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Proyecto: Metrologia Química

Validation and Estimation of Measurement Uncertainty in Microbiology . Proyecto: Metrologia Química. Validation of test methods in chapter 4. EA Guide 4/10 . Use of natural products or spiked materials. Qualitative tests. Relative truness, comparison against reference method.

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Proyecto: Metrologia Química

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  1. Validation and Estimation of Measurement Uncertainty in Microbiology Proyecto: Metrologia Química

  2. Validation of test methods in chapter 4 EA Guide 4/10 Use of natural products or spiked materials Qualitative tests Relative truness, comparison against reference method Specifity, false positives, false negatives Repeatability Reproducibility

  3. Validation of test methods in chapter 4 EA Guide 4/10 Use of natural products or spiked materials Quantitative tests Relative truness, comparison against reference method Specifity, false positives, false negatives LOD Repeatability Reproducibility

  4. Incertidumbre de las mediciones in Microbiología Muestreo Lecturas (personal) Uniformidad de la distribución de los microbios Cálculo de cfu‘s, Distribución de Poisson Pesas usadas + volumenes + diluciones Fuentes de incertidumbre en Microbiología Coeficiente de confirmación Mezclado adecuado Condiciones de incubación Desempeño del medio Efectos de la muestra Equipos, e.g. spiral plater, exactitud Tiempo hasta la inoculación

  5. MU – Normal- and Poisson distribution In chemical testings quite often the normal distribution is used to quantify measurement uncertainties … Pay attention: Microorganisms hate statistics …

  6. But as singles we are behaving as .... Poissoins.

  7. Normas Legales Referencias bibliográficas aceptadas Resultado de ensayos de aptitud Gráficos de control y datos de validación Cuantificación de acuerdo a la Niemela et al., Finland Experiencia de expertos

  8. Incertidumbre de las mediciones en Microbiología Ideas Acreditación de laboratorios microbiológicos (EA-Guide 4/10) ISO 7218 Microbiología de alimentos y alimentos para animales - Reglas generales para exámenes microbiológicos ISO/TS 19036 No. 19036, released 2006-02 Microbiology of food and animal feeding stuffs - Guide on estimation of measurement uncertainty for quantitative determinations

  9. Incertidumbre de las mediciones en Microbiología Ideas ISO 7218 Microbiología de alimentos y alimentos para animales - Reglas generales para exámenes microbiológicos Provides statistical data for colony counts and MPN

  10. cfu per plate Confidence level 95% 1 < 1 to 6 (-97% bis +457%) 2 < 1 to 7 (-88% bis +261%) 3 < 1 to 9 (-79% bis +192%) 4 1 to 10 (-73% bis +156%) 5 2 to 12 (-68% bis +133%) 10 5 to 18 (-52% bis +84%) 15 8 to 25 (-44% bis +65%) Uncertainties for colony counts for single plate measurements according to ISO 7218

  11. and in addition ISO/TS 19036….. ISO / Technical Specification No. 19036 – 2006-02 Microbiology of food and animal feeding stuffs - Guide on estimation of measurement uncertainty for quantitative determinations

  12. Philosophy of ISO/PDTS 19036 Global approach for the estimation of measurement uncertainty 5.1 Combined Standard Uncertainty 5.2 SD of reproducibility 5.4 General rules for the estimation of SD of reproducibility 5.3 Expanded Uncertainty • Intra-laboratory SD • of reproducibility • Reproducibility SD of method • derived from an ILC study Expression of measurement uncertainty in test reports

  13. Maximum r = 0,27 Avarage r = 0,087 Avarage r = 0,087

  14. Incertidumbre de las mediciones en microbiología Ejemplo: Resultado de EAs, cuentas totales a 36 °C No. Fecha Valor asignado (cfu/ml) Resultado Lab (cfu/ml) Valor medio derecuperación en % Operador 01 02 03 04 05 06 07 08 09 10 11 12 31.12.93 22.02.94 30.05.94 17.01.95 02.06.95 28.06.96 18.12.96 21.05.97 29.04.98 11.12.98 01.03.99 07.06.99 J J J JJJJJJJJJ 132 136 66 149 30 33 273 49 83 299 80 256 140 149 78 120 26 26 230 48 74 301 98 260 106 110 118 81 87 79 84 98 89 101 123 102

  15. Incertidumbre de las mediciones en microbiología Ejemplo: Resultado de EAs, cuentas totales a 36 °C No. Fecha Operador Resultados Lab (cfu/ml) Valor asignado (cfu/ml) Valor medio derecuperación en % 13 14 15 16 17 18 19 20 21 22 07.12.99 21.02.00 19.05.00 29.08.00 02.03.01 20.06.01 21.09.01 30.11.01 13.03.02 27.05.02 J J J JJJJJJJ 118 15 18 22 139 172 15 52 51 39 132 12 20 22 150 174 18 55 45 46 112 80 111 100 108 101 120 106 88 118 98 %  15 % Incertidumbre combinada:  15 % Incertidumbre expandida :  30 % Rec.: SD:

  16. Incertidumbre en Microbiología 20 medidas de la misma muestra Ejemplo: Conteo total de bacterias en hamburguesas Resultados Log10 1.1 x 104 4.041.1 x 104 4.041.1 x 104 4.041.1 x 104 4.041.1 x 104 4.043.0 x 105 4.474.8 x 104 4.682.7 x 105 5.437.8 x 103 3.896.7 x 104 4.831.9 x 104 4.288.0 x 103 3.90 2.2 x 104 4.341.2 x 105 5.101.2 x 104 4.101.5 x 105 5.181.7 x 104 4.233.0 x 104 4.480.5 x 104 3.702.0 x 104 4.30 Media 4.4 x 104 cfu/g SD 0.52 x 104 cfu/g SD Relativa Incertidumbre Expandida Uexpanded = ± 24% Resultado 4.4 ± 1.1 x 104 cfu/g 4.4 x 104± 24% cfu/g

  17. Estimated Measurement Uncertaintiesfrom Food Industry • Listeria monocytogenes: ± 0.20 log CFU/ml • Aerobic mesophilic counts: ± 0.15 log CFU/ml • Bacillus cereus: ± 0.20 log CFU/ml • Coliforms: ± 0.25 log CFU/ml • Enterobacteriacea: ± 0.25 log CFU/ml • Staphylococcus aureus: ± 0.20 log CFU/ml • Yeast: ± 0.20 log CFU/ml • Aerobic mesophilic spores: ± 0.25 log CFU/ml • Clostridium perfringens: ± 0.20 log CFU/ml

  18. Incertidumbre de medición estimadas en la industria alimentaria • Listeria monocytogenes: ± 0.20 log CFU/ml • Aerobic mesophilic counts: ± 0.15 log CFU/ml • Bacillus cereus: ± 0.20 log CFU/ml • Coliforms: ± 0.25 log CFU/ml • Enterobacteriacea: ± 0.25 log CFU/ml • Staphylococcus aureus: ± 0.20 log CFU/ml • Levadura: ± 0.20 log CFU/ml • Esporas Aeróbicas mesofilas: ± 0.25 log CFU/ml • Clostridium perfringens: ± 0.20 log CFU/ml

  19. Once woke up, now also mikrobiologists have ideas ... about measurement uncertainties …

  20. Incertidumbre de las mediciones in Microbiología Muestreo Lecturas (personal) Uniformidad de la distribución de los microbios Cálculo de cfu‘s, Distribución de Poisson Pesas usadas + volumenes + diluciones Fuentes de incertidumbre en Microbiología Coeficiente de confirmación Mezclado adecuado Condiciones de incubación Desempeño del medio Efectos de la muestra Equipos, e.g. spiral plater, exactitud Tiempo hasta la inoculación

  21. = + + + + + + + + 2 2 2 2 2 2 2 2 2 w w w w w w w w w w y K K K K K F T X V H S A M L Multiple-plate instrument A microbial test result uncertainty calculation might be concidered „completely corrected“ if the following factors have been applied (of course, if relevant) KH=personal yield coefficient KS = sample stability coefficient KA = yield of medium coefficient KM = material / environment coefficient KL = overlap correction factor F=dilution factorT = total reading X = sum of confirmed counts V = total test portion volume A systematic component has no effect if the coefficientis 1.0 and its uncertainty is getting 0.

  22. Thank you very much for your attention!!!

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