194 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 



the nature of the antibiotic action of streptomycin and its utilization for 

 chemotherapeutic purposes is presented elsewhere (pp. 287-296). 



It was at first suggested (947) that 3 units of activity be recognized 

 for measuring streptomycin: an S unit, or the amount of material that 

 will inhibit the growth of a standard strain of E. coU in i ml. of glu- 

 cose-free nutrient agar or broth j an L unit, inhibition on a liter basis j a 

 G unit, inhibition on a dry weight basis of crystalline material. Since 

 streptomycin base was found to be 1,000 S units per i mg., it was de- 

 cided to accept the weight of streptomycin as a basis of standardization: 

 I S unit is thus equivalent to i microgram of the pure base. 



Streptomycin can be isolated from the medium by several proce- 

 dures. In one method (115), culture filtrates of S. griseus assaying 100 

 to 180 units of streptomycin per ml. served as the starting material. 

 Several common adsorption agents, such as charcoal, can be used to re- 

 move the active material from the culture. The substance is then eluted 

 with hydrochloric acid in 95 per cent ethanol. Anhydrous hydrogen 

 chloride in methanol is a more convenient reagent, since the crude 

 streptomycin can be precipitated directly from the methanol solution 

 with ether. The filtrate is clarified at pH 2 with 0.5 per cent carbon j 

 this is followed by removal of the streptomycin, at ^H 7, with i per 

 cent carbon, which is washed successively with water, neutral ethanol, 

 and neutral methanol, and the streptomycin is eluted by two or three 

 extractions with o.i N methanolic hydrogen chloride. The alcoholic 

 extracts are combined and 2 to 3 volumes of ether added, precipitating 

 the crude streptomycin chloride as a light-brown amorphous powder. 

 When the methanol solution contains much water, a sticky gum results. 

 The recovery of the streptomycin by this method varies from 30 to 50 

 per cent, the product assaying from 150 to 300 micrograms. 



For further purification, a faintly acid solution of crude streptomycin 

 chloride in 70 to 80 per cent methanol is percolated over a sulfuric 

 acid-washed alumina column (^H 5 to 6) j an inactive fraction giving 

 a positive Sakaguchi test first appears, followed by a Sakaguchi-negative 

 fraction. This test parallels the antibiotic action of the fractions. A small 

 amount of active material remains on the column and can be washed 

 through by lowering the methanol content of the solvent. This ma- 

 terial contains sulfate ion but no chloride. The streptomycin sulfate 



