 0.9998

F10 - + 5464444171 Escherichia coli 0.9998 F11 - + 5424444171 Escherichia coli 0.9997 F12 - + 4464444171 Escherichia coli 0.9813 F13 - + 5424444171 Escherichia coli 0.9997 F14 - + 5464444171 Escherichia coli 0.9998 F15 - + 5464444171 Escherichia coli 0.9998 F16 - + 5425444571 Escherichia coli 0.9679 F17 - + 5464444171 Escherichia coli 0.9998 F18 - + 5464444171 Escherichia coli 0.9998 F19 - + 5464444171 Escherichia coli 0.9998 F20 - + 5464444171 Escherichia coli 0.9998 F21 - + 5424444171 Escherichia coli 0.9997 F22 - + 5464444171 Escherichia coli 0.9998 F23 - + 5424444171 Escherichia coli 0.9997 F24 - + 5465444161 Escherichia coli 0.9976 F25 - + 5465444161 Escherichia coli 0.9976 F26 - + 5464044171 Escherichia coli 0.999 F27 - + 5465446071 Escherichia coli 0.9945 F28 - + 5464444171 Escherichia coli 0.9998 F29 - + 5464444171 Escherichia coli 0.9998 F30 - + 5464444171 Escherichia coli 0.9998 F31 - + 5464444171 Escherichia coli 0.9998 F32 - + 5425444571 Escherichia coli 0.9679 F33 - + 5464444171 Escherichia coli 0.9998 F34 - + 5464444171 Escherichia coli 0.9998 F35 - + 5424444171 Escherichia coli 0.9997 F36 - + 5464444171 Escherichia coli 0.9998 F37 - + 5464044171 Escherichia coli 0.999 F38 - + 5464444171 Escherichia coli 0.9998 F39 - + 5464444171 Escherichia coli 0.9998 F40 - + 5465464171 Escherichia coli 0.991 F41 - + 5464444171 Escherichia coli 0.9998 F42 - + 5464444171 Escherichia coli 0.9998 F43 - + 5464444171 Escherichia coli 0.9998

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F44 - + 5464444171 Escherichia coli 0.9998 F45 - + 5424444171 Escherichia coli 0.9997 F46 - + 5464444171 Escherichia coli 0.9998 F47 - + 5665444171 Escherichia coli 0.9962 F48 - + 5464444171 Escherichia coli 0.9998 F49 - + 5465404171 Escherichia coli 0.9314 F50 - + 5464444171 Escherichia coli 0.9998 F51 - + 5465464171 Escherichia coli 0.991 F52 - + 5464444171 Escherichia coli 0.9998 F53 - + 5464444171 Escherichia coli 0.9998 F54 - + 5464444171 Escherichia coli 0.9998 F55 - + 5424444171 Escherichia coli 0.9997 F56 - + 5464444171 Escherichia coli 0.9998 F57 - + 5464444171 Escherichia coli 0.9998 F58 - + 5464444171 Escherichia coli 0.9998 F59 - + 5464444171 Escherichia coli 0.9998 F60 - + 5464444171 Escherichia coli 0.9998 F61 - + 5464444171 Escherichia coli 0.9998 F62 - + 5464444171 Escherichia coli 0.9998 F63 - + 5464444171 Escherichia coli 0.9998 F64 - + 5464444171 Escherichia coli 0.9998 F65 - + 5424444171 Escherichia coli 0.9997 F66 - + 5665444171 Escherichia coli 0.9962 F67 - + 5464444171 Escherichia coli 0.9998 F68 - + 5464444171 Escherichia coli 0.9998 F69 - + 5435444171 Escherichia coli 0.9803 F70 - + 5464444171 Escherichia coli 0.9998 F71 - + 5464444171 Escherichia coli 0.9998 F72 - + 4464444171 Escherichia coli 0.9813 F73 - + 5464444171 Escherichia coli 0.9998 F74 - + 5724444571 Escherichia coli 0.9999 F75 - + 5465464171 Escherichia coli 0.991 F76 - + 5424444171 Escherichia coli 0.9997 F77 - + 5424444171 Escherichia coli 0.9997 F78 - + 5464444171 Escherichia coli 0.9998 F79 - + 5464444171 Escherichia coli 0.9998 F80 - - 5464444171 Escherichia coli 0.9998 F81 - + 5424444061 Escherichia coli 0.8035 F82 - + 5464044171 Escherichia coli 0.999 F83 - + 5465446071 Escherichia coli 0.9945 F84 - + 5464444171 Escherichia coli 0.9998 F85 - + 5464444171 Escherichia coli 0.9998 F86 - + 5564044171 Escherichia coli 0.9911 F87 - + 5464444171 Escherichia coli 0.9998 F88 - + 5464444171 Escherichia coli 0.9998 F89 - + 5464444171 Escherichia coli 0.9998 F90 - + 5424444171 Escherichia coli 0.9997

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F91 - + 5464444171 Escherichia coli 0.9998 F92 - + 5464444171 Escherichia coli 0.9998 F93 - + 5424444171 Escherichia coli 0.9997 F94 - + 5420444571 Escherichia coli 0.9983 F95 - + 5425444571 Escherichia coli 0.9679 F96 - + 5464444171 Escherichia coli 0.9998 F97 - + 5464444171 Escherichia coli 0.9998 F98 - + 5464444171 Escherichia coli 0.9998 F99 - + 5465464171 Escherichia coli 0.991

F100 - + 5765475577 Klebsiella oxytoca 0.7486

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Appendix A3 Identification of isolates from negative wells from Colilert®-18/Quanti-Tray®

Isolate Ref β-Gal β-Gluc Oxidase Indole Crystal E/NF

profile Identification Confidence

N1 NO GROWTH N2 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 N12 NO GROWTH N13 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

15

N43 NO GROWTH N44 NO GROWTH N45 NO GROWTH N46 NO GROWTH N47 NO GROWTH N48 NO GROWTH N49 NO GROWTH N50 NO GROWTH N51 NO GROWTH N52 NO GROWTH N53 NO GROWTH N54 NO GROWTH

16

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

A Read 1

Analyst A

Read 2

Analyst B

Read

Analyst C

Read Repeatability 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

17

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

A Read 1

Analyst A

Read 2

Analyst B

Read

Analyst C

Read Repeatability Reproducibility

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 0.0000 Sum RSD 0.0014 RSDc 0.0000 RSDc 0.0069

18

Appendix C Replicate counts from suspensions of E. coli, Klebsiella pneumoniae and Citrobacter freundii from Colilert®-18/Quanti-Tray® after 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 freundii Day post manufacture 18 h 22 h %

change 18 h 22 h % change 18 h 22 h %

change 8 2

4 7

2 4 7

0 0 0

12 11 15

12 11 15

0 0 0

11 14 16

11 14 16

0 0 0

15 9 16 8

9 16 8

0 0 0

9 19 13

9 19 13

0 0 0

2 5 7

3 5 7

+ 50 % 0 0

24 14 9

10

14 9

10

0 0 0

14 12 16

14 12 16

0 0 0

14 16 18

14 16 18

0 0 0

30 13 17 11

13 17 11

0 0 0

18 16 13

18 16 13

0 0 0

18 18 8

18 18 8

0 0 0

36 8 13 14

8 13 14

0 0 0

18 13 17

18 13 17

0 0 0

16 15 16

16 15 16

0 0 0

44 8 5 9

8 5 9

0 0 0

24 14 19

24 14 19

0 0 0

11 16 10

12 18 11

+ 9 % + 13 % + 10 %

56 13 4 4

13 4 4

0 0 0

10 16 14

10 16 14

0 0 0

6 7

11

7 14 11

+ 17 % + 100 %

0 84 10

8 8

10 8 8

0 0 0

14 16 19

14 16 19

0 0 0

11 8

12

11 8

12

0 0 0

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 7 11 12

7 11 12

0 0 0

15 20 22

16 20 22

+ 7 % 0 0

16 20 30

16 21 30

0 + 5 %

0 205 10

8 14

10 8

14

0 0 0

17 20 18

17 20 18

0 0 0

18 18 22

18 18 22

0 0 0

269 8 10 10

8 10 10

0 0 0

13 26 19

13 26 19

0 0 0

14 17 14

14 17 14

0 0 0

319 13 18

13 18

0 0

15 17

15 17

0 0

4 13

5 15

+ 25 % + 15 %

19

13 13 0 11 11 0 13 14 + 8 % 367 2

11 10

2 11 10

0 0 0

12 21 20

12 21 20

0 0 0

17 20 21

17 20 21

0 0 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 %

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% Confidence Intervals Theoretical repeatability No. of wells

giving positive reaction

MPN per 100 ml sample

Lower Upper

Repeatability standard deviation Log10SD

Relative standard

uncertainty CV%

0 <1.0 0.0 3.7 - - 1 1.0 0.3 5.6 0.4343 100.0 2 2.0 0.6 7.3 0.3071 70.7 3 3.1 1.1 9.0 0.2508 57.7 4 4.2 1.7 10.7 0.2172 50.0 5 5.3 2.3 12.3 0.1943 44.7 6 6.4 3.0 13.9 0.1774 40.9 7 7.5 3.7 15.5 0.1643 37.8 8 8.7 4.5 17.1 0.1537 35.4 9 9.9 5.3 18.8 0.1450 33.4

10 11.1 6.1 20.5 0.1376 31.7 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 14 16.4 9.8 27.5 0.1166 26.8 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 19 23.8 15.3 37.3 0.1005 23.1 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 23 30.6 20.4 46.3 0.0919 21.2 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 27 38.4 26.4 56.6 0.0856 19.7 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 31 47.8 33.4 69.0 0.0809 18.6 32 50.4 35.4 72.5 0.0799 18.4

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 19.8 49 165.2 115.2 272.2 0.0929 21.4 50 200.5 135.8 387.6 0.1094 25.2 51 > 200.5 146.1 infinite - -