PROCEDURE FOR ASSAY OF ANTIBIOTICS BY DIFFUSION METHOD

1.0 PURPOSE :
This SOP provides the details for the various steps involved in estimation of antibiotics by its diffusion in the agar, containing specific culture. The diameter of the inhibition zone is dependent on the concentration of antibiotics and provides basis for the assay.
2.0 SCOPE :
This SOP covers the procedure for assay of Antibiotics in Raw-materials & Finished products.
3.0 RESPONSIBILITY :
Microbiologist.
a) Reference USP 24 Page No. 1823 to 1828.
b) Reference BP 2001 Appendix XIV A. Biological Assay of Antibiotics.
4.0 TEST ORGANISM :
Refer Appendix (User should prepare the appendix)

4.1 Preparation of culture suspension: Use fresh transfer slant every time. Add 10ml of sterile saline. Use 0.1% of culture suspension to prepare assay plates the transmittance of culture suspension should be around 25%.
4.2 Assay medium: Antibiotic Assay Medium No. 11 of Hi-media M004.


Suspend 30.5 grams in 1000ml distilled water. Boil to dissolve the medium completely.
4.3 Preparation of assay plates:
Follow the instructions of the manufacturers for reconstitution.
For rehydration, use clean undamaged glass ware and distilled water.
Weigh the appropriate amount of medium in a clean dry flask 2-3 times larger than the final volume of the prepared medium.
Add only part of the required amount of the distilled water and swirl to dissolve.
Gradually add the remaining water from the sides of the container.
Majority of the media are clear and dissolve at this stage.
The pH should be adjusted to the value specified and it is corrected by adding 0.1M sodium hydroxide or 0.1M Orthophosphoric acid.
For complete dissolving heat the medium using an open flame, hot plate or boiling water bath taking care to avoid excessive heating or scorching of the medium.
Cover the flask with cotton plug and aluminum foil.
Sterilize at 121°C , 15 psi for 20 minutes in an validated Autoclave.
Transfer the flask containing the sterilized medium in a water bath and allow it to cool to 47°C.
Add 1.0ml suspension of culture to the assay media and mix thoroughly but gently to avoid air bubbles.
Distribute the inoculated media to each of the numbered petri dishes to provide a depth of 3mm.
Allow to set and transfer to the refrigerator until use.
Remove the plate 30 minutes before use and bore 6 cups of 8 mm diameter with a borer.
5.0 : Preparation of Standard: Refer Appendix.
6.0 : Preparation of Sample: Refer Appendix.
7.0 : Plating out solution
Introduce 50 ul of Standard high/low and Test high/low in the prepared cups of Assay plates in alternate cups, to get 3 reading of each dilution. (Refer, Diagram for addition of assay solution in cups for three readings) Maintain the plates at room temperature for 1 hour and the incubate at 37°C, for 16 to 18 hours.
8.0 : Measurement of zones
Invert the plate and measure the diameter of the zone of inhibition produced by different concentrations of standard and test samples with the digital vernier calipers. (Precision of 0.01mm) Calculate the Percentage potency by statistical method. Also calculate the fiducial limits of assay by using computer programme.
Percent Potency = Antilog (2+ a log I)

Where a = (TH+TL) – (SH+SL) / (TH -TL) + (SH-SL)



SH = Sum of standard high doses average diameter
SL = Sum of standard low doses average diameter
TH = Sum of test high doses average diameter
TL = Sum of test low doses average diameter
i = Ratio of high doses concentration to low doses concentration



9.0 : Fiducial limit
The fiducial limits of error must be between 95% to 105%
10.0 : Validity of the assay
If the determined potency is lower than 50% or greater than 150% of the standard, the test is invalid and should be repeated using higher or lower dilution or the case may be.
11.0 : Preparation of buffer:
pH 8 buffer
Buffer solution is prepared by dissolving 16.73 gms of dipotassium hydrogen orthophosphate(K2HPO4) and 0.523 gms of potassium dihydrogen orthophosphate (KH2PO4) in about 750ml of water. pH 8 is adjusted with 0.1 M sodium hydroxide or 0.1 M Orthophosphoric acid and diluting to 1000ml with water.
12.0 Diagram of addition of assay solution in caps (for three readings) For one sample



Diagram of addition of assay solution in caps (for three readings)
For two sample


Appendix