Methods for Standardisation of Antibiotics

Antibiotics are the agents that kill or reduce the bacterial growth. They belong to a class of antimicrobials (a larger group also comprising of anti-viral, anti-fungal, and anti- . parasitic drugs). Antibiotics are chemicals either derived from or produced by the microorganisms (i.e., bugs or germs such as bacteria and fungi).

The methods of microbiological assay of antibiotics are based on the following principles:

Method A (Serial Dilution Method): The dilution of test antibiotic (which will inhibit the growth of a susceptible organism) is compared with the dilution of the standard antibiotic preparation having an identical effect. Through the potency of standard, the strength of the unknown can be calculated.

Method B (Diffusion Method): Due to the ease with which quantitative results can be obtained, this method is preferred over serial dilution method.

Dilutions of the test and standard antibiotic preparations are made in geometric proportions.

Test organism is inoculated on the agar plates, which are then placed on a horizontal surface so that agar occupies a constant depth of the plate. The test organism is either mixed with agar and then poured on the plates or is later applied on the agar surface already set on plates.

Antibiotic solutions are applied in:

Cups in medium using a sterile cork borer of 10mm diameter; a vacuum device or a splayed-out pen nib is used for removing the cut agar disc.

Cylinders of stainless steel, glazed porcelain, Pyrex glass, or sterilisable plastic of 8mm external diameter and 10mm height are slightly warmed so that they sink to a constant depth when placed on the agar.

Filter paper or cellulose discs absorb a fixed volume of solution.

Standard ceramic insulation beads (or fish-spine beads) are when touched on the solution surface attract a definite volume of it. When using this method, the agar medium surface should be dry.

Curve of Diameter of Zone of Inhibition versus Log Concentration of Antibiotics

Thereafter the agar plates are left for 2 hours at room temperature so that the antibiotic solution diffuses throughout the medium for further growth of organism.

Growth inhibition is observed after incubation as a clear zone of inhibition around each container. Diameter of this zone of inhibition is proportional to the log of antibiotic concentration.

The diameters of zone of inhibition are best measured using an ‘antibiotic zone reader’ in which an optical system forms an image of plate on a large grid. Two diameters at right angles are used to check the ellipticity of zone.

On plotting log concentration of the standard antibiotic against the diameter of zone of inhibition, a standard curve (figure) is obtained.

Principle of Antibiotics

The microbiological assay of antibiotics as per I.P. relies on the principle that the inhibition of bacterial growth by the measured concentration of test antibiotic is compared with the inhibition of bacterial growth by the known concentration of standard antibiotic preparation having a known activity.

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Santhakumar Raja

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