Principal and procedure involved in the microbial assay of penicillin

The microbial assay of penicillin is based on its antibacterial activity and is typically performed using a method called the agar diffusion method (also known as the zone of inhibition method). The principle and procedure are described below:

Principle

The assay works on the principle of measuring the ability of penicillin to inhibit the growth of a test microorganism, such as Staphylococcus aureus. When penicillin is placed in contact with a bacterial culture, it diffuses through the agar medium, inhibiting bacterial growth in a concentration-dependent manner. The extent of growth inhibition is proportional to the concentration of penicillin.

Procedure

Materials Required:

1. Penicillin standard solution
2. Test solution (containing the sample of penicillin)
3. Test microorganism (Staphylococcus aureus or other susceptible bacteria)
4. Sterile nutrient agar medium
5. Sterile petri dishes
6. Sterile cork borer or well puncher
7. Incubator

Step-by-step procedure:

1. Preparation of Standard and Test Solutions:

   • Prepare a series of dilutions of the standard penicillin solution.
   • Prepare the test sample solution to be assayed.

2. Preparation of Nutrient Agar Medium:

   • Sterilize the nutrient agar and pour it into sterile petri dishes. Allow it to solidify.
   • Seed the agar plates with a known concentration of the test organism (e.g., Staphylococcus aureus). This can be done by inoculating the agar surface with a bacterial suspension.

3. Application of Solutions:

   • Use a sterile cork borer or well puncher to create small wells in the agar plate.
   • Fill these wells with the different concentrations of the penicillin standard solution and the test sample.
   • Ensure that each well is filled with an equal volume of the respective solutions to avoid discrepancies in results.

4. Incubation:

   • Incubate the plates at 37°C for 18–24 hours to allow bacterial growth and the diffusion of penicillin into the medium.

5. Measurement of Zones of Inhibition:

   • After incubation, observe and measure the zones of inhibition (the clear areas around the wells where bacterial growth has been prevented).
   • The diameter of these zones is directly related to the concentration of penicillin in the solution.

6. Calculation:

   • Compare the zones of inhibition produced by the test sample with those produced by the standard penicillin solutions.
   • Using a standard curve, determine the concentration of penicillin in the test solution by comparing the zone diameters.

Interpretation:

• The concentration of penicillin in the test solution can be determined based on the relationship between the size of the inhibition zone and the known concentrations of the standard solution.

This method is widely used due to its accuracy and ability to measure the biological potency of penicillin.