78 ANTIBIOTIC ACTION OF ANTAGONISTS 



smooth to the rough phase there is a marked increase in resistance to 

 certain penicillins but not to others. This has a bearing upon a knowl- 

 edge of the chemical entities present in the penicillin preparations. 



This method is also very convenient for measuring the activity of 

 streptothricin and streptomycin. A standard curve is obtained by filling 

 the cups in quadruplicate with dilutions of the standard containing lO, 

 20, 40, 60, 80, and 100 |jg/ml. The dilution of the unknown contains 

 about 50 Mg/ml. After overnight incubation at 30° C, the inhibition 

 zones around the cups are measured and plotted to give a standard 

 curve. The concentrations of the unknowns are read off this curve by 

 projecting the value of the inhibition zones. 



A standard streptomycin agar was developed (582) consisting of 

 6 gm. peptone, 1.5 gm. beef extract, 3.0 gm. yeast extract, 15.0 gm. 

 agar, 1,000 ml. distilled water, -pH after sterilization 7.9 ±0.1. The 

 test strain B. subtilis is grown on agar or in submerged liquid medium. 

 The cells are suspended in sterile 0.5 M potassium phosphate buffer, 

 ^H 7.0, and pasteurized to kill the vegetative cells. The spore suspen- 

 sion is counted by plating and is stored at 2° to 4° C. Twenty-milliliter 

 portions of sterile agar are first poured into the plates 5 the agar is 

 allowed to harden and is then covered with 4 ml. of the seeded agar 

 containing about 250,000 spores per ml. of agar. The plates may be 

 stored at 2° to 4° C. for several days. The test sample is diluted with 

 equal volume of 0.2 M potassium phosphate buffer, ^H 7.9, and all 

 subsequent dilutions are made with o. i M buffers. Either paper discs or 

 cups may be used, 4 to 6 per plate ; in the case of discs 0.08 ml. of sample 

 is added rapidly to each disc after it has been placed on the agar. A 

 standard is used on each plate. The plates are incubated at 30° C. for 15 

 to 30 hours. At 37° C. the zones develop after 4 to 6 hours' incubation. 

 A typical curve is shown in Figure 8. 



Turbidimetric Method 



End-point methods have long been recognized as having many limi- 

 tations. Since it is difficult to determine accurately the end point and 

 since it takes a relatively much larger amount of an antibiotic substance 

 to inhibit completely the growth of the test organism as compared with 

 only 50 or 99 per cent inhibition, the suggestion has been made that 



