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%