1.0 OBJECTIVE : To laid down procedure for Microbiological assay for determining the potency of the drug.
2.0 SCOPE : This SOP is applicable for testing of microbiological assay of antibiotics and vitamins of raw materials and finished products.
3.0 RESPONSIBILITY : Microbiologist, Q.C. In charge.
4.0 REQUIREMENTS : Volumetric flasks, Petri plates, pipettes, Media, hand glows and gowns, 0.2µ filtered 70% I.P.A., microorganisms, autoclave, borer, -laminar air flow bench and incubator etc.
5.0 PROCEDURE :
5.1.1 Prepare the plate in the leveled horizontal place, of uniform thickness.
5.1.2 Properly mix the appropriate culture in the media, to get proper zones.
5.1.3 Measure the zones of inhibition/exhibition accurately.
5.1.4 Take care during the boring of media plates; it should be circular and uniform.
5.1.5 After preparation of standard and sample plates, kept these plates carefully in incubator at appropriate temperature.
5.1.6 During mixing and pouring of media take care no air bubble should be formed.
5.2 MICROBIOLOGICAL ASSAY PROCEDURE:
5.1 Prepare appropriate agar media as per respective method of analysis, as per media preparation.
5.2 Enter in the MLT Testing area as per respective area entry procedure and bring autoclaved media in MLT room, and cool down the media up to 40° to 50°C.
5.3 Prepare the specified culture dilution mentioned in respective method of analysis, as per Sub culturing and preparation of working dilutions.
5.4 Now add the appropriate calculated quantity of the culture as per respective test procedure in the media at 40° to 50°C and mix well take care air bubble should not be formed.
5.5 Transfer 20 to 25 ml of mixed cultured media in sterile Petri plates and slowly rotate the Petri plates to uniform mix and settle the content.
5.6 Close the lid of Petri plates and allow to solidify the media fro about 1 hour.
5.7 Now take the 8mm SS borer and sterile the borer in flame, and cool.
5.8 Bore the solidified prepared plates in four apposite directions in equal distance for standard higher and lower dilutions and sample higher and lower dilutions.
5.9 Prepare atleast 4 such types’ media plates for one sample assay analysis.
5.10 Now prepare the standard and sample higher and lower dilutions as per the individual respective product test procedure.
5.11 Mark the each cavity of plates as standard higher (S1), Standard lower (S2) and Test higher (U1), Test lower (U2).
5.12 Pipette out about 100micro-liters of standard high & 100 micro-liters of standard low dilution in opposite direction in four plates.
5.13 Now pipette out 100 micro-liters of test high & 100 micro-liters of test low dilution in opposite direction in all four plates and close the lid of plates.
5.14 Allow the assay plates after pipette out at room temperature for 1 to 4 hour for diffusion.
5.15 Care fully transfer these plates in incubator; maintained at 37 °+ 1 °C, incubate for 18 to 24 hours without disturbance.
5.16 After 18 to 24 hours measure zone of inhibition in case of product is antibiotics and zone of exhibitions in case of product is vitamins.
5.17 Note down the measurement in the Microbiological Assay in analysis protocol report.
5.18 Sum the diameters of the zones of each dilutions of standard higher (S1), Standard lower (S2) and Test higher (U1), Test lower (U2).
5.19 Calculate the % potency of the sample from the following equation :
% of Potency = Antilog (2 + a log I)
Were a may have positive or negative value and should be used algebraically.
Where a = (U1 + U2) – (S1 + S2) ; and I = Ratio of Dilutions
(U1 – U2) + (S1 – S2)
U1 and U2 are the sums of the zone diameters with solutions of the unknown of high and low levels, and S1 and S2 are the sums of the zone diameters with solutions of the standard high and low levels.
5.20 If the potency of the sample is lower than 60% or greater that 150% of the standard, the assay is invalid and should be repeated using higher or lower dilutions of the same dilution.
5.21 Now from the % potency we can calculate the potency of the unknown sample from the following formula & record the result in analysis protocol
Potency in µg =
% Potency x standard concentration x potency of the standard in µg x 1000
100 x sample concentration x 1000