Antibiotic Potency Test by

Microbiological Assay
Marlia Singgih Wibowo
School of Pharmacy ITB
 Why antibiotic should be analysed ?

The use of antimicrobial agent

are increasing rapidly
resistency of many pathogenic
microbes
New Microbes and viruses : HIV,
Avian Flu Virus
To confirm the Effectivity of
available antimicrobial agent
against new strains
 Definition of
concentration vs potency

Concentration amount per unit volume

Potency measurement of power to kill
or inhibit the growth of certain
microorganism
 Why we have to analyse the
potency using microbiology?

Different Response of microbes

Specific
Sensitive
Spectrum of antibiotics
Principle of antibiotic potency test based on
Farmakope Indonesia edisi IV 1995

Estimation of antibiotic potency through

direct comparison between sample (antibiotic
to be tested) and standard antibiotic which is
valid, calibrated and used as references.
 Objective of the test
As a standard to confirm the potency of
antibiotic to kill or inhibit the growth of
certain microorganism
 General Method
1. Agar difusion

2. Turbidimetric
 Agar Diffusion Method
Metal cyllinder or paper disc containing
antibiotic are place on the solid agar which
contain certain microorganism

Observe the growth of microorganism

after incubation
 Turbidimetric Method

Inhibition of microbial growth in solution

placed in tube

Turbidimetric Method is suitable for antibiotic

which is not soluble in water , for example :
Gramisidin
 Thermostatic Control
During incubation for both diffusion
and turbidimetric method

For diffusion 0,5C

For turbidimetric 0,1C

Note : temperature can be from air or
water circulation
 Diffusion method

1. Petri dish 20 x 100 mm

2. Stainless steel cyllinder or porcelain with
diameter : outer 8 mm, inside 6 mm, height
10 mm
3. Wash with Nitric acid 2 N if necessary
 Turbidimetric method

1. Reaction Tube made from glass or

plastic with same height
2. Spectrofotometric tube has to be sterile
3. All residues are removed and sterilized
before and after use.
 Media and Buffer Solution
Media Buffer solution
Media 1 pH 6,6 Buffer nomor 1 pH 6,0
Media 2 pH 6,6 Buffer nomor 3 pH 8,0
Media 3 pH 7,0 Buffer nomor 4 pH 4,5
Media 5 pH 7,9 Buffer nomor 10 pH 10,5
Media 8 pH 5,9 Buffer nomor 16 pH 7,0
Media 9 pH 7,2
Media 10 pH 7,2 Solution :
Media 11 pH 8,3 Distilled water

Media 13 pH 5,6 Formaldehyde

Media 19 pH 6,1 Injection solution : NaCl

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

Potency of antibiotic can be expressed in

unit or g of activity per mg of dried
material, as stated in Pharmacopeia (BPFI).
g of activity is based on single active
ingredient
unit is used when there are more than one
active ingredient in the antibiotic
Preparation of Standard
Use table 1 to dissolve antibiotic
Store in refrigerator for certain period of time
In the day of analysis, prepare the dilution
with concentration 1 : 1.25
Note :
For Ampicilin : the standard and dilution sholud
be made at once

Zink Bacitrasin : every dilution should contain

chloric acid

In general, drying process using vacuum oven

at 5 mmHg, 60C, for 3 hrs
Preparation of samples
Prepare the solution for samples and
standard according to Pharmacopeia

Assay : prepare 1 dose of test = standard

dose with code S3
Dose for test (U) = Dose for Standard S3
 Tested Microorganisms
See Table 2, all microorganisms are
maintained in agar slant, incubate it according
to Table 3
Tested Microbes should be pure culture and
transferred every week.
If using Klebsiella pneumoniae, use the strain
with no capsule
 Design of Assay
In diffusion assay, parameter used is
diameter of inhibition formed around the
disc, after incubation
In turbidimetric assay, parameter used :
turbidity formed after incubation
Suggestion : use one dose, and dilute it to
get 5 concentrations
 Turbidimetric Method
1 ml of test solution and standard solution of each doses placed
in 3 tubes (triplo) randomly

make 2 control tubes

Add 9 ml of inocula into each tubes

Place tubes in waterbath or incubator at (36-37,5)C;

For 2 hrs

After incubation, add 0.5 mL of dilute formaldehyde

Transmittance or Absorption at 530 nm

 Agar Diffusion Method
In Sterile Petri dish

Add 4.0 ml of microbes inoculla

Place 6 cylinder on surface of agar in radius of 2.8 cm;

Height 12 mm

Add solution with (S3); make triplo

Inkubate at 32-35C for16-18 hrs

Measure the diameter of inhibition formed on agar

Design of test
2+2 : one standard and one sample, with two doses
for each in one petri dish.

3+3 : one standard and one sample, with three

doses for each, in one petri dish

5+1 : one standard and one sample, with 5 doses for

standard, and dose for sample is adjusted near to
middle dose of standard (S3), in one petri dish
Dosis baku

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 :

S1 (a) = Y1 + (Y3T Y31)

S2 (b) = Y2 + (Y3T Y32)
S3 (c) = Y3T
S4 (d) = Y4 + (Y3T Y34)
S5 (e) = Y5 + (Y3T Y35)
For standard curve, measure diameter of
the lowest and the highest :

(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