AFNOR CERTIFICATION VALIDATION OF THE METHOD

Transcription

1 NF VALIDATION AFNOR CERTIFICATION VALIDATION OF THE METHOD COLILERT 8 / QUANTI TRAY (ref. Colilert 8 : 6 7 4/ Quanti tray: 6 3 7) Attestation number: IDX 33/ /9 For the enumeration of Escherichia coli and coliforms Protocol for human consumption waters (except bottled waters) SUMMARY REPORT DECEMBER 6 Expert laboratory : Manufacturer : I.S.H.A. IDEXX Laboratories, Inc. 5, avenue de la République IDEXX Drive 93 MASSY Westbrook FRANCE Maine 49 USA This report of analysis concerns only objects subjected to analysis. Its reproduction is authorized only in the form of complete photographic facsimile. It contains 48 pages. Only some assays reported in this document are covered by the accreditation of the Section Laboratory of COFRAC. They are identified by the symbol (*). Assays realized at ISHA : 5 avenue de la République, 93 Massy, France.

2 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Table of contents. Introduction Validation history Validation repositories Alternative method Application scope Reference method (*) Method comparison study Relative accuracy Number and nature of samples Raw results Statistical exploitation Conclusion Linearity Contamination level Raw results Statistical exploitation Conclusion Detection and quantification limits Protocol Results Conclusion Selectivity Protocols Results Conclusion Conclusion Interlaboratory study Study organization Participating laboratories Absence of coliforms in the matrix Strain stability in the matrix Samples preparation and spiking Samples labeling Samples shipping, reception and analysis Results Temperature and state of the samples Enumeration of culturable microorganisms Expert and collaborative laboratories results Interpretation of the results Bias calculation Accuracy profile Conclusion... 6 Institut Scientifique d'hygiène et d'analyse /48

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4 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6. Introduction.. Validation history The method Colilert 8 / QUANTI TRAY has been validated by AFNOR Certification in November 9 with the trademark NF Validation under the attestation number IDX 33/ /9 for the enumeration of Escherichia coli and coliforms. A renewal of the method, without modifications, was conducted in October 3.. Validation repositories The initial validation study of the method Colilert 8 / Quanti Tray was performed according to the validation protocol for an alternative commercial method as compared to a reference method (revision July 8). The renewal study of 3 was performed according to the version of the validation protocol..3. Alternative method Colilert 8 with Quanti Tray is a colorimetric method for detection and enumeration of Escherichia coli and coliforms β glucuronidase positive in human consumption waters. The protocol of the alternative method is as follows and also in appendix. Figure : protocol of the alternative method Step : Add the Colilert 8 reagent to ml of sample Shake until dissolved Pour sample/ reagent mixture into a Quanti Tray Step : Seal in a Quanti Tray Sealer. Step 3 : Place the sealed tray in an incubator at 36± C for 8 to hours Step 4 : Count the number of yellow wells and refer to the MPN table Express results in MPN of coliforms / ml of sample Count the number of yellow and fluorescent wells with a UV lamp at 365 nm and refer to the MPN table Express results in MPN of Escherichia coli / ml of sample.3. Application scope The application scope of the alternative method concerns waters for human consumption..4. Reference method (*) The NF EN ISO 938 (): Water quality Detection and enumeration of Escherichia coli and coliform bacteria Part : Membrane filtration method was used as reference method. The protocol of the reference method is presented in appendix. Institut Scientifique d'hygiène et d'analyse 4/48

5 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6. Method comparison study In 9, two categories of waters have been tested with the reference method and with the alternative method (analysis in duplicate for each method) : tap waters and fountain waters. The two categories were grouped in the category "waters for human consumption" in the revision of the protocol of validation. Thus, only the category waters for human consumption is presented for the relative accuracy parameter. The statistical exploitations in this report doesn t take into account samples with a concentration inferior to 4 CFU/ ml, as stated in the standard ISO Relative accuracy The relative accuracy is the degree of correspondence between the response obtained by the reference method and the response obtained by the alternative method on the same samples.... Number and nature of samples Different types of samples analyzed are summarized in table. Table : number and nature of samples analyzed Targert microorganism Water type Number of samples analyzed Number of samples used Tap water 3 Coliforms Fountain water 9 4 Total 6 44 Tap water 36 Escherichia coli Fountain water 34 7 Total 7 49 Total Escherichia coli and other coliforms 3 93 Globally, 3 samples were analyzed and 93 results were used. No naturally contaminated sample was analyzed. The samples have been artificially contaminated. The contamination levels used cover the entire measurement range of the alternative method.... Raw results Figure presents the two dimensional graphs for the two couples matrix/strain tested and for whole category human consumption waters, respectively in CFU per test portion and in log CFU per test portion. The y axis is reserved for the alternative method and the x axis for the reference method. Raw results are presented in appendix 3. The statistical calculations and graphics were established from results obtained after 48 hours of incubation for the reference method Petri dishes. The representation of a line of equation y = x figures dashed on the graphs. Institut Scientifique d'hygiène et d'analyse 5/48

6 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Figure : two dimensional graphs for relative accuracy (black line: y=x)..3. Statistical exploitation The relationship of relative accuracy between the reference method and the alternative method is evaluated with the linear model: 'y = a + bx '. This formula corresponds to the equation of the linear regression drawn from raw results obtained by experimentation, y representing the alternative method and x the reference method. There is a perfect accuracy (or there is no systematic bias) between the two methods if this equation is equal to the theoretical 'y = x' equation, which applies in the ideal model where the two methods behave similarly. The intercept is theoretically zero in this ideal model (hypothesis [a = ]). The estimated intercept obtained with the two methods is checked using p {a = }. If the alternative method is a systematic bias against the reference method, the probability p {a = } is less than α =.5. Institut Scientifique d'hygiène et d'analyse 6/48

7 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 The 'b' slope is theoretically equal to in the ideal model (hypothesis [b = ]). The estimated slope obtained with the two methods should pass by p {b = }. Statistically, if the alternative method does not give the same values as the reference method, the probability p {b = } is less than α =.5. The linear regression method is chosen over the value of the robustness of the ratio R of overall repeatability standard deviation: If Rob.R >, linear regression by least squares (OLS ) is used with the x axis for the reference method, if Rob.R <.5, a linear regression by least squares (OLS ) is used with the x axis for the alternative method, If.5 < Rob.R <, orthogonal regression (GMFR) is used with the x axis to the reference method. Table : statistical data for the enumeration of E. coli and coliforms Parameter Data Rob.R Regression Probabilities (%) T a t(a) b t(b) Intercept at Slope at used critical E. coli Raw.33 GMFR Log.6 GMFR Coliforms Raw.9 GMFR Log.584 GMFR Table 3 : bias and repeatability of the two parameters Bias (D) Repeatability Parameter Data r Rob.r Mean Median RM AM RM AM Raw E. coli Log Raw Coliforms Log Conclusion The equations for the regression line and the correlation coefficient are as follows: E. coli / Water for human consumption: Alt. =.75 Ref log Alt. =.6 log Ref. +.8 Coliforms / Water for human consumption: Alt. =.57 Ref. 3.3 log Alt. =.6 log Ref..9 The hypothesis [a= and b= ] is accepted for the parameter Escherichia coli. For the parameter coliforms, the hypothesis [a=] is accepted but the hypothesis [b=] is rejected. For this parameter, results obtained for the regression line are corrects, as well as correlation coefficients (.95 in raw data and.939 in log ). The bias calculated between the alternative method and the reference method are at.6 CFU / ml and at.36 log CFU / ml for the parameter E.coli and at.3 CFU / ml and at.6 log CFU / ml for the parameter coliforms. The relative accuracy for the alternative method is satisfactory... Linearity The linearity is the ability of the method when used with a given matrix to give results that are in proportion to the amount of analyte present in the sample, that is an increase in analyte corresponds to a linear or proportional increase in results.... Contamination level The matrix / strain couples are presented in Table 4. For each couple, four contamination levels were tested in duplicate by the reference method and the alternative method. Institut Scientifique d'hygiène et d'analyse 7/48

8 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Table 4 : couples matrix / strain analyzed Strain Matrix Target level (CFU/mL) Escherichia coli ESC.. Tap water Citrobacter freundii CIT..4 Fountain water Raw results Graphs of figure 3 show the values of each sample obtained by the alternative method and the reference method. The y axis is reserved for the alternative method and the x axis for the reference method. Raw results and statistical calculations are summarized in appendix 4. The representation of a line of equation y = x figures dashed on the graphs. Figure 3 : two dimensional graphs for the linearity of the couple Escherichia coli ESC.. / tap water and Citrobacter freundii CIT..4 / fountain water Institut Scientifique d'hygiène et d'analyse 8/48

9 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December Statistical exploitation Statistical interpretations are carried out in accordance with the requirements of standard EN ISO 64. The linear regression method is chosen over the value of the robustness of the ratio R of overall repeatability standard deviation: If Rob.R >, linear regression by least squares (OLS ) is used with the x axis for the reference method, if Rob.R <.5, a linear regression by least squares (OLS ) is used with the x axis for the alternative method, If.5 < Rob.R <, orthogonal regression (GMFR) is used with the x axis to the reference method. Remark: for the couple coliforms / fountain water, the first level of contamination was not exploited because one of the two repetitions presented a result at CFU/ ml with the reference method and gave a result equal to zero after transformation in logarithm. Table 5 : statistical data of couples matrix / strain Strain / Regression Data Rob. R matrix used E. coli / Tap water Coliforms / Fountain water F critical Rob. F P (Rob.F) Correlation coefficient (r) Raw.96 GMFR Log.966 OLS Raw.4 GMFR Log.39 GMFR The relationship between the methods is not linear: if Rob.F > critical F or, if P (Rob.F) < α (=,5)...4. Conclusion The relationship between the two methods, for couples matrix / strain tested, is linear. Correlation coefficients are also satisfactory. The linearity of the alternative method is satisfactory..3. Detection and quantification limits The critical level is defined as the smallest amount that can be detected, but not quantified as an exact value. The detection limit is defined as the level above the critical level. The quantification limit is defined as the smallest amount of analyte that can be measured and quantified with an accuracy and precision defined under the experimental conditions..3.. Protocol The limits of detection and quantification were determined by analyzing a pure culture of Escherichia coli ESC.. and a pure culture of Citrobacter freundii CIT.. by the alternative method..3.. Results Raw results are presented in appendix 5 and the summary in the following tables. Table 6 : data (s and x ) for the enumeration of Escherichia coli Level (CFU/ ml) Number of positive samples Standard deviation (s ) Bias (x ) Institut Scientifique d'hygiène et d'analyse 9/48

10 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Table 7 : LC, LOD and LOQ values of the alternative method for the enumeration of Escherichia coli Parameter Formula Values obtained Critical level (CL).65 s + x.85 Limit of detection (LOD) 3.3 s + x.7 Limit of quantification (LOQ). s + x 5.6 Table 8 : data (s and x ) for the enumeration of coliforms Level (CFU/ ml) Number of positive samples Standard deviation (s ) Bias (x ) Table 9 LC, LOD and LOQ values of the alternative method for the enumeration of coliforms Parameter Formula Values obtained Critical level (CL).65 s + x.4 Limit of detection (LOD) 3.3 s + x.3 Limit of quantification (LOQ). s + x Conclusion The limits of detection and the limits of quantification of the alternative method are satisfactory..4. Selectivity Specificity is defined as the ability of the method to accurately measure a given analyte, or quantity in the sample without interference from non target components. Selectivity is defined as the ability of the method to measure the analyte only..4.. Protocols Fifty target strains, including twenty strains of Escherichia coli, for inclusivity and thirty non target strains for exclusivity (from the collections of national, international and internal of ISHA) were analyzed. The tests were performed according to the protocol of the alternative method. The contamination rates used for inclusivity are between 3 and CFU/ ml. For exclusivity, they are 3 to 5 times the detection concentration of the alternative method (about 4 CFU / ml)..4.. Results Raw results are in appendix 6. For thirty coliform strains tested, twenty five were detected by the alternative method. The five undetected coliform strains by the alternative method were not detected or were not characteristic on the media of the reference method (TTC tergitol). Only one strain of Escherichia coli among twenty strains tested were not detected by the alternative method. Nevertheless this strain was not detected by the reference method. Identifications realized on strains giving discordant results confirmed the identity of strains tested. No non target strain showed cross reaction with the alternative method Conclusion The selectivity of the method is satisfactory. Institut Scientifique d'hygiène et d'analyse /48

11 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6.5. Conclusion The linearity and the relative accuracy of the alternative method are satisfactory. The bias between the reference method and the alternative method is acceptable. The alternative method is specific and selective. Institut Scientifique d'hygiène et d'analyse /48

12 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 3. Interlaboratory study 3.. Study organization 3... Participating laboratories The interlaboratory study was realized by the expert laboratory and thirteen participating laboratories Absence of coliforms in the matrix Before spiking, the absence of coliforms was verified in the batch of water used, according to the reference method Strain stability in the matrix The stability of a Escherichia coli strain in tap water was evaluated for 3 days at 4± C. The strain (code ESC..3) was isolated from an aquatic environment. The numeration results on TTC tergitol after filtration of ml are summarized in table. Table : results of Escherichia coli by the reference method (CFU/ ml ) between D and D Day Level Level Level 3 D 64 D 65 D Samples preparation and spiking The matrix was inoculated with the target strain suspension to obtain four contamination levels: level : CFU / ml, level : from to CFU / ml, level : from to 5 CFU / ml, level 3 : from 5 to CFU / ml The matrix was distributed at ml in sterile bottles. Every bottle was individually spiked and homogenized. Eight samples per laboratory were prepared ( samples per contamination level). Each laboratory received 8 samples to analyze, sample to quantify the endogenous microflora and water sample containing a temperature probe. The results of the enumerations of the heterophilic flora, the target levels and the real levels of contamination are presented in table. Table : target level, real level and culturable microorganisms of the matrix Escherichia coli ESC..3 (CFU/ ml) Level Flora (CFU/mL) Target level Real level to < to to Samples labeling The labelling of the bags was realized as follows: a code to identify the laboratory: from A to M (cf. table ) and a code to identify each sample, only known by the expert laboratory. The samples and the temperature control vials (water sample with a temperature probe) were stored at 4 C before shipping. Institut Scientifique d'hygiène et d'analyse /48

13 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Table : sample code by contamination level Contamination level (CFU / ml) Sample code 3 / 4 / 8 5 / / Samples shipping, reception and analysis The samples were shipped in a coolbox on September 4 th, 9. The coolboxes were received on March 5 th, 9 by the thirteen participating laboratories. The control temperature was recorded upon receipt of the package and the temperature probe sent to the expert laboratory. The samples were analyzed on March 5 th. The expert laboratory concurrently analyzed a set of samples under the same conditions with both methods. 3.. Results 3... Temperature and state of the samples The temperature readings upon reception, the one recorded by the probe and the state of the samples are shown in table 3. Table 3 : temperature and state of the samples upon reception and during the shipping Laboratory Temperature ( C) recorded by the Temperature upon State of the samples probe reception Mean SD A 8, C Good 3,,7 B 6,5 C Good,3, C 3,5 C Good,9,6 D 3, C Good,5,5 E 3, C Good 3,5,5 F 3,3 C Good 3,,7 G 6, C Good,8,7 H 4,4 C Good 3,3,5 I 5,4 C Good,9,6 J 3,9 C Good 3,4,3 K,9 C Good 3,,5 L 3,5 C Good 3,3,8 M 5, C Good,8,8 Temperatures are correct for all laboratories (inferior at 8.4 C) except for the laboratory K. This laboratory showed temperature equal to.9 C. Nevertheless the temperature probe indicated a mean temperature at 3. C. These data are included in the statistical analysis. The analysis of thermal profiles of probes showed, for all participants, mean temperatures during the shipment between.3 and 3.5 C Enumeration of culturable microorganisms For the whole laboratories, the enumeration of culturable microorganisms at C was inferior to CFU/mL. Concerning the enumerations at 36 C, the results were varying between < and CFU/mL Expert and collaborative laboratories results The overall results are presented in table 4 and in the appendix 7. The results of the reference method are presented for a reading of the Petri dishes after 48 h of incubation at 37± C. Institut Scientifique d'hygiène et d'analyse 3/48

14 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 The reading of wells with the alternative method showed an equivalent result between the yellow wells and the fluorescents wells for each sample. Results of alternative method are presented for a reading of yellow and fluorescent wells after incubation 8 hours at 37± C. Data of laboratories A and G were excluded from the final analysis of results: For laboratory A, reading results for fluorescent wells were not reported on the result form. For laboratory G, plates of TTC tergitol superior to CFU have not been counted. Laboratories A and G have been contacted for a second reading but Petri dishes and Quanti Tray had been already eliminated. Table 4 : E. coli enumeration results in CFU/ ml (RM) and MPN/ ml (AM) (RM: reference method, AM: alternative method, R: replicate and R: replicate, *: results of the yellow well only) Level Level Level Level 3 Laboratory RM AM RM AM RM AM RM AM R R R R R R R R R R R R R R R R A* < < > B < < > 3 C < < D < < E < < F < < G < < > > 44 3 H < < I < < J < < K < < > L < < > 3 M < < Expert < < Interpretation of the results The data presented in the following paragraphs were calculated from the raw and logarithmic results, in the same way as for the presentation of the results of the preliminary study Bias calculation Table 5 shows the target value, the mean, the relative bias and the bias of each level of contamination. Table 5 : calculation of the bias of the alternative method Raw data Data log Low Medium High Low Medium High Target value Mean Relative bias % 3.% 4.5% Bias The accuracy of the method is estimated by the bias which varies from.3 to.34 CFU/ ml and from.4 to.93 log CFU/ ml. Institut Scientifique d'hygiène et d'analyse 4/48

15 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December Accuracy profile Table 6 shows the values of tolerance and the tolerance limits of the alternative method for probability of tolerance values of 8%. Figures 4 and 5 show the accuracy profiles. Table 6 : Values and tolerance limits of the alternative method Values in Probability of tolerance Limits of acceptability Level Low Medium High CFU/ ml log CFU/ ml 8% 9% 8%,35 Low tolerance value High tolerance value Low tolerance limit 63% 73% 75% High tolerance limit 6% 6% 88% Low acceptability limit % % % High acceptability limit 9% 9% 9% Low tolerance value High tolerance value Low tolerance limit High tolerance limit Low acceptability limit High acceptability limit Figure 4: Accuracy profile of the alternative method with tolerance probability (β) at 8% and an acceptability limit at 9% % Biais Limite haute tolérance (différence) Limite d'acceptabilité haute Limite basse tolérance (différence) Limite d'acceptabilité basse 5% Biais (%) % Niveaux,, 4, 6, 8,,, 5% % Institut Scientifique d'hygiène et d'analyse 5/48

16 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Figure 5 : Accuracy profile of the alternative method with tolerance probability (β) at 8% and an acceptability limit at 9% (with value in CFU /,7,5 Biais Limite haute tolérance (différence) Limite d'acceptabilité haute Limite basse tolérance (différence) Limite d'acceptabilité basse,3 Biais (%), -,,8,,,4,6,8,, -,3 -,5 -,7 Niveaux The bias of the alternative method increases slightly from the low level to the high level of contamination. For all the contamination levels, the tolerance interval is comprised between the acceptability limits Conclusion The accuracy profile obtained from the results of the reference method and the alternative method shows that the bias of Colilert 8 method with Quanti Tray for the enumeration of β glucuronidase positive Escherichia coli and β galactosidase positive coliforms in drinking water is acceptable. The tolerance limits of the alternative method for a probability of 8% are included within the limits of acceptability of 9% (data: CFU/ ml) or of.35 (data: log CFU/ ml). Institut Scientifique d'hygiène et d'analyse 6/48

17 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 4. Practicability The practicability was evaluated according to the 3 criteria defined by AFNOR Technical Committee. Mode of packaging of test components The Colilert 8 are packaged in single capsule. The Quanti Tray devices are conditioned par ten in aseptic bag. Volume of reagents The Colilert 8 are conditioned par or capsules. 3 Storage conditions of components and shelf life of unopened products The Colilert 8 reagent should be conserved between and 5 C. The Quanti Tray devices should be conserved between 4 and 3 C. 4 Modalities after first use Each Colilert 8 and Quanti Tray serves a unique analysis and should not be reused. 5 Equipment and specific local requirements Quanti Tray Sealer model X. Wood lamp. 6 Reagents ready to use or for reconstitution None. 7 Training period for operator with no experience with the method The duration of training is estimated to be hour. 8 Handling time and flexibility of the method in relation to the number of samples The duration of analysis with the reference method is more important than the duration of analysis with the alternative method. 9 Time required for results The time to results for the alternative method is hours. Concerning the reference method, the time to results for negative samples is between 4 and 48 hours and for positive samples, the time to results is between 48 and 7 hours. Operator qualification Identical as necessary for the reference method Steps common with the reference method None. Traceability of analysis results None. 3 Maintenance by laboratory None. Institut Scientifique d'hygiène et d'analyse 7/48

18 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 5. Conclusion The Colilert 8 / Quanti Tray method for the enumeration of β glucuronidase positive Escherichia coli and β galactosidase positive coliforms in drinking water was compared to the method described in the standard ISO 938 (). Results obtained for the comparative study showed that : the linearity between the two methods is satisfactory, the relative accuracy of the alternative method is satisfactory: values of repeatability obtained are.47 log (E. coli) and.36 log (coliforms) for the alternative method and.3 log (E. coli) and.93 log (coliforms) for the reference method. The bias between the two methods is.36 log for enumeration of E. coli and.6 log for enumeration of coliforms. the selectivity of the alternative method is satisfactory. Results obtained for the interlaboratory study showed that : the bias of the alternative method is satisfactory, the tolerance limits of the alternative method for a tolerance probability at 8 % are comprised in the acceptability limits of 9% (data in CFU/ ml) or of.35 (data in log CFU/ ml). Massy, December 7 th, 6 Olivier Mathia Research Engineer Unit Innovation Biology Institut Scientifique d'hygiène et d'analyse 8/48

19 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Appendix Alternative method Institut Scientifique d'hygiène et d'analyse 9/48

20 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Alternative method Step : Add the Colilert 8 reagent to ml of sample (at room temperature) Close the vial and shake until dissolved Pour sample / reagent mixture into a Quanti Tray Step : Seal the Quanti Tray with a Quanti Tray Sealer Step 3 : Incubate at 36± C for 8 to h Step 4 : For coliforms: Count the number of yellow wells. Refer to the MPN table provided with the trays to obtain a Most Probable Number For E. coli : Count the number of yellow and fluorescent wells Refer to the MPN table provided with the trays to obtain a Most Probable Number Step 5 : Expression of the results : Express the results in MPN of coliforms or E. coli / ml of water Institut Scientifique d'hygiène et d'analyse /48

21 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Appendix Reference method Institut Scientifique d'hygiène et d'analyse /48

22 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Reference method Membrane filtration Filter ml of sample on a membrane Place each membrane on a TTC tergitol 7 agar plate Incubation Incubate at 36± C for ± 3 h + (4±) h if the colonies are not characteristic Reading and confirmation Characteristic colonies : lactose positive Confirm isolated colonies with : Oxydase test and production of indole Expression of the results : CFU/ ml Institut Scientifique d'hygiène et d'analyse /48

23 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Appendix 3 Relative accuracy results Institut Scientifique d'hygiène et d'analyse 3/48

24 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 N Strain Origin Sample ISO 938- Colilert 8/ Quanti-tray 4H 48H Coliforms detection CFU CFU/ ml CFU CFU/ ml E.coli detection (fluo) (yellow) A B A B A B A B A B A B ESC..3 Escherichia coli industrie laitière FR ,7 3,4 ESC..3 Escherichia coli industrie laitière FR45 8 9, 9, 3 ESC..3 Escherichia coli industrie laitière FR ,7 7,5 4 ESC..3 Escherichia coli industrie laitière FR , 6,4 5 ESC..4 Escherichia coli ATCC 8739 FR ,5 88,5 6 ESC..4 Escherichia coli ATCC 874 FR ,6 7 ESC.. Escherichia coli EPA QC, 3594 FB ,7, 8 ESC.. Escherichia coli EPA QC, 8688 FB ,7 9, 9 ESC.. Escherichia coli English, III-8BS FR 3 / ,5,3 ESC.. Escherichia coli English, III-8BS FR ,4 36,4 ESC.. Escherichia coli English, III-8BS FR ,4 3,7 ESC.. Escherichia coli EPA QC, 8688 FR6 75, ,5 8,4 3 ESC.. Escherichia coli EPA QC, 8688 FR ,8 69,7 4 ESC.. Escherichia coli EPA QC, 8688 FR ,4 5,4 5 ESC.. Escherichia coli EPA QC, 8688 FR ,7 5,4 6 ESC.. Escherichia coli CIP 547 FR , 9, 7 ESC.. Escherichia coli CIP 547 FR ,8 78, 8 ESC.. Escherichia coli CIP 547 FR49 3 / , 9,8 9 ESC.. Escherichia coli CIP 547 FR5 & , 5,4 ESC.. Escherichia coli CIP 547 FR5 & ,8 3,8 ESC.. Escherichia coli CIP 547 FR53 & ,9 9, ESC..4 Escherichia coli 3 ESC..4 Escherichia coli 4 ESC..4 Escherichia coli Pâte blanche sortie mélange Pâte blanche sortie mélange Pâte blanche sortie mélange E.coli FOUNTAIN WATERS Target Concentration CFU/mL dilution FR54 6 / ,,5 FR ,9 59, FR ,4, 5 ESC..7 Escherichia coli Eau de puits FR ,3 83, 6 ESC..7 Escherichia coli Eau de puits F ,7 3,8 7 ESC..7 Escherichia coli Eau de puits FB ,7 4, 8 ESC..7 Escherichia coli Eau de puits FB , 6,4 A: repetition B: repetition Institut Scientifique d'hygiène et d'analyse 4/48

25 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 E.coli TAP WATERS N Strain Origin Sample A B A B A B A B A B A B ESC..4 Escherichia coli ATCC 874 ED ,4 8,8 ESC..4 Escherichia coli ATCC 874 ED , 6,4 3 ESC..9 Escherichia coli 4 ESC..9 Escherichia coli 5 ESC..9 Escherichia coli 6 ESC..9 Escherichia coli 7 ESC..37 Escherichia coli 8 ESC..37 Escherichia coli 9 ESC..37 Escherichia coli ESC..37 Escherichia coli Eau de distribution : douchette bac préparation Eau de distribution : douchette bac préparation Eau de distribution : douchette bac préparation Eau de distribution : douchette bac préparation Déchets de pâte recyclés Déchets de pâte recyclés Déchets de pâte recyclés Déchets de pâte recyclés ED , ED ,8 7, ED ,,7 ED ,3 6,4 ED ,3 53, ED ,7 ED , ED ,3 ESC.. Escherichia coli English, III-8BS ED ,4 ESC.. Escherichia coli English, III-8BS ED , 9, 3 ESC.. Escherichia coli English, III-8BS ED3 7 3, 3, 4 ESC.. Escherichia coli EPA QC, 359 ED ,,3 5 ESC.. Escherichia coli EPA QC, 3593 ED ,8 38,4 6 ESC..7 Escherichia coli Eau de puits D6 b , 6,4 7 ESC.. Escherichia coli Effluent secondaire D ,4 3 8 ESC.. Escherichia coli Effluent secondaire D ,7 94,5 9 ESC.. Escherichia coli Effluent secondaire D ,5 9,8 ESC..3 Escherichia coli Eau de puits D8 8 3, 5,3 ESC..39 Escherichia coli ESC..39 Escherichia coli 3 ESC..39 Escherichia coli 4 ESC..39 Escherichia coli 5 ESC..3 Escherichia coli 6 ESC..3 Escherichia coli 7 ESC..3 Escherichia coli 8 ESC..3 Escherichia coli Crevette crue décortiquée (isol. TBX) Crevette crue décortiquée (isol. TBX) Crevette crue décortiquée (isol. TBX) Crevette crue décortiquée (isol. TBX) Noix de St-Jacques (isol. TBX) Noix de St-Jacques (isol. TBX) Noix de St-Jacques (isol. TBX) Noix de St-Jacques (isol. TBX) Target Concentration CFU/mL dilution D4 4 / ,4, D5 99 / 5,3 7,5 D7 65 / 5 8,7 D8 / 5 7,5 D4 5 / , 65, D5 /,3 9,8 D7 / 65, 8,4 D ,,3 9 ESC.. Escherichia coli Eau de fontaine D7 85 / 73,8 65,9 3 ESC.. Escherichia coli Eau de fontaine D8 56 / ,4 53, 4H ISO H CFU CFU/ ml CFU CFU/ ml Coliforms detection (yellow) Colilert 8/ Quanti-tray E.coli detection (fluo) A: repetition B: repetition Institut Scientifique d'hygiène et d'analyse 5/48

26 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Coliformes FOUTAIN WATERS ENTB.. Enterobacter aerogenes ISO 938- Colilert 8/ Quanti-tray 4H 48H Coliforms detection CFU CFU/ ml CFU CFU/ ml E.coli detection (fluo) (yellow) A B A B A B A B A B A B Industrie laitière ED 5 5,3 5,3 < < CIT.. Citrobacter diversus CIP 8.87T F ,8 47,8 < < 3 CIT.. Citrobacter diversus CIP 8.87T F , 65, < < 4 BUT.. 5 ENTB.3. Buttiauxella agrestis Enterobacter sakazakii Eau de puits F < < CIP F ,7, < < 6 CIT.. Citrobacter diversus CIP 8.87T F , 7, < < 7 CIT.. Citrobacter diversus CIP 8.87T F ,4 47,8 < < 8 CIT.. Citrobacter diversus CIP 8.87T F ,,3 < < 9 ESC.. Escherichia hermanii CIP 376 F ,7 9, < < KLE.. Klebsiella oxytoca Effluent secondaire F65 95 / , 65, < < KLE.. Klebsiella oxytoca Effluent secondaire F ,9 73,8 < < ENTB ENTB ENTB ENTB.3.3 N Strain Origin Sample Enterobacter sakazakii Enterobacter sakazakii Enterobacter sakazakii Enterobacter sakazakii Target Concentration CFU/mL dilution CIP 358 F64 / ,5 59, < < CIP 358 F ,8,7 < < CIP 358 F ,7 5,4 < < CIP 358 F ,,4 < < 6 KLE.. Klebsiella oxytoca Salade de soja F7 / , 65, < < 7 KLE.. Klebsiella oxytoca Salade de soja F , 69,7 < < 8 KLE.. Klebsiella oxytoca Salade de soja F ,9 4,6 < < 9 KLE.. Klebsiella oxytoca Salade de soja F ,4, < < KLE.3. Klebsiella planticola Saucisse F7 9 / , 44,5 < < KLE.3. Klebsiella planticola Saucisse F ,4 73,8 < < KLE.3. Klebsiella planticola Saucisse F ,8 3,4 < < 3 KLE.3. Klebsiella planticola Saucisse F ,, < < 4 KLU.. Kluyvera sp. EPA F7 33 / ,9 < < 5 KLU.. Kluyvera sp. EPA F7 / ,9 < < A: repetition B: repetition Institut Scientifique d'hygiène et d'analyse 6/48

27 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Coliformes TAP WATERS ENTB.. ENTB.. 3 ENTB.. 4 ENTB.. 5 ENTB.. 6 ENTB.. 7 ENTB.. 8 ENTB..3 9 ENTB..3 ENTB..3 ENTB.. ENTB.. 3 ENTB.. 4 ENTB.. Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter aerogenes Enterobacter cloacae Enterobacter cloacae Enterobacter cloacae Enterobacter cloacae ISO 938- Colilert 8/ Quanti-tray 4H 48H Coliforms detection CFU CFU/ ml CFU CFU/ ml E.coli detection (fluo) (yellow) A B A B A B A B A B A B Industrie laitière ED 5 5,3 5,3 < < Industrie laitière ED ,3 < < Industrie laitière ED3 3 3, 4, < < CIP 6.86T ED , 6,4 < < CIP 6.86T ED ,7 9,9 < < CIP 6.86T ED7 6 9,9 7,5 < < CIP 6.86T ED ,3 4, < < 466:8 Thames isolate # :8 Thames isolate # :8 Thames isolate #358 ED , 3,7 < < ED ,4 6,4 < < ED 4 < < Eaux usagées ED , 4,6 < < Eaux usagées ED ,7 < < Eaux usagées ED ,9 9,9 < < Eaux usagées ED , 4, < < 5 CIT.. Citrobacter koseri CIP 7. ED9 8 / ,5 9,8 < < 6 CIT.. Citrobacter koseri CIP 7. ED ,8 7,8 < < 7 CIT.. Citrobacter koseri CIP 7. ED 9 / ,8 94,5 < < 8 CIT.. Citrobacter koseri CIP 7. ED ,8 38,4 < < 9 BUT.. BUT.. Buttiauxella agrestis Buttiauxella agrestis Eau de puits ED ,5,5 < < Eau de puits ED , 44,5 < < CIT.. Citrobacter freundii ATCC 89 ED 5 / ,3 65, < < CIT.. Citrobacter freundii ATCC 89 ED ,9 83, < < 3 CIT.. Citrobacter freundii ATCC 89 ED , 69,7 < < 4 EWI.. 5 EWI.. N Strain Origin Ewingella americana Ewingella americana JMM3, env. Sample, 4569: JMM3, env. Sample, 4569: A: repetition B: repetition Sample Target Concentration CFU/mL dilution D3 / ,5,3 < < D34 67 / ,3,3 < < Institut Scientifique d'hygiène et d'analyse 7/48

28 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Exactitude relative - Escherichia coli - Eau de consommation humaine Méthode de référence (NF ISO 938-) Méthode alternative (Quanti-tray ) Ech. Rep. Rep. M SD Rep. Rep. M SD Différence Est. y Dév ,5,,7 3,4 6,55 8,73-4,95 33,95-7,4,,44 9, 9, 9,, 8,,95 7, , 5,657 3,7 7,5,6 4,384,6 8,679, , 4,43,5 88,5 44,5 79,96 6,5 89,333 55, ,5, 56 4,6 48,3,889,8 5,8 -, ,5 4,95,7,,45,6 3,95 8,895, ,5,,7 9, 9,95,6-4,55 6,43-6, , 4,4 8,5,3 9,9,6 49,9 64,599 45, ,,88 36,4 36,4 36,4, -5,6 66,749-3, ,5 7,778,4 3,7 3,5,99 -,45 5,669 -, , 7,7 44,5 8,4 3,45 8,455-53,55 99, -67, ,5 3,536 73,8 69,7 7,75,899 9,5 67,87 4, ,5,77 5,4 5,4 5,4, -8, 36, -, ,5 3,536 3,7 5,4 9,55 8,73,5 8,895, ,5 7,778 65, 9, 37,5 39,669 9,65 6,433, ,5 3,536 73,8 78, 76, 3, 5,5 75,89, 7 3 5,,3 65, 9,8 47,5 5,3 3,5 3,745 3, , 5,657 53, 5,4 5,75,99 -,5 58,46-6, ,5, 8,8 3,8 6,3 3,536 3,8 4,7,8 6 9, 4,43 9,9 9, 4,55 6,576-4,45,58-5, , 4,43 65,,5 8,85 4,96 3,85 7,6, , 5,657 65,9 59, 6,5 4,88 4,5 6,448, ,5 6,364 3,4, 7,3 7,,8 7,498 -, ,5 7,778 45,3 83, 64, 6,79 4,7 64,6,39 5 8,,88,7 3,8,5,9,5,583, ,,44 3,7 4, 8,95 6,78-7,5 7,8-8, ,,44 4, 6,4 5,3,556 -,7 8,679-3, ,5,77 36,4 8,8 3,6 5,374, 4,7 8, ,,88, 6,4 9,3 4,,3 9,754 9, , 7, , 57,55,9 6,55 54,9, ,5 7,778 8,8 7, 7,95,,45 9,649 -, ,,88,,7 5,9 6,788 3,9,98, ,, 5,3 6,4 5,85,778,85 4,377, , 7,7 5 3,7 4,35,99,35 5,3 -, ,5, 5 6,4 5,7,99, 5,669, ,,88, 9, 5,5 5,78 6,5 9,754 5, ,5, 9,,3 5, 5,55 4,7 97,398 7, ,5 6,364 8,8 38,4 33,6 6,788-4,9 5,3-8, ,, 5,4 3 7,7 3,53 8,7,58 7, , 4,43 69,7 94,5 8, 7,536 3, 63,53 8, ,5 6,364 94,5 9,8,5 4,96 4,65 5,68-3, ,,3,4,,75,99-3,5 6,96-5, 43 5, 7,7 5 8,7,85 4,455-3,5 6,6-4, , 35,355 65, 65, 65,, -9,8 9,776-44, , 7,7,3 9,8 5,55,53,55 3,745-8,95 46, 4,4 65, 8,4 4,8 33,93 3,8 8,368 3, , 7,7 83,,3 9,,869 33, 63,53 8,677 48,, 73,8 65,9 69,85 5,586-3,5 7,64-37, , 7,7 5,4 53, 5,75,99 6,75 48,468 3,8 Mx= 5, My= 56,6 4,4 MEDx= 33,5 MEDy= 3,6,6 q= 49 SDbx= 46,739 SDby= 49,83 Biais n= MEDwx = 4,43 MEDwy = 4,88 N=qn= 98 SDwx= 8,698 SDwy= 6,693 rob. SDwx= 6,9 rob. SDwy= 7,9 Choix de la méthode GMFR R=,99 rob. R=,33 Sx= 46,97 Sy= 5,443 r=,94 b=,75 Res. SEM= 9,574 a=,76 Res. SD= 7,68 S(b)=,6 S(a)= 4,4 p(t;b=)=,4 p(t;a=)=,986 t(b)=,5 t(a)=,8 Répétabilité MR MA r 4,356 46,74 rob. r 7,6 9,96 Méthode alternative (log NPP/mL) Les points représentés correspondent aux moyennes des répétitions de chaque échantillon Une courbe d'équation y = x figure en pointillés Méthode de référence ( log UFC/mL) Institut Scientifique d'hygiène et d'analyse 8/48

29 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Exactitude relative - Escherichia coli - Eau de consommation humaine Méthode de référence (NF ISO 938-) Méthode alternative (Quanti-tray ) Ech. Rep. Rep. M SD Rep. Rep. M SD Différence Est. y Dév.,477,59,498,9,36,5,43,38 -,85,55 -,,79,,4,56,83,83,83,,44,64,9 3,6,79,84,337,37,875,6,85,65,864,4 4,934,93,99,,3,947,5,5,6,949,76 5,68,653,667,,748,69,678,99,,696 -,7 6,46,3,34,5,36,346,33,,97,6,7 7,36,45,388,38,36,83,3,3 -,89,45 -,5 8,845,699,77,3,74,6,4,48,68,8,39 9,86,778,79,,56,56,56, -,3,8 -,6,954,3,8,45,93,37,5,3 -,3,53 -,38,55,79,67,7,6,73,7,6 -,5,99 -,83,83,778,796,5,868,843,856,8,6,85,3 3,53,59,55,9,45,45,45, -,,553 -,48 4,76,3,39,88,37,45,7,9,3,64,7 5,49,9,7,9,8,38,8,7,58,,7 6,833,863,848,,868,893,88,8,33,877,3 7,,4,57,8,8,3,66,74,9,88,78 8,763,699,73,46,75,7,74,6 -,7,76 -,46 9,3,38,35,4,459,377,48,59,67,378,4,4,34,73,98,996,83,39,3 -,34,99 -,6,93,,,,8,3,6,59,59,33,7,73,79,76,4,89,77,795,33,33,79,4 3,477,3,4,,5,346,48,6,9,46, 4,83,73,773,57,656,9,788,86,5,8 -,4 5,55,34,99,6,36,377,346,43,47,35, 6,3,76,3,38,37,63,88,363 -,33,9 -,349 7,845,954,9,77,63,86,75,9 -,85,93 -,9 8,36,34,35,4,56,459,5,7,58,378,3 9,4,845,943,39,346,5,8,93,337,967,34 3,748,663,75,6,748,77,76,7,54,734,6 3,34,59,43,5,459,433,446,9,6,457 -, 3,,46,73,3,45,36,8,9,8,98,83 33,6,6,6,,74,86,765,58,63,64,4 34,954,79,6,9,76,37,56,8,4,4,5 35,4,4,59,64,76,5,95,7,36,84, 36,4,845,943,39,45,83,64,68,,967,97 37,94,996,955,58,38,6,,3,67,985,37 38,74,643,684,57,459,584,5,88 -,6,7 -,9 39,79,79,79,,45,477,44,5,6,35,36 4,79,748,77,3,843,975,99,93,39,799, 4,49,3,3,6,975,3,44,97,3,6 -,7 4,6,,3,46,93,45,69,34 -,3,37 -,58 43,,3,5,3,76,94,58,67 -,93,76 -,8 44,3,34,86,79,8,8,8, -,68,39 -, 45,79,4,6,7,6,3,59,76 -,,9 -,3 46,79,,4,56,8,73,46,,6,7,75 47,86,73,769,5,9,6,963,6,93,798,64 48,,,,,868,89,843,35 -,57,3 -,87 49,699,6,65,69,7,75,74,6,63,679,35 Mx=,536 My=,564,8 MEDx=,55 MEDy=,5,36 q= 49 SDbx=,44 SDby=,48 Biais n= MEDwx =,56 MEDwy =,59 N=qn= 98 SDwx=,5 SDwy=, rob. SDwx=,83 rob. SDwy=,88 Choix de la méthode GMFR R=,97 rob. R=,6 Sx=,4 Sy=,43 r=,945 b=,6 Res. SEM=,4 a=,8 Res. SD=,99 S(b)=,48 S(a)=,77 p(t;b=)=,896 p(t;a=)=,86 t(b)=,3 t(a)=,33 Répétabilité MR MA r,3,3 rob. r,3,47 Méthode alternative (log NPP/mL) Les points représentés correspondent aux moyennes des répétitions de chaque échantillon Une courbe d'équation y = x figure en pointillés Méthode de référence ( log UFC/mL) Institut Scientifique d'hygiène et d'analyse 9/48

30 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Exactitude relative - Coliformes - Eau de consommation humaine Méthode de référence (NF ISO 938-) Méthode alternative (Quanti-tray ) Ech. Rep. Rep. M SD Rep. Rep. M SD Différence Est. y Dév.,5,77 5,3 5,3 5,3, -6,,94-4, , 9,899 59, 69,7 64,4 7,495 -,6 74,55 -,5 3 5,5 3,536 7, 6,4,75 7,566 -,75 3, -, ,5,77 8,7 9,9 9,3,849-5, 3,765-4, ,5,77 5,3 4, 4,75,778,5,94, ,,44 9, 3,7 6,45 3,889 -,55 7,85 -, ,, 6,4 6,4 6,4, -,6 6,44, , 49,497 44,5,3,9 3,547 -, 4,63-8, ,5 7,778 53, 4,6 46,85 8,839-3,65 55,4-8,57 6 8,,88 5 8,7,85 4,455-6,5 7,85-5, ,5 4,95 9,9 9,9 9,9,,4 6,8 3, , 7,7,5 9,8 65,5 49,99 3,5 53,, ,5, 7,8 7,8 7,8, 7,3 9,37 8, , 35,355 73,8 94,5 84,5 4,637 9,5 83,77, ,5, 47,8 38,4 43, 6,647,6,78, ,,44,5,5,5, 74,5 4,787 57, ,5 3,536 9, 44,5 6,8 5,3 73,3 58,894 67, , 35,355,3 65, 33,5 45,84 -,75 53, -9, ,5 9,9 65,9 83, 74,5,6-3, 98,37-3, , 7,7,3,3,3, 6,3 83,77 7,58,5,77 5,3 5,3 5,3, -6,,94-4, , 4,43 73,8 47,8 6,8 8,385 -, 79,44-8, ,, 65, 65, 65,, 3, 84,444-9, ,, ,5 3,536-5,5 6,63-3, , 7,7,7, 5,9 6,788-4, 3,7-5, ,5,77 9, 7, 3,5 5,586 8,65 3,765 9, , 4,43 36,4 47,8 4, 8,6 6, 7,7 5, ,5,77 78,,3 89,75 6,334 35,5 6,5 9, ,,44 8,7 9, 3,95 7,45 -,5 4,344 -, , 4,46 9, 65, 37,5 39,669 7,5 35,844, , 8,485 65,9 73,8 69,85 5,586 5,85 7,44 -, , 4,4 88,5 59, 73,8,789-6, 89,558-5, ,, 8,8,7 4,75 5,78-3,5 4,958-6, , 5,657,7 5,4 3,5 3,33,5,444, , 5,657,,4,75,99,75 9,75, , 49,497 65, 65, 65,, 5, 3,58 35, ,5 3,536 53, 69,7 6,4,738 4,9 6,365 -, ,,44 34,4, 8,3 8,67,3 9,387 -, , 56,569 65, 44,5 54,85 4,637-5,5 5,7-5, ,5,77 6,4 73,8 68, 8,6 8,6 65,837, ,5 7,778 3,8 3,4 8, 6,8 7,6,78 7, ,5,,,,, -4,4 4,9-3, ,, 56 65,9 6,95 7,,95 54,844 6, , 7,7 56 4,9 49,45 9,63-5,55 7, -,75 Mx= 55,39 My= 6,96 5,667 MEDx= 5,5 MEDy= 44,975,75 q= 44 SDbx= 46,5 SDby= 54,4 Biais n= MEDwx = 3,536 MEDwy = 6,78 N=qn= 88 SDwx= 7,433 SDwy= 5,96 rob. SDwx= 5,4 rob. SDwy= 9,959 Choix de la méthode GMFR R=,877 rob. R=,9 Sx= 47,59 Sy= 54,998 r=,95 b=,57 Res. SEM=,6 a= -3,3 Res. SD= 9,785 S(b)=,68 S(a)= 4,9 p(t;b=)=, p(t;a=)=,54 t(b)=,35 t(a)=,65 Répétabilité MR MA r 48,83 4,88 rob. r 4,677 7,886 Méthode alternative (log NPP/ ml) Les points représentés correspondent aux moyennes des répétitions de chaque échantillon Une courbe d'équation y = x figure en pointillés Méthode de référence (log UFC/ ml) Institut Scientifique d'hygiène et d'analyse 3/48

31 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Exactitude relative - Coliformes - Eau de consommation humaine Méthode de référence (NF ISO 938-) Méthode alternative (Quanti-tray ) Ech. Rep. Rep. M SD Rep. Rep. M SD Différence Est. y Dév.,79,4,6,7,74,74,74, -,336, -,77,869,778,84,64,77,843,87,5 -,6,86 -,54 3,3,398,349,69,433,5,34,54 -,6,37 -,3 4,76,46,6,,94,996,968,4 -,94,5 -,48 5,699,6,65,69,74,63,674,7,3,54,34 6,3,79,55,34,83,37,,4 -,45, -, 7,93,93,93,,86,86,86, -,97,85 -,8 8,4,954,79,77,6,6,83,9,4,49 -,66 9,653,748,7,67,75,69,667,8 -,34,73 -,56,4,3,53,69,76,94,58,67 -,95,8 -,6,699,79,889,69,996,996,996,,7,89,87,4,46,3,3,3,3,8,34,78,6, 3,954,79,7,88,5,5,5,,34,953,98 4,699,,849,3,868,975,9,76,7,89,3 5,79,34,3,45,679,584,63,67,3,84,348 6,97,4,,5,3,3,3,,,73,9 7,748,78,78,9,38,6,99,86,37,753,345 8,4,4,3,5,6,8,,5 -,,98 -,86 9,973,98,94,46,89,9,869,7 -,7,993 -,4,93,845,874,4,6,6,6,,3,98,87,79,4,6,7,74,74,74, -,336, -,77,833,869,85,6,868,679,774,33 -,77,89 -,8 3,,,,,8,8,8,,8,96 -,78 4,73,748,74,,55,43,468,5 -,7,767 -,99 5,544,398,47,3,36,45,8,9 -,9,464 -,84 6,76,46,6,,83,433,358,6,97,5,43 7,46,36,4,7,56,679,6,84,8,398, 8,74,73,736,6,893,6,949,79,3,763,86 9,46,4,75,4,94,83,,43 -,64,3 -, 3,76,954,65,57,38,8,8,7,63,33 -,5 3,845,763,84,58,89,868,843,35,39,839,4 3,845,954,9,77,947,77,859,4 -,4,947 -,88 33,58,58,58,,459,36,388, -,9,587 -,99 34,55,45,335,3,36,45,36,63,5,3,49 35,845,76,,34,45,93,69,34,59,946,4 36,76,93,4,93,8,8,8,,78,4,4 37,73,77,75,7,75,843,784,84,33,78,4 38,43,46,447,,537,346,44,34 -,5,437,4 39,34,46,44,39,8,6,89,4 -,55,335 -,46 4,778,77,775,5,795,868,83,5,57,86,6 4,45,76,96,69,377,5,444,95,48,67,77 4,46,3,88,6,45,45,45, -,43,46 -, 43,699,699,699,,748,89,784,5,85,7,63 44,845,778,8,47,748,63,69,8 -,,848 -,57 Mx=,567 My=,573,5 MEDx=,7 MEDy=,649,6 q= 44 SDbx=,44 SDby=,468 Biais n= MEDwx =,47 MEDwy =,74 N=qn= 88 SDwx=,97 SDwy=,94 rob. SDwx=,69 rob. SDwy=,9 Choix de la méthode GMFR R=,97 rob. R=,584 Sx=,48 Sy=,47 r=,939 b=,6 Res. SEM=,65 a= -,9 Res. SD=,34 S(b)=,6 S(a)=,98 p(t;b=)=,4 p(t;a=)=,5 t(b)=,9 t(a)=,967 Répétabilité MR MA r,7,64 rob. r,93,36 Méthode alternative (log NPP/ ml) Les points représentés correspondent aux moyennes des répétitions de chaque échantillon Une courbe d'équation y = x figure en pointillés Méthode de référence (log UFC/ ml) Institut Scientifique d'hygiène et d'analyse 3/48

32 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Appendix 4 Linearity results Institut Scientifique d'hygiène et d'analyse 3/48

33 IDEXX - Colilert-8 / Quanti-Tray Summary report - V December 6 Linéarité - Résultats bruts Linéarité Escherichia.coli Taux NF ISO 938- d'inoculation Matrice Souche Origine Répétition Répétition Répétition Répétition Moyenne (UFC/ ml) UFC/ boîte UFC/ boîte UFC/ ml UFC/ ml Eau de Escherichia coli ESC.. Effluent secondaire 5 distribution 3 3 7, ,5 Taux Quanti-tray d'inoculation Matrice Souche Origine Répétition Répétition Moyenne (UFC/ ml) UFC/ ml UFC/ ml 5, Eau de 69,7 94,5 94,5 Escherichia coli ESC.. Effluent secondaire 5 distribution 94,5 9,8 9,8,5,5,5 Taux NF ISO 938- d'inoculation Matrice Souche Origine Répétition Répétition Répétition Répétition Moyenne (UFC/ ml) UFC/ boîte UFC/ boîte UFC/ ml UFC/ ml Eau de fontaine Citrobacter freundii CIT.4 Eau de puits Taux Quanti-tray d'inoculation Matrice Souche Origine Répétition Répétition Moyenne (UFC/ ml) UFC/ ml UFC/ ml,5 3 7,8 3,7 5,75 Eau de fontaine Citrobacter freundii CIT.4 Eau de puits 5, 6,4 9,3 5, , Institut Scientifique d'hygiène et d'analyse 33/48