METHODS OF MEASURING ANTIBIOTIC ACTIVITY 73 



24 hours) is important. The plates are incubated at 28° or 37° C. for 

 16 to 24 hours, and readings are made. The highest dilution at which 

 the test organism fails to grow is taken as the end point. Activity is ex- 

 pressed in units, using the ratio between the volume of the medium and 

 the end point of growth or the dilution at which growth is inhibited 



(964). 



The bacteriostatic and fungistatic activity of several antibiotic sub- 

 stances is shown in Table 7. 



Serial Dilution Method 



Once a substance is characterized as regards its selective action upon 

 specific bacteria, its activity or concentration can be measured more ac- 

 curately by the liquid dilution or titration method. One test organism is 

 selected, usually a strain of S. aureus. Different strains may vary in their 

 action. Definite volumes of the test medium are placed in test tubes and 

 sterilized (sterility is essential in this method), and various dilutions of 

 the active substance are added. The dilutions can range in order of 3 :i, 

 2:1, or even narrower, namely in series of 1.2:1, 1.5:1, etc. The tubes 

 are inoculated with the test organism and incubated for 16 to 24 hours. 

 In some cases the medium is inoculated before it is distributed into the 

 tubes. The highest dilution of the antibiotic giving complete inhibi- 

 tion of growth, as expressed by a lack of turbidity of medium, is taken 

 as the end point. Activity is expressed in units as above. 



The dilution method has several disadvantages: every assay takes 

 much time 5 during chemical fractionation, the substance may become 

 contaminated with bacteria not sensitive to the active substances 5 only 

 one organism can be used in a single series of tests. 



One modification of the method has been adapted for measuring the 

 activity of penicillin. Several dilutions of the active agent are prepared 

 and 0.5 ml. portions added to 4.5 cm. quantities of liquid medium in 

 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 (1,5)5 ^ "^i" 

 croscopic examination (311) indicated defective fission of the bacteria, 



