Microbiological Assay of Anitbiotics

Microbiological Assay of Anitbiotics here i am siting one example of Penicillin
The group of methods for assay of antibiotics include
1] Cylinder plate.
2] Cup plate and
3] Paper disc method.
The term Cylinder plate is applied to the method in which small cylinders are placed on a surface of the agar plates and term.
Cup plate in which cavities or cups one drilled out on the agar in the paper disc method, a small filter paper disc soaked in the antibiotic solution is placed on the agar. The surface of agar plate is seeded with susceptible micro organism, and antibiotic of different cone is placed on the cylinders,
cups or disc (soaked in the solution). It diffuses thro the agar and the zone of the inhibition of growth of bacteria are produced around each antibiotic Comparison of diameter of each zone gives an idea of the concentration and potency of contibiotic.
Requirement:- Sterile Petri dishes, sterile pipettes, nutrient agar [ Peptone 1%, beef extract 1%, Nacl 0.5%, Agar 2%, PH 7.2 ] 24hrs old culture of s.aureus,Std. peniatlin solution, filter paper dishes.

How to do Preparation: Prepare the plates by adding exactly 15ml of nutrient agar to each sterile, Petri dish aseptically. Allow it to set, Incubate the plate for 24 hrs as a test for sterility. Inoculate sterile nutrient agar which has been melted and cooled to 500c with a 24hrs broth (culture grown in liquid medium). Culture or s.aurem in proportion of (1:100)
Mix well and pour in the Petri dish.Place petridishes on horizontal surface until agar stets.
Use the plates for assay of penicillin by any of the following 3 methods.
1] Cylinder plate method: – Place (4) sterile cylinder in each Petri dish by dropping them at a height of 1/8th of inch in agar surface. Place each cylinder equidistance from center. By sterile pipettes fill each cylinder with standard solution penicillin containing 0.5, 1, 1.5, 2, and 2.5. I units/ml respectively. Fill other cylinder with sample to be tested. Place the plates carefully and incubate of 370c for 18hrs. Measure diameter of zone of inhibition. Take average measurements of stds and the test sample.
2] Cup plate method: – In the seeded agar plates cut out cylindrical cups by means of cork borer (diameter about 3.5mm). Remove dishes by means of small pointed spheres. Place identical volume (0.2ml) of various test solution and standers in the cups. Incubate at 370c for 18hrs.compare diameters of zone of inhibition as before.

3] Paper disc methods :- Cub small circular filter paper, soak them in corresponding concentration of std and sample solution of penicillin and allow to dry place this disc aseptically in each plate in a circular and equidistant from center. Incubate and proceed as before.
Determine the zone of inhibition
Plot a graph of Log to the base 10 of concentration Vs Zone of inhibition ,
Extrapolate the zone of inhibition obtained from graph to find out the concentration of unknown potency of given antibiotic
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