Antibiotic Potency Test by Microbiological Assay

Marlia Singgih WibowoSchool of Pharmacy ITB

Why antibiotic should be analysed ?

The use of antimicrobial agent are increasing rapidly resistency of many pathogenic microbesNew Microbes and viruses : HIV, Avian Flu VirusTo confirm the Effectivity of available antimicrobial agent against new strains

Definition of concentration vs potency

Concentration amount per unit volumePotency 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 SpecificSensitive

Spectrum of antibiotics

Estimation of antibiotic potency through direct comparison between sample (antibiotic to be tested) and standard antibiotic which is valid, calibrated and used as references.

Principle of antibiotic potency test based on Farmakope Indonesia edisi IV 1995

Objective of the test

As a standard to confirm the potency of antibiotic to kill or inhibit the growth of

certain microorganism

General Method1. 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

Inhibition of microbial growth in solution placed in tube

Turbidimetric Method is suitable for antibiotic which is not soluble in water , for example : Gramisidin

Turbidimetric Method

During incubation for both diffusion and turbidimetric method

For diffusion ± 0,5°C

For turbidimetric ± 0,1°CNote : temperature can be from air or water circulation

Thermostatic Control

Diffusion method

1. Petri dish 20 x 100 mm2. 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

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.

Turbidimetric method

Media and Buffer Solution Media Buffer solution

Media 1 pH 6,6Media 2 pH 6,6Media 3 pH 7,0Media 5 pH 7,9Media 8 pH 5,9Media 9 pH 7,2Media 10 pH 7,2Media 11 pH 8,3Media 13 pH 5,6Media 19 pH 6,1Media 32 pH 6,6Media 34 pH 7,0Media 35 pH 7,0Media 36 pH 7,3Media 39 pH 7,9

Buffer nomor 1 pH 6,0Buffer nomor 3 pH 8,0Buffer nomor 4 pH 4,5Buffer nomor 10 pH 10,5Buffer nomor 16 pH 7,0

Solution :Distilled waterFormaldehyde Injection solution : NaCl

Unit and Reference for Antibiotic Potency test

Potency of antibiotic can be expressed in “unit” or “µg” of activity per mg of dried material, as stated in Pharmacopeia (BPFI).“µg” of activity is based on single active ingredient “unit” is used when there are more than one active ingredient in the antibiotic

Definition

Preparation of StandardUse 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

For Ampicilin : the standard and dilution sholudbe made at once

Zink Bacitrasin : every dilution should contain chloric acid

In general, drying process using vacuum oven at 5 mmHg, 60°C, for 3 hrs

Note :

Preparation of samples

Prepare the solution for samples and standard according to Pharmacopeia

Assay : prepare 1 dose of test = standard dose with code S3Dose for test (U) = Dose for Standard S3

Tested Microorganisms See Table 2, all microorganisms are maintained in agar slant, incubate it according to Table 3Tested 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 incubationIn turbidimetric assay, parameter used : turbidity formed after incubationSuggestion : 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 MethodIn 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 32-35°C 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 μL. The discs are on agar.Pre-incubate for 1 hr, then incubate at 35-37 ºC for 18-24 hrsMeasure the diameter of inhibition zone formed.

Pattern of discs on design of (5+1)

Dose S3

Other dosesS3

S1S3

S2

S3

S4

S3

S5

S3

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 :

YT =(3e + 2d + c – a )

5

YR =(3a + 2b + c – e )

5

YR = diameter of inhibition of the lowest

YT = diameter of inhibition of the highest

Prepare standard curve on semilog paper : X axis : log of dosesY axis : diameter of inhibition Connect the points for S1 (YR) to S5 (YT)

Correction of diameter of sample U:YU correction = YS + (YU – Y3U)Y3U = average diameter of S3 on agar UYU = average diameter of U on agar UYS = interpolation of S3 on std curve

Calculation for potency of sample

Do interpolation of YU to X axis XU

Dose of U = XU/S3 x dose S3Potency of U = dose U x dilution factor

Diameter of inhibition zone

Metal cyllinder