1.0 PURPOSE
To lay down the procedure for testing for the microbial assay to determine the potency of the drug under test.2.0 SCOPE
This SOP shall be applicable to the microbiology lab.3.0 RESPONSIBILITY
Microbiologist4.0 ACCOUNTABILITY
Head of Department5.0 PROCEDURE
5.1 Precaution to be taken
5.1.1 Glassware / Cup borer used shall be sterilized.5.1.2 Molten agar temperature should be between 45 – 50°C at the time of inoculation.
5.1.3 All equipment is to be thoroughly cleaned before and after each use.
5.1.4 For assay cylinders, use stainless steel or porcelain cylinders with the following dimensions, each dimension having a tolerance of ± 0.1 mm: outside diameter 8 mm; inside diameter 6 mm, and length 10 mm.
5.2 Test procedure
5.2.1 Prepare the media used in assay according to SOP5.2.2 Preparation of Phosphate buffer.
5.2.2.1 Prepare the potassium phosphate buffer required for the antibiotic under assay. The buffers are sterilized after preparation, and the pH specified in each case is the pH after sterilization.
5.2.2.2 Potassium phosphate buffer 0.1 m, pH 7.0 – Dissolve 13.6 g of dibasic potassium phosphate and 5.0 g of monobasic potassium phosphate in 1000 ml of water. Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to a pH of 7.0 ± 0.2.
5.3 Preparation of the standard
To prepare a stock solution, dissolve a quantity of the USP Reference Standard / Working standard of a given antibiotic, accurately weighed, or the entire contents of a vial of USP Reference Standard, where appropriate, in the solvent specified in that table, and then dilute to the required concentration as indicated. Store in a refrigerator and use within the period indicated. On the day of the assay, prepare from the stock solution five or more test dilutions, the successive solutions increasing stepwise in concentration, usually in the ratio of 1:1.25 for a cylinder-plate assay. Use the final diluent specified and a sequence such that the middle or median has the concentration designated.5.4 Preparation of the sample
From the information available for the preparation to be assayed (the “Unknown”), assign to it an assumed potency per unit weight or volume, and on this assumption prepare on the day of the assay a stock solution and test dilution as specified for Each antibiotic but with the same final diluent as used for the USP Reference Standard. The assay with five levels of the Standard requires only one level of the Unknown at a concentration assumed equal to the median level of the Standard.5.5 Preparation of Inoculum
5.5.1 Remove the growth from a recently grown slant or culture of the organism, with 3 ml of sterile peptone saline and sterile glass beads.5.5.2 Inoculate the surface of 250 ml of the soybean digest agar medium on the flat side of a Roux bottle.
5.5.3 Spread the suspension evenly over the surface of the agar with the aid of sterile glass beads, and incubate at the temperature shown above. After completion of incubation, prepare the stock suspension by collecting the surface growth in 50 ml of medium 35.
5.5.4 Determine the dilution factor which will give 25 % light transmission at about 530 nm.
5.5.5 For the cylinder-plate assay, determine by trial the proportions of stock suspension be incorporated in the inoculum, starting with the volumes indicated in, that result in the satisfactory demarcation of the zones of inhibition and giving a reproducible dose relationship. Prepare the inoculum by adding a portion of stock suspension to a sufficient amount of agar medium that has been melted and cooled to 45 to 50, and swirling to attain a homogeneous suspension.
5.6 Procedure
5.6.1 In a cylinder-plate assay, the essential comparisons are restricted to relationships between zone diameter measurements within plates, exclusive of the variation between plates in their preparation and subsequent handling.5.6.2 Cylinder-Plate Method
Add approximately 1 ml of inoculum as prepared above in Medium 35 pour the medium into a sterile petri dish and allow it to harden.
5.6.3 Two-level factorial assay.
5.6.3.1 Prepare parallel dilutions containing 2 levels of both the standards (S1 and S2) and the unknown (U1 and U2) on each of four plates. Fill each of its four cylinders with different test dilution, alternating standard and unknown.
5.6.3.2 Incubate the plates at the upright position at 30-35 °C for 24 to 48 hours.
5.6.3.3 Keep the plate upright at room temperature and measure the diameters of the zones of inhibition.
5.6.4 Estimation of potency
Sum the diameters of the zones of each dilution and calculate the % potency of the sample (in terms of the standard) from the following equation:
% Potency = Antilog (2.0 + a log I)
Wherein a may have a positive or negative value and should be used algebraically and
Where a = (U1+U2) - (S1+S2)
(U1+U2) + (S1-S2)
U1 and U2 are the sums of the zone diameters with solutions of the unknown of high and low levels.
S1 and S2 are the sums of the zone diameters with the solution of the standard of high and low levels.
‘I’ is the ratio of dilutions.
5.6.5 Limit: As per the respective product specification.
5.6.6 The potency of the sample may be calculated from the expression
% Potency x assumed potency of the sample
100
6.0 ABBREVIATIONS
6.1 SOP – Standard operating procedure6.2 °C – Degree Centigrade
6.3 M – Morality
6.4 G – Gram
6.5 Ml – Milliliter
6.6 U – Unit
6.7 ATCC – American Type of Culture Collection
6.8 % – Percentage
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