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<81> Antibiotic Microbial Assays

Marlia Singgih Wibowo


School of Pharmacy ITB

Why we have to determine the


potency of antibiotic?

Resistance Effect : the use of


antimikrobial agents are tends to
increase every year risk of
various resistance problems,
especially for pathogenic microbes
New strains of pathogens and
new viruses : HIV, Avian Flu Virus
Effectiveness of inhibition of an
antimicrobial agent is depends on
the concentration of the active
ingredient

Antibiotic preparation (dosage form)


containing not only single compound
It can be multi-component from similar
active ingredient or different kind of
activity
Example : Bactrim (trimethoprim and
sulphonamide), polymyxin B (polymyxin
B1, B2 dan B3)

Definition of concentration vs
potency

Concentration amount of the active


ingredient per certain weight/volume
Potency activity of inhibit/kill certain
microorganisms

Why using microbiology assay?

Response of microbes against


antibiotic are various
Specific
Sensitive

Spectrum of several antibiotics

Scheme of diseases
analysis stage

ELISA for analysis of specific


antigen in pathogenic microbes
Tested microbes using in
antibiotic assay are used to
determine the MIC of the antibiotic

Principles of antibiotic assay based on


Farmakope Indonesia edisi IV 1995
and USP 30-2007
Estimation of antibiotic potency
through direct comparison between
sample (antibiotic being tested) and
standard and calibrated antibiotic

General Method
1. Cylinder Plate
2. Turbidimetric

Cylinder Plate Method


Difussion of antibiotic from the cylinder
on agar in petri dish containing
microbes culture

Microbial growth are inhibited

Turbidimetric Method

The antibiotic will inhibit the growth of


microbes in liquid media in test tubes.
Turbidity intensity of the culture will express
the population of microbes

Standard Dose
Sample dose

Design for assay: 5+1 (FI ed IV, 1995)

Reference solution is prepared in several


potencies : S1, S2, S3, S4 and S5 with
combination level for each potency at 1.25
times. Example : Median potency is 10
IU/mL, then: S2=8.0 IU/mL, S1=6.4 IU/mL,
S4=12.5 IU/mL dan S5=15.6 IU/mL

Sample solution is prepared in several


dilution similar to the Reference
solution.
S3 solution (middle potency) is placed
in every plates used. 100 L of S3 The
solution are put on the agar and
alternately, place other solutions (S1,
S2, S4, S5) and sample solution (U) ,
repeat the experiment 3x

Pre-incubate the agar plates for 1 hour


at room temperature, and then incubate
at 35-37 C for 18-24 hrs.
After incubation, the inhibition zone
appreared surround the discs are
measured, and calculate the potency of
the sample.

Pattern of assay design (5+1)


S3

S3

S1

S2

S3
S5

S4

S3
U

S3

S3
Other solution

CALCULATION

Calculate average of diameter of S3 on


all plates (Y3T)
Calculate average of S3 on each plates
with S1,2,4 and 5 (Y31, Y32, Y34 and Y35)
Calculate average of diameter of S1,2,4
and 5 (Y1, Y2, Y4 dan Y5)

Correction on each solution :

S1
S2
S3
S4
S5

(a) = Y1 + (Y3T Y31)


(b) = Y2 + (Y3T Y32)
(c) = Y3T
(d) = Y4 + (Y3T Y34)
(e) = Y5 + (Y3T Y35)

Make reference curve :

YR =

(3a + 2b + c e )
5

YT

(3e + 2d + c a )
5

YR = diameter of the
lowest potency
YT = diameter of the
highest potency

Plot on semilog graphic paper :


Axis X : log potency
Axis Y : diameter hambat
Plot S1 (YR) to S5 (YT)

How to calculate the potency of


sample
YU correction = YS + (YU Y3U)
Y3U = average diameter S3 on plate with
U (sample)
YU = average diameter U
YS = interpolation S3 on ref.curve

The Potency of U (sample) is calculated by


plotting YU to the axis X (get the value of
XU) U = XU/S3 x potency of S3
Potency U = U x dilution factor

Diameter