Pour Plate method

STEP 1: Specification

Determination of heterotrophic

bacteria per mℓ reticulation water

using the pour plate method

Specification

using the pour plate method

Measurement procedure

sampling

shake sample

2 x 1 ml aliquots2 x 1 ml aliquots

pour with agar

incubate

count colonies

Formula

221 RR

R+=2

R =

Pour plate method

STEP 2: Identification of the

uncertainty sourcesuncertainty sources

cfu/mL

sample preparationmedia

sampling homogeneity

aliquotchlorine removal

Mgross

Mtare

prec cal

volume

preccaltemp

selectivity

purity

weighing

pH and conductivitymixingmoisture content

Fishbone diagram

cfu/mL

incubationcounting

sterilisation

sample bottles

pipettespetri

dishesair

temp

time

humidity

target colonies

medium

Pour plate count

STEP 3: Quantifying the

uncertainty componentsuncertainty components

Quantifying uncertainty components

�Best feasible estimation of the run-to-

run variation of the analytical process

�Best possible estimation of the overall

Three major steps from the in-house method

development and method validation studies:

�Best possible estimation of the overall

bias (Recovery) and its uncertainty

�Other uncertainties

cfu/mL

sample preparationmedia

sampling homogeneity

aliquotchlorine removal

Mgross

Mtare

cal

volumecaltemp

selectivity

purity

weighing

pH and conductivitymixingmoisture content

Fishbone diagram with repeatability branch

cfu/mL

incubationcounting

sterilisation

sample bottles

pipettespetri

dishesair

temp

time

humidity

target colonies

medium

precision

Mgross

aliquotMtare

sterilisationmedia

incubationcounting

Precision study

Analyst R1 R2 R

A 105 107 106

B 99 102 101

C 97 98 98

D 95 101 98D 95 101 98

E 101 102 102

F 98 100 99

Average 101

Std deviation 3

%RSD 2.97

cfu/mL

sample preparationmedia

sampling homogeneity

aliquotchlorine removal

Mgross

Mtare

cal

volumecaltemp

selectivity

purity

weighing

pH and conductivitymixingmoisture content

Fishbone diagram

cfu/mL

incubationcounting

sterilisation

sample bottles

pipettespetri

dishesair

temp

time

humidity

target colonies

medium

precision

Mgross

aliquotMtare

sterilisationmedia

incubationcounting

Bias study

�CRM

�Proficiency testing scheme

�Spiking study

Bias study

�CRM

�Proficiency testing scheme

�Spiking study

X

Spiking study

�Sample with ‘known’ amount of

cfu’s/mL

�Each analyst must analyse the sample�Each analyst must analyse the sample

�Spike a typical sample with different

amounts

Spiking study

Analyst Sample 1 mL 2 mL 3 mL 4 mL 5 mL

A 155 200 245 295 351

B 150 201 250 293 345

C 160 205 255 301 356C 160 205 255 301 356

D 149 203 254 298 344

E 151 204 256 302 353

F 145 195 254 295 352

Spiking study

y = 48.4x + 101.8R² = 0.9981

200

250

300

350

400

0

50

100

150

200

1 2 3 4 5 6

Spiking study

Analyst Sample 1 mL 2 mL 3 mL 4 mL 5 mL

A 102 155 200 245 295 351

B 103 150 201 250 293 345

C 109 160 205 255 301 356

D 104 149 203 254 298 344

E 103 151 204 256 302 353

F 94 145 195 254 295 352

Average 103

Std

deviation

5

Bias study

Analysts

reproducibility

Spiking

study

Average 101 103

Std deviation 3 5Std deviation 3 5

Comparison of the means of two

samples• Step 1: Compare the two standard deviations (F-test)

22

210 : σσ =H

22

211 : σσ ≠H

the variances are not significantly different

the variances are significantly different211 : σσ ≠H

22

21

s

sFcalc =

05,0;; 21 ννFFcrit =

the variances are significantly different

=2,78

=5,0503

Comparison of the means of

two samples• No significant difference

( ) ( )2

11

21

222

2112

−+−+−=

nn

snsns pool

=17

21

21

21

11nn

s

xxt

pool

calc

+

−=

221 −+= nnDF

= 0,84

81,105,0;10 == ttcrit

Uncertainty in the bias

( )653 22

22

=+=

+= spikingilityreproducib ssbiasu

653 22 =+=

cfu/mL

sample preparationmedia

sampling homogeneity

aliquotchlorine removal

Mgross

Mtare

cal

volumecaltemp

selectivity

purity

weighing

pH and conductivitymixingmoisture content

Fishbone diagram

cfu/mL

incubationcounting

sterilisation

sample bottles

pipettespetri

dishesair

temp

time

humidity

target colonies

medium

precision

Mgross

aliquotMtare

sterilisationmedia

incubationcounting

Pour plate count

STEP 4: Calculating the

combined standard combined standard

uncertainty

Combine standard uncertainties

( )7

22

=

+= biasusu ilityreproducibc

7=

%RU (k=2) = 14%