SUBSTANCES PRODUCED BY FUNGI 177 



If the solutions are adjusted to /)H 6.8, it retains its potency for 

 3 months. The crude penicillin does not dialyze through a collodion 

 membrane and resists heating at 6o° to 90° C. for short periods j it 

 remains active when heated at 100° C. for 5 minutes but not for 10 min- 

 utes (737). 



Fleming first reported that penicillin is insoluble in ether. This was 

 found (135) to be due to the alkaline reaction of the filtrate 5 for at fH 

 2.0 ether removes completely the antibacterial substance. The ether 

 extract is evaporated with some water in vacuo at 40° to 45° C, the 

 residual water containing the active substance, which is extremely labile. 



For practical purposes, penicillin is extracted from the acidified cul- 

 ture by means of different organic solvents, such as ether or amyl ace- 

 tate (7, 8). It is then removed from the solvent by shaking with phos- 

 phate buffer or with water at /)H 6.7. Since penicillin is rapidly de- 

 stroyed at a high acidity, the first extraction must be carried out very 

 quickly and at a low temperature. In the presence of the solvents, peni- 

 cillin is stable for several days. The aqueous extract may be partly de- 

 colorized by shaking with charcoal and filtering. The solution is cooled, 

 acidified, and extracted several times with ether or amyl acetate j the 

 extracts are passed through an adsorption alumina column, or through 

 a 2.5 per cent precipitate of an alkaline earth carbonate on silica gel 

 (109). Water may often contain a pyrogenic or heat-producing sub- 

 stance that must be removed from the penicillin. 



The following four main zones were recognized in the chromato- 

 grams, beginning from the top : 



1 . A dark brownish-orange layer, the depth of which is inversely propor- 



tional to the amount of charcoal used for the decolorization ; this zone 

 contains some penicillin 



2. A light yellow layer containing most of the penicillin but none of the 



pyrogen 



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 



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

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



