SOP for Microbiological Antibiotic assay  

This Document describes (Standard Operating procedure) Sop for Microbiological Antibiotic Assay

Sop for Microbiological Antibiotic Assay

I. Purpose & Scope:

  • The purpose of this SOP is to lay down the procedure for Microbiological Antibiotic assay
  • This Standard operating procedure shall be applicable for Microbiological Antibiotic assay to determine the potency of drug under test in the Microbiology section of Quality Control Department

II. Responsibilities:

• All Quality Control and Quality Assurance personnel’s shall be responsible to follow and implement this SOP.

III : Introduction and Procedural Part :

Precautions to be taken during microbiological Antibiotic assay :

  • Cup borers shall be sterilized.
  • Molten agar temperature should be between 45-50°C at the time of inoculation.
  • All equipment’s shall be thoroughly cleaned before and after each use.

Requirements : 

  • Antibiotic assay medium11.
  • Potassium phosphate buffer pH 7.0/ pH 8.0
  • Microbial cultures ( Staphylococcus epidermidis ATCC12228, Bacillus subtilis ATCC 6633)
  • Stainless steel cup borer.
  • Solvents such as Chloroform, Acetone, Methanol.

Preparation of Buffer :

Preparation of Phosphate buffer:
  • Prepare the potassium phosphate buffer required for the antibiotic assay. The buffers shall be sterilized after preparation and pH specified in each case is the pH after sterilization.
  • Phosphate buffer pH 8.0: Dissolve 16.73 gm of K2HPO4 and 0.523 gm of KH2PO4 in 1000 ml of water. Adjust the pH with either Phospharic acid or Potassium Hydroxide to 8.0 ±0.2.
  • Potassium phosphate buffer 0.1M pH 7.0: Dissolve 13.6 gram of dibasic potassium phosphate ( K2HPO4 ) and 4.0 gram of monobasic potassium phosphate( KH2PO4 ) in 1000 ml of water. Adjust with phosphoric acid and potassium hydroxide to a pH of 7.0±0.2

Preparation of Standard:

  • To prepare a stock solution, dissolve a specified quantity of the USP Reference standard / working standard of a given antibiotic in the specified solvent and then dilute to the required concentration as indicated in STP.
  • Store in refrigerator and use within the period indicated.
  • On the day of assay, prepare from the stock solution i.e prepare the standard high (SH) and standard low (SL) solution.

Preparation of Sample :

  • From the information available for the preparation to be assayed (Unknown) assign to it an assumed potency per unit weight or volume and on this assumption prepare on the day of assay a stock solution.
  • Make an entry of the sample in Antibiotic assay analysis register, refer Annexure II.
  • Prepare the test high (TH) and Test low (TL) solution for assay.

Preparation of Inoculum and agar plates: 

  • Inoculate the growth from recently grown working culture slant of the organism in sterile peptone or saline. Homogenize by swirling or by using vortex mixture.
  • For cylinder plate / cup plate assay method, prepare the inoculums by adding a portion of stock suspension to a sufficient amount of sterile agar medium that has been melted and cooled to 45 to 50°C and swirling to attain a homogeneous suspension.
  • Use culture of Staphylococcus epidermidis ATCC12228 for Genamycin and Neomycin assay. Use Bacillus subtilis ATCC 6633 for Erythromycin assay.
  • For erythromycin, neomycin and gentamycin use ‘Antibiotic assay Medium-11’.
  • Cylinder plate method: Prepare assay plates using sterile petri-plates, pour approximate 20 ml of assay medium in each of required plate and allow it to solidify into a smooth base layer of uniform depth. Drop four assay cylinders on the inoculated surface from height of 12 mm in each plate.
  • Cup plate method: Prepare assay plates using sterile petri-plates, pour 20 to 30 ml of assay medium in each of required plate and allow it to solidify into a smooth base layer of uniform depth. Make four wells into the each agar plate by using sterile borer.

Testing Procedure for Microbiological Antibiotic assay :

  • Label each cylinder or well as appropriate dilution i.e. Standard High(SH), Standard Low(SL), Test High(TH) and Test Low(TL).
  • Fill the four cylinders/wells of each plate with 0.1 ml of individual standard and test dilutions of respective strength (high & low).
  • Incubate the plates in upright position at 30 to 35°C for 18 to 24 hours.
  • After incubation observe the plates for zones of growth inhibition. The zones will be formed due to antimicrobial activity.
  • During observation, measure and record the diameter of each zone of growth inhibition in mm. Measure the zone diameter using Antibiotic zone reader or vernier caliper.
  • Calculate the average diameters of the zones for each dilution
  • Estimation of Potency: calculate the % Potency of the sample (in term of standard) from the following equation;

% Potency = Antilog (2+a logI)

Where ‘a ‘may have positive or negative value and should be used algebraically

(TH+TL) – (SH+SL)

a = ———————————-

         (TH-TL) + (SH-SL)

Where, TH and TL are the average of the zones diameter with solution of the test of high and low level respectively.

SH and SL are the sum of the zone diameter with solution of the Standard of high and low level respectively.

Where, I = ratio of dilution.

  • From this calculate the assay, record the result in Annexure I.

IV: Annexure microbiological Antibiotic assay :

Annexure I: Report for Microbiological Antibiotic assay

Annexure II: Microbiological Antibiotic assay analysis register

Annexure – I

  Report for Microbiological Antibiotic Assay                                                             


Name of Sample                Date Of Analysis    
Batch No.                           A.R. Number            
In house Media Lot No.    Culture Used           
Incubation Temp & Time                               Completed  on         


  1. Sample Preparation:


     Standard Solution: ———————————————————————————–



      Test Solution: ———————————————————————————-




  1. B) Observation (in mm):



  Relative % Potency = Antilog (2 + a log I)

I = Ratio of dilution


(TH+TL) – (SH+SL)

a = ———————————-

          (TH-TL) + (SH-SL)


= ———————————–



= ———————————-




% Potency = Antilog (2+a log I)






Assay       =





Acceptance criteria :


Remark: —————————————————————————————————-


Analyzed By:                                         Checked By:                                  Approved By:

Date:                                                       Date:                                               Date:



Annexure- II

Microbiological Antibiotic assay analysis register 


Date Name of Product Batch Number A.R. Number Assay in % Assay Remark Completed On Analyzed by Checked by

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