Methods for Standardization of Antibiotics (Microbiological Assay)

• Standardization of antibiotics using microbiological assays involves determining the potency and efficacy of the antibiotics based on their ability to inhibit the growth of specific microorganisms.

• Here are the main methods used for microbiological assays in the standardization of antibiotics:

Methods for Standarization of Antibiotics

1) Agar Diffusion Assay (Kirby-Bauer Method)

Principle:

• Based on the diffusion of antibiotics through an agar medium, inhibiting the growth of a bacterial lawn.

Procedure:

1. Preparation: Inoculate an agar plate with a standardized bacterial suspension.

2. Application: Place antibiotic samples on paper discs or in wells on the agar surface.

3. Incubation: Incubate the plates to allow bacterial growth and diffusion of the antibiotic.

4. Measurement: Measure the diameter of the inhibition zones around the discs or wells.

5. Comparison: Compare the inhibition zones to those produced by standard antibiotic concentrations to determine potency.

Applications:

• Commonly used for antibiotic potency testing and sensitivity testing.

2) Tube Dilution Assay

Principle:

• Determines the minimum inhibitory concentration (MIC) by diluting the antibiotic in a liquid growth medium.

Procedure:

1. Preparation: Prepare a series of test tubes with decreasing concentrations of the antibiotic.

2. Inoculation: Add a standardized bacterial suspension to each tube.

3. Incubation: Incubate the tubes to allow bacterial growth.

4. Measurement: Measure bacterial growth, typically by optical density (OD) at a specific wavelength or by visual inspection.

5. Comparison: Compare the MIC to the MIC of a standard reference antibiotic to determine potency.

Applications:

• Used to determine the effective concentration of antibiotics and compare the potency of different batches.

3) Turbidimetric Assay

Principle:

• Measures the turbidity (cloudiness) of a culture medium as an indicator of microbial growth.

Procedure:

1. Preparation: Inoculate a liquid medium with bacteria and add the antibiotic samples.

2. Incubation: Incubate the mixture to allow microbial growth.

3. Measurement: Measure the turbidity using a spectrophotometer at a specific wavelength.

4. Comparison: Compare the growth inhibition caused by the test antibiotic to that of standard antibiotic solutions to determine potency.

Applications:

• Suitable for antibiotics that have a known effect on the growth rate of specific microorganisms.