SUBSTANCES PRODUCED BY FUNGI 203 



3. An orange layer which contains some penicillin and some or all of the 



pyrogen 



4. A brownish or reddish-violet layer which contains almost no penicillin; 



the pigment disappears on exposure to light 



The fourth fraction is discarded, and the others are eluted with 

 M/15 phosphate buffer (-pH 7.2). The penicillin is again extracted 

 with ether, then with water, sodium hydroxide being used to adjust the 

 fH. Since penicillin is destroyed readily in alkaline solution, care must 

 be taken in adding the alkali. The "nonpyrogenic" or "therapeutic" 

 fraction, which contains about 80 per cent of the penicillin, is extracted 

 with pyrogen-free water. It is a deep reddish-orange liquid, yellow in 

 dilute solution, with a characteristic smell and bitter taste. 



Another method for obtaining penicillin has been suggested (638). 

 In this method, the culture medium was adjusted to fH 3 to 4, satu- 

 rated with ammonium sulfate and extracted with chloroform. The con- 

 centrated chloroform extract was treated with phosphate buffer at fH 

 7.2 to remove the active substance. This process was repeated, the less 

 active substance being separated from the active fraction by extraction 

 with chloroform at different ranges. By precipitating the concentrated 

 extracts from petroleum ether, the free acid form of penicillin was ob- 

 tained. By saturating the chloroform-benzol solution with dry am- 

 monia gas, an ammonium salt was obtained which gave a dark yellow 

 microcrystalline powder. The salt was more stable than the acid form. 

 By acetylating or benzoylating the ammonium salt a further increase in 

 stability was obtained. This penicillin was strongly dextrorotatory and 

 had an adsorption maximum of 2,750 A°. The preparation had an ac- 

 tivity of 32,000,000 dilution units against hemolytic streptococci, 

 which corresponds to about 240 Oxford units per milligram. 



When ether is used, the medium is adjusted to fH 3, extracted sev- 

 eral times, the ether extract treated with dilute NaHCOa, the aqueous 

 solution acidified and again extracted with ether j this is followed by 

 shaking with excess of BaCOo, separating aqueous phase, filtering, and 

 evaporating in frozen state (145). 



By the adsorption method, activated charcoal or fuller's earth is used 

 (20 gm./L). The solution is first acidified to /)H 3.6, filtered, neutral- 

 ized, treated with charcoal, and filtered. Ethanol is used to remove the 



