Microbial Assay of Antibiotics

Microbial Assay of Antibiotics
STANDARD PREPARATION AND UNITS OF ACTIVITY
Preparation of media
Buffer solutions
Standard solution
Sample solution
Test organisms
Preparation of inoculum� Method -1
Method 2
Method 3
Method 4
Determination of Inoculum
Apparatus
Assay design
Assay method�
cylinder plate method
One level assay with standard curve
Estimation of potency
Turbidimetric method

1. Microbial assay of antibiotics 1 Prepared by: ADITYA SHARMA NIPER Guwahati

2. Microbial assay of antibiotics It is based upon the comparison of the inhibition of growth of microorganisms by measured concentration of the antibiotics under examination with that produced by known concentration of standard preparation of antibiotic having a known activity. Two general methods are usually employed 2 Method A Cylinder plate method or Plate(USP) Cup-plate (IP) Diffusion method (BP) Method B Turbidimetric method or Tube assay

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4. STANDARD PREPARATION AND UNITS OF ACTIVITY A standard preparation is a authentic sample of the appropriate antibiotic, for which potency has been precisely determined by the reference to an appropriate international standard. The potency of the standard preparation may be expressed in International Units or in µg per mg of the pure antibiotic. µg of activity is based on single active ingredient. Unit is used when there are more than one active ingredient in the antibiotic. 4

5. Preparation of media Preparation of test microorgnism inocula from the ingrdients listed in the following table. 5 Dissolve the ingredients in sufficient water to produce 1000ml . Add sufficient 1M sodium hydroxide or hcl, as required, so after sterilization the pH is as given.

6. Buffer solutions Dissolve given quantities of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in water to produce 1000ml. Adjust the pH with 8M phosporic acid or 10M potassium hydroxide, after sterilization. 6

7. Standard solution 7 Dissolve standard preparation of antibiotic in the solvent specified. Dilute to required concentrations Store in a refregirator and use within the period indicated. From th stock solutions prepare 5 or more test dilutions the succesive solutions increasing in stepwise in concentrations in the ratio of 1:2.5 for method A and smaller for method B

8. Sample solution Assign assumed potency per unit weight or volume. On the day of assay prepare a stock solution and test dilution as specified for each antibiotic. Dilute the sample stock solution in the specified final diluent to obtain a nominal concentration equal to median concentration of the standard 8

9. Test organisms Maintain a culture on slants of the medium and under the incubation conditions specified in Table, and transfer weekly to fresh slants. 9

10. Preparation of inoculum 10

11. 11 Preparation of inoculum Method -1

12. 12 Method 2

13. Method 3 Maintain the test organism on 10 ml agar slants of Medium G. Incubate at 32º to 35º for 24 hours. Inoculate 100 ml of nutrient broth. Incubate or 16 to 18 hours at 37º and proceed as described in Method I. 13

14. Method 4 Proceed as described in Method 1 but wash the growth from the nutrient surface using 50 ml of Medium 1 (prepared without agar) in place of saline solution . 14

15. Determination of Inoculum The inoculum prepared above is inoculated into 100ml of each type of the medium. Assay is performed as indicated in the monograph. In cylinder-plate assays, double-layer plates may be prepared by pouring a seed layer (inoculated with the desired microorganism) over a solidified uninoculated base layer. 21 ml of base layer and 4 ml of the seed layer may be generally suitable. Each cylinder was filled with the median concentration of the antibiotic and Then incubate the plates. After incubation, examine and measure the zones of inhibition. The volume of suspension that produces the optimum zones of inhibition with respect to both clarity and diameter determines the inoculum to be used for the assay. 15 For method A-

16. Determination of Inoculum Proceed as described for Method A . The specific antibiotic assay was performed by running only the high and low concentrations of the standard response curve. Read the absorbance's of the appropriate tubes. Determine which inoculum produces the best response between the low and high antibiotic concentrations Use that inoculum for the assay. 16 For method B-

17. Apparatus All equipment is to be thoroughly cleaned before and after each use. Glassware for holding and transferring test organisms is sterilized by dry heat or by steam . Thermostatic control is required at several stages of a microbial assay, when culturing a microorganism and preparing its inoculum and during incubation in a plate assay. Temperature control during the conduct of the experiment is done by maintaining a continuous heat through air or water 17

18. Apparatus A spectrophotometer in which a 580nm(for inoculum preparation) or a 530 nm(for tube assay) filter for allowing only required wavelengths. For the instrument to carry over the assay a flow through cell and a drain are arranged to communicate the exchange of medium components whenever necessary. Set the instrument to zero using uninoculated broth. 18

19. Apparatus Rectangular trays or glass or plastic petri dishes of particular dimensions are used Aga r is made as gel and into which bores are made cylindrically into which antibiotic solution is dropped on or paper discs wetted with antibiotic was placed 19 Cylinder-plate Assay Receptacles

20. Apparatus Test tubes of particular dimension with uniformity in length, width and thickness were maintained along with blemish and scratch free properties. The receptacles are cleaned with 2M nitric acid or chromic acid to take off the left residues. 20 Turbidimetric Assay Receptacles.

21. Assay design Normal assay design : For a cylinder plate assay, comparative study of inhibition zone diameters. For a turbidimetric assay, comparison of turbidities Alternative assay designs: A two level(or 3 level) factorial assay. Single level assay with a standard curve. 21

22. 22 For factorial assay , prepare 2 or 3 test dilutions of both standard and test on the day of assay. For single level assay , prepare 5 dilutions of standard and a single test solution such that it falls in the median value of the corresponding standard preparations. If the computed potency is less than 60% or greater than 150% repeat the assay by adjusting the assumed potencies. Smaller independent assays carried over large number of days is more reliable in potency estimation than a single large assay carried using same plates and tubes .

23. 23 Assay method cylinder plate method

24. One level assay with standard curve prepare from the stock solution, 5 dilution (solutions S 1to S 5 ) representing 5 test levels of the standard and increasing stepwise in the ratio of 4:5. From the information available, assign to antibiotic an assumed potency per unit weight or volume . a stock solution with same solvent as used for the standard. Prepare from this stock solution a dilution to a concentration equal to the median level of the standard to give the sample solution. 24 Standard solution Sample solution

25. 25 A.One level assay with standard curve (cont.) METHOD

26. Estimation of potency Calculate average inhibition zones of each tested samples per plate and the inhibition zones of S3 of 12 plates. The average value of S3 is considered as correction factor. From the averages of each sample’s concentration the correction factor is subtracted . Plot these values on a semi log paper using concentration in mcg/ml on ordinate and zone diameter on abscissa. Highest and lowest zone diameters are calculated and the concentrations obtained after averages are plotted through them. 26

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28. 28 If the averages of the zone sizes of the sample solutions is greater than that of the average of the reference S3 then add the difference to the avg zone diameter of the reference. If the value is smaller then subtract the difference between them from the avg of the reference. Check for the concentrations corresponding to these corrected values from the response line. From the dilution factor potencies can be calculated.

29. Turbidimetric method The method has the advantage of a shorter incubation period for the growth of the test organism (usually 3 to 4 hours) The presence of solvent residues or other inhibitory substances affects this assay more than the cylinder plates assay Care should be taken to ensure freedom from such substances in the final test solutions. This method is not recommended for cloudy or turbid preparations. 29

30. 30 At the same time prepare three control tubes, one containing the inoculated culture medium (culture control), another identical with it but treated immediately with 0.5 ml of dilute formaldehyde solution (blank) and a third containing uninoculated culture medium.

31. Estimation of potency Plot the average absorbance's for each concentration of the standard on semi-logarithmic paper with the absorbance's on the arithmetic scale and concentrations on the logarithmic scale. Construct the best straight response line through the points either by inspection or by means of the following expressions: 31

32. Thankyou... 32