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Proyecto: Metrologia Química

Validation and Estimation of Measurement Uncertainty in Microbiology

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EA Guide 4/10 EA Guide 4/10

Validation of test methods in chapter 4Validation of test methods in chapter 4

Use of natural products or spiked materials

Qualitative tests

Specifity, falsepositives,

false negatives

Specifity, falsepositives,

false negatives

Relative truness, comparison againstreference method

Relative truness, comparison againstreference method

RepeatabilityRepeatability

Reproducibility Reproducibility

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EA Guide 4/10 EA Guide 4/10

Validation of test methods in chapter 4Validation of test methods in chapter 4

Use of natural products or spiked materials

Quantitative tests

Specifity, falsepositives,

false negatives

Specifity, falsepositives,

false negatives

Relative truness, comparison againstreference method

Relative truness, comparison againstreference method

RepeatabilityRepeatability

Reproducibility Reproducibility

LODLOD

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Fuentes de incertidumbre en

Microbiología

Incertidumbre de las mediciones in Microbiología

Lecturas(personal)

Muestreo Uniformidad de la distribuciónde los microbios

Pesas usadas+ volumenes + diluciones

Mezcladoadecuado

Desempeño delmedio

Tiempo hastala inoculación

Efectosde la muestra

Condiciones de incubación

Cálculo de cfu‘s,

Distribución de Poisson

Coeficiente deconfirmación

Equipos, e.g.spiral plater, exactitud

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In chemical testings quite often the normal distribution is used to quantify measurement uncertainties …

In chemical testings quite often the normal distribution is used to quantify measurement uncertainties …

Pay attention:

Microorganisms hate statistics …

Pay attention:

Microorganisms hate statistics …

Normalverteilung

MU – Normal- and Poisson distribution MU – Normal- and Poisson distribution MU – Normal- and Poisson distribution MU – Normal- and Poisson distribution

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

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NormasLegales

Referenciasbibliográficas

aceptadas

Gráficos de control y datos de validación

Cuantificación de acuerdo a la

Niemela et al., Finland

Resultado deensayos de aptitud

Experiencia de expertos

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Incertidumbre de las mediciones en MicrobiologíaIncertidumbre de las mediciones en Microbiología

Acreditación de laboratorios microbiológicos(EA-Guide 4/10)

Acreditación de laboratorios microbiológicos(EA-Guide 4/10)

Microbiología de alimentos y alimentos para animales -Reglas generales para exámenes microbiológicos

Microbiología de alimentos y alimentos para animales -Reglas generales para exámenes microbiológicos

ISO 7218 ISO 7218

IdeasIdeas

ISO/TS 19036No. 19036,

released 2006-02

ISO/TS 19036No. 19036,

released 2006-02

Microbiology of food and animal feeding stuffs -

Guide on estimation of measurement uncertainty for quantitative determinations

Microbiology of food and animal feeding stuffs -

Guide on estimation of measurement uncertainty for quantitative determinations

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Incertidumbre de las mediciones en MicrobiologíaIncertidumbre de las mediciones en Microbiología

Microbiología de alimentos y alimentos para animales -Reglas generales para exámenes microbiológicos

Microbiología de alimentos y alimentos para animales -Reglas generales para exámenes microbiológicos

ISO 7218 ISO 7218

IdeasIdeas

Provides statistical data forcolony counts and MPN

Provides statistical data forcolony counts and MPN

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Uncertainties for colony counts for single plate measurements

according to ISO 7218

8 to 25 (-44% bis +65%)15

5 to 18 (-52% bis +84%)10

2 to 12 (-68% bis +133%)5

1 to 10 (-73% bis +156%)4

< 1 to 9 (-79% bis +192%)3

< 1 to 7 (-88% bis +261%)2

< 1 to 6 (-97% bis +457%)1

Confidence level 95%cfu per plate

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and in addition ISO/TS 19036 …..and in addition ISO/TS 19036 …..

ISO / Technical SpecificationNo. 19036 – 2006-02

ISO / Technical SpecificationNo. 19036 – 2006-02

Microbiology of food and animal feeding stuffs -Guide on estimation of measurement uncertainty

for quantitative determinations

Microbiology of food and animal feeding stuffs -Guide on estimation of measurement uncertainty

for quantitative determinations

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Philosophy of ISO/PDTS 19036Philosophy of ISO/PDTS 19036

Global approach for the estimation of measurement uncertaintyGlobal approach for the estimation of measurement uncertainty

5.1 Combined Standard Uncertainty

5.1 Combined Standard Uncertainty

5.2 SD of reproducibility 5.2 SD of reproducibility

5.3 Expanded Uncertainty

5.3 Expanded Uncertainty

5.4 General rules for the estimation of SD of reproducibility

5.4 General rules for the estimation of SD of reproducibility

6 Intra-laboratory SD of reproducibility

6 Intra-laboratory SD of reproducibility

7 Reproducibility SD of method derived from an ILC study

7 Reproducibility SD of method derived from an ILC study

Expression of measurement uncertainty in test reports

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Analytical Laboratory Ltd.[Company Logo]

Management FormControl chart

Code: … Date: …Revision: 01 Page: 2 of 2

Compiled [Date][Signature]

Checked [Date][Signature]

Valid from [Date][Signature]

Maximum r = 0,27

0,30

0,25

0,20

0,15

0,10

0,05

0,00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Avarage r = 0,087

Avarage r = 0,087

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Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología

Ejemplo: Resultado de EAs, cuentas totales a 36 °C

FechaNo. Operador Valor asignado(cfu/ml)

Resultado Lab(cfu/ml)

Valor medio derecuperación en %

010203040506070809101112

31.12.9322.02.9430.05.9417.01.9502.06.9528.06.9618.12.9621.05.9729.04.9811.12.9801.03.9907.06.99

JJJJJJJJJJJJ

13213666

1493033

2734983

29980

256

14014978

1202626

2304874

30198

260

106110118818779849889

101123102

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Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología Incertidumbre de las mediciones en microbiología

FechaNo. Operador Resultados Lab(cfu/ml)

13141516171819202122

07.12.9921.02.0019.05.0029.08.0002.03.0120.06.0121.09.0130.11.0113.03.0227.05.02

JJJJJJJJJJ

118151822

13917215525139

132122022

15017418554546

11280

11110010810112010688

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98 % 15 %

Rec.:SD:

Incertidumbre combinada: 15 %Incertidumbre expandida : 30 %

Valor asignado(cfu/ml)

Ejemplo: Resultado de EAs, cuentas totales a 36 °C

Valor medio derecuperación en %

25

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Ejemplo: Conteo total de bacterias en hamburguesas

20 medidas de la misma muestra

20 medidas de la misma muestra

Incertidumbre en MicrobiologíaIncertidumbre en Microbiología

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 3.902.2 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

MediaMedia 4.4 x 104 cfu/g4.4 x 104 cfu/g

SDSD 0.52 x 104 cfu/g0.52 x 104 cfu/g

SD RelativaSD Relativa %12%100104.41052.0

4

4

IncertidumbreExpandida

IncertidumbreExpandida Uexpanded = ± 24%Uexpanded = ± 24%

ResultadoResultado 4.4 ± 1.1 x 104 cfu/g4.4 x 104 ± 24% cfu/g

4.4 ± 1.1 x 104 cfu/g4.4 x 104 ± 24% cfu/g

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Estimated Measurement Uncertaintiesfrom Food Industry

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

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

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Incertidumbre de medición estimadas en laindustria alimentaria

Incertidumbre de medición estimadas en laindustria 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

• 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

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Once woke up, now also

mikrobiologistshave ideas ...

about measurement uncertainties

…

Once woke up, now also

mikrobiologistshave ideas ...

about measurement uncertainties

…

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Fuentes de incertidumbre en

Microbiología

Incertidumbre de las mediciones in Microbiología

Lecturas(personal)

Muestreo Uniformidad de la distribuciónde los microbios

Pesas usadas+ volumenes + diluciones

Mezcladoadecuado

Desempeño delmedio

Tiempo hastala inoculación

Efectosde la muestra

Condiciones de incubación

Cálculo de cfu‘s,

Distribución de Poisson

Coeficiente deconfirmación

Equipos, e.g.spiral plater, exactitud

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A microbial testresult uncertaintycalculation mightbe concidered„completely corrected“ if thefollowing factorshave been applied(of course, if relevant)

A microbial testresult uncertaintycalculation mightbe concidered„completely corrected“ if thefollowing factorshave been applied(of course, if relevant)

A systematic component has no effect if the coefficientis 1.0 and its uncertainty is getting 0.

A systematic component has no effect if the coefficientis 1.0 and its uncertainty is getting 0.

KH = personal yield coefficient KS = sample stability coefficientKA = yield of medium coefficientKM = material / environment coefficientKL = overlap correction factorF = dilution factorT = total readingX = sum of confirmed countsV = total test portion volume

KH = personal yield coefficient KS = sample stability coefficientKA = yield of medium coefficientKM = material / environment coefficientKL = overlap correction factorF = dilution factorT = total readingX = sum of confirmed countsV = total test portion volume

Multiple-plate instrumentMultiple-plate instrument

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LMASH

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Thank you very muchThank you very muchfor your attention!!!for your attention!!!