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MICROBIOLOGICAL ASSAY
METHOD
Slamet Ibrahim, Marlia Singgih
School of Pharmacy ITB
INTRODUCTION
(Good Analytical Practices) :
a. Test is done to fulfill a specific objective
or user need
b. Test is done using validated method,
procedure and instruments to guarantee
the result that suitable with the objective
c. Test should be carried out by qualified
personnel with highly competent skill
Continued.
Objective :
Evaluate the method performances :
sensitivity, selectivity, accuracy, precision,
etc., also to evaluate the weaknesses, and
limitation of the method
Check factors that influence the
performances of method and how it
influence the results
Verify or Proof the analytical method which
is used in the laboratory
Requirement of Validation
for Analytical Method
Using Calibrated instruments and tools
Prepared by competent personnel
2 Method Validation
Primary Validation for new method, or
modified of standard method.
Secondary Validation for verification
process, if the lab adopted a validated
method
Parameter of Validation in
microbiology assay
Acurracy
Precision (repeatability, reproducibility
and intermediate precision)
Sensitivity
Selectivity and Specificity
Linearity
Acceptable range (upper and lower limit)
Robustness of method
Microbiological Assay
1. QUALITATIVE
2. QUANTITATIVE
Qualitative Method
In qualitative method, its response is
presence or absence of parameter
detected, either direct or indirect to
samples
Quantitative Method
In Quantitative Method, its response is in
the form of analite amount, that can be
measured directly (i.e. enumeration of
microbes) or indirectly (i.e : Absorbance
value, Intensity, Impedancy, etc.)
Qualitative:
9
9
9
9
Direct identification
Morphological analysis :macroskopic and microskopic
Alternative method (Dye-reduction Test, Electrical Methods, ATP
Determination)
Rapid method (immunochemical, molecular techniques)
Quantitative :
9
9
9
9
9
9
Identification of Microorganism
Escherichia coli
Salmonella thypi
Validation Preparation
Before validation is done, the laboratory should
prepare some important matters :
1. Microorganism of target or reference (see SR-02
:
persyaratan tambahan untuk akreditasi
laboratorium, Pengujian Kimia dan Biologi
SNI 17025, DP.01.16, Januari 2004)
2. Equipment and Instrument which regularly
calibrated
3. Competent Personnels
4. Statistic Program for calculation, evaluation and
result interpretation
Parameter of Validation
Accuracy (kecermatan) in
microbiological assay
Definition :
The accuracy of an analytical procedure
expresses the closeness of agreement
between the value which is accepted
either as a conventional true value or an
accepted reference value and the value
found
Accuracy
Recovery = in % = H/A x 100
Relative Deviation : (H A)/A x 100
H = result of assay method
A = result of real assay from microorganism
target
Relative Recovery : H/B x 100
H = result of assay method
B = result of standard method
Accuracy
Method of assay :
Spiked-placebo Recovery Method
Standard Addition Method
Use 9 times measurement ( 3 level
concentration with 3 replication)
Theory
125
125
125
125
127
119
120
125
125
132
125
131
125
Calculation
% Recovery = Result of assay method/theory x
100%
= 125/125 x 100% = 100%
% Relative Recovery = Assay Method/std method
x
100% = 125/125 x 100% =
100%
Precision (keseksamaan)
Definition
The precision of an analytical procedure
expresses the closeness of agreement (degree
of scatter) between a series of measurements
obtained from multiple sampling of the same
homogeneous sample under the prescribed
conditions
Parameter of Precision : repeatability,
reproducibility, intermediate precision
Precision
Relative Standard Deviation (RSD) for
intermediate precision :
logaritm
a
b
(II/I)2
avgr (I)
diff (II)
93
86
1,9685
1,9345 1,9515 0,0340
0,000303
34
30
1,5315 1,4771 1,5043 0,0544
0,001306
98
73
1,9912 1,8633 1,9273 0,1279
0,004404
89
83
1,9494 1,9191 1,9342 0,0303
0,000246
116
104
2,0645 2,0170 2,0407 0,0475
0,000540
168
156 2,2253 2,1931 2,2092 0,0322
0,000212
62
56
1,7924 1,7482 1,7703 0,0442
0,000623
38
28
1,5798 1,4472
1,5135 0,1326
0,007679
330
300
2,5185 2,4771
2,4978 0,0414
0,000275
2300 2040
3,3617 3,3096
3,3357 0,00521 0,000244
= 0,015832
RSD = 0,015832/2 x 10 = 0,0281
CV = 100 x RSD = 2,81 %
Upper limit
The highest test result which indicated by
deviation of analysis 15.0% from average (min
measurement 3times)
2.LC HC-1
1,96
[ 2.LC HC ]1/2
Linearity
(for antibiotic potency assay)
Linearity : samples concentration and the assay
response are proportional , directly or by math
equation
Reference curve is made from at least 3
concentration between 50 150% of actual conc
(FDA)
To determine concentration in the sample : use
80, 100 and 120% of target concentration
Parameter of Linearity
Reference curve
Analysis sensitivity : F = y/ x
Residual deviation /residual regression
line
Sy/x = [(y-)2/n-2]1/2
where y = analite response
= calculated from regression line
Linearity
Coefficient of variation : regression
function
, (Vx0 2%)
Vx0 = S y/x . 100%
b.x
Coefficient of correlation ( r 0,999)
D = b log C + a
log Cs = Ds a/ b, where :
Diameter
of inhib
x
x
x
Ds = Diameter of inhibition
b. Use 1 ref.soln and blank
log Cs = (Ds a)/(Db a) . log Cb
where :
Cb = conc. analit in ref/std
Log Ci
Robustness of Method
The robustness of an analytical procedure
is a measure of its capacity to remain
unaffected by small, but deliberate
variations in method parameters and
provides an indication of its reliability
during normal usage.
Robustness of method
Should be done during the development of
method and depends on factors that
influence the assay
If the assay is very sensitive to
environment, the assay should be
controlled carefully
Robustness of method
Many conditions should be controlled :
Stability of sample
Temperature of incubation
Time of incubation
Aerobic or anaerobic
media (nutrition), etc