74 ANTIBIOTIC ACTION OF ANTAGONISTS 



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 zone around the cylinder 

 is measured to the nearest 0.5 mm. by means of pointed dividers. 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 fH of 

 the test material, the thickness of the agar, or the sterility of the ma- 

 terial. 



The "Oxford unit," as determined by this method, is the amount of 

 penicillin that will just inhibit completely the growth of the test strain 

 of 5. aureus in 50 ml. of medium. Thus, a preparation containing one 

 unit of penicillin per milligram of material just inhibits the growth of 

 the test organism in a dilution of 1:50,000 (7, 273, 385). 



One of the modifications of this method (285) consists in using a 

 spore suspension of B. subtilis 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. 



This method is also very convenient for measuring the activity of 

 streptothricin. A standard curve is obtained by filling the cups in quad- 

 ruplicate with dilutions of the standard containing 10, 20, 40, 60, 80, 

 and 100 streptothricin units per milliliter. The dilution of the unknown 

 contains about 50 units per milliliter. After overnight incubation at 

 30° C, the inhibition zones around the cups are measured and plotted 

 to give a standard curve. The units of the unknowns are read off this 

 curve by projecting the value of the inhibition zones. 



