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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:
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
Preparation: Inoculate an agar plate with a standardized bacterial suspension.
Application: Place antibiotic samples on paper discs or in wells on the agar surface.
Incubation: Incubate the plates to allow bacterial growth and diffusion of the antibiotic.
Measurement: Measure the diameter of the inhibition zones around the discs or wells.
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
Preparation: Prepare a series of test tubes with decreasing concentrations of the antibiotic.
Inoculation: Add a standardized bacterial suspension to each tube.
Incubation: Incubate the tubes to allow bacterial growth.
Measurement: Measure bacterial growth, typically by optical density (OD) at a specific wavelength or by visual inspection.
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
Preparation: Inoculate a liquid medium with bacteria and add the antibiotic samples.
Incubation: Incubate the mixture to allow microbial growth.
Measurement: Measure the turbidity using a spectrophotometer at a specific wavelength.
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.
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