METHODS OF MEASURING ANTIBIOTIC ACTIVITY 73 



test tubes. These are inoculated with a standard drop (0.04 ml.) of a 

 24-hour culture of the test organisms. Complete or partial inhibition is 

 shown by the absence of turbidity after 24 hours of incubation at 37° C. 

 Dilutions higher than those required for complete or partial inhibition 

 gave, after 24 hours of incubation, only a retarding effect (2, 7) j a nii- 

 croscopic examination (308) indicated defective fission of the bacteria, 

 even though the macroscopic appearance of the culture did not show any 

 inhibition. Pneumococci and S. viridans show marked strain differences 

 by this method. In one experiment with Salmonella tyfhi, partial in- 

 hibition was obtained in a dilution of i : 10,000 j however, elongation 

 of the cells was detected in a dilution of i : 6o,000, a concentration 

 which was considered as a therapeutic possibility (Table 9). 



The Agar Diffusion or "Agar Cuf" Method (7, 284, 285, 385) 



This method, first employed by Reddish (735) and by Ruehle (773) 

 largely for qualitative purposes, was later developed (7, 385) for 

 quantitative use. A suitable agar medium is inoculated with the test or- 

 ganisms {S. aureus or B. subtilis), the active agent being placed upon 

 the agar, within a groove or in a special small glass cup with an open 

 bottom from which the substance diffuses into the medium. The rate of 

 diffusion of the active substance is parallel to its concentration. By meas- 

 uring the zone of inhibition and comparing it with that of a known 

 standard preparation, the potency of the active substances can be calcu- 

 lated. This method has the advantage of simplicity and convenience, 

 since it does not require sterile material and several preparations or 

 duplicates can be tested on the same plate. The method also possesses 

 certain disadvantages, however, since it cannot be used for comparing 

 different substances but is limited to the measurement of activity of only 

 one type of substance ; it cannot be used for the study of unknowns until 

 a standard has been established for each unknown. 



Nutrient agar containing 5 gm. NaCl, 3 gm. meat extract, 5 gm. 

 peptone, 15 gm. agar, 1,000 ml. tap water, and adjusted to ^H 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 

 I : so 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 



