METHODS OF MEASURING ANTIBIOTIC ACTIVITY 77 



peptone, 15 gm. agar, 1,000 ml. tap water, and adjusted to fH 6.8, is 

 poured into plates to a depth of 3 to 5 mm. The plates are seeded thor- 

 oughly with the test organism (S. aureus) by flooding with i: 10 or 

 1:50 dilution of i6-to-24-hour-old broth culture in sterile water. The 

 excess fluid may be removed with a pipette. The surface of the agar is 

 allowed to dry somewhat in the 37° C. incubator for i to 2 hours, the 

 lids of the plates being raised about i cm. above the bottoms of the 

 dishes. Sterile short glass cylinders (5 mm. inside diameter) are placed 

 on the agar, the lower edge of the cylinder sinking into the agar, and are 

 filled with the test solution. Several cylinders may be placed in one dish. 



For measuring the activity of penicillin, the plates are incubated for 

 12 to 16 hours at 37° C. The diameter of the clear zone around the 

 cylinder is measured with pointed dividers to the nearest 0.5 mm. The 

 relation of concentration of penicillin in the solution to the zone of in- 

 hibition, or the "assay value," is expressed by a curve which is obtained 

 with standard solutions. This curve tends to flatten out above 2 units of 

 penicillin per milliliter. The assay value is not influenced by the -pH of 

 the test material, thickness of the agar, or sterility of the material. 



The Oxford unit (O.U.), as determined by this method, is the 

 amount of penicillin that will just inhibit completely the growth of the 

 test strain of S. aureus in 50 ml. of medium. Thus, a preparation con- 

 taining one unit of penicillin per milligram of material just inhibits the 

 growth of the test organism in a dilution of 1 150,000. 



An international standard for penicillin, based upon crystalline ma- 

 terial, has been adopted. 



In one of the modifications of the agar diffusion method, a spore sus- 

 pension of B. suhtills is used as the test organism. It is grown for sev- 

 eral days under forced aeration, and the cultures are pasteurized in or- 

 der to destroy the vegetative cells. The spore suspension is stored in the 

 cold and used as the stock inoculum j it is titrated in order to determine 

 the optimum amount for seeding purposes. The lowest level (usually 

 0.1 to 0.2 ml. per 100 milliliters of agar) that gives a dense, continuous 

 growth of the organism under the assay conditions is selected as the 

 optimum. 



It has been reported (839) that when B. suhtilis changes from the 



