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Microbiological Assay
Marlia Singgih Wibowo
School of Pharmacy ITB
Why antibiotic should be analysed ?
2. Turbidimetric
Agar Diffusion Method
Metal cyllinder or paper disc containing
antibiotic are place on the solid agar which
contain certain microorganism
Media 32 pH 6,6
Media 34 pH 7,0
Media 35 pH 7,0
Media 36 pH 7,3
Media 39 pH 7,9
Unit and
Reference for
Antibiotic Potency test
Definition
Dosis sampel
Design test 5+1
Standard solutions are prepared as S1,
S2, S3, S4 and S5 with proportion of
1.25.
Middle Dose is prepared, for example
10 IU/mL, so : S2=8.0 IU/mL, S1=6.4
IU/mL, S4=12.5 IU/mL and S5=15.6
IU/mL
Each solution put into cyllinder or absorb on the
paper disc, volume : 100 L. The discs are on
agar.
Pre-incubate for 1 hr, then incubate at 35-37 C
for 18-24 hrs
Measure the diameter of inhibition zone formed.
Pattern of discs on design of
(5+1)
Dose S3
S3 S3 S3
S1 Other doses
S2 S4
S3 S3
S5 U
How to calculate :
Measure the average diameter of S3 in
all petri dishes (Y3T)
Measure the diameter of S3 in each
petri of S1,2,4 and 5 (Y31, Y32, Y34 and
Y35)
Measure the diameter of S1,2,4 and 5
(Y1, Y2, Y4 and Y5)
Do correction of diameter of each
standard solution :
(3a + 2b + c e ) YR = diameter of
YR = inhibition of the lowest
5
(3e + 2d + c a ) YT = diameter of
inhibition of the
YT =
highest
5
Prepare standard curve on semilog
paper :
X axis : log of doses
Y axis : diameter of inhibition
Connect the points for S1 (YR) to S5
(YT)
Calculation for potency of sample
Correction of diameter of sample U:
YU correction = YS + (YU Y3U)
Y3U = average diameter of S3 on agar U
YU = average diameter of U on agar U
YS = interpolation of S3 on std curve
Do interpolation of YU to X axis XU
Dose of U = XU/S3 x dose S3
Potency of U = dose U x dilution factor
Diameter of inhibition zone
Metal cyllinder