Validation of Colilert®-18/Quanti-Tray® for the Enumeration of E. coli and Coliform Bacteria from Water

January 2008

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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-nitrophenylβ-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 4methyl-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 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 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. βGalactosidase 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 βglucuronidase 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) 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

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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 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

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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 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

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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 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

8

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

9

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

β-Gal

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

β-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 Colilert®18/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

200.5

146.1

infinite

-

-

21