THE VITAMINES IN THE VEGETABLE KINGDOM 61 



shown by these investigators that a medium of amino acids and salts 

 did not suffice and that an addition of creatine, creatinine, xanthine 

 and hypoxanthine had no effect. Only when a 0.5 per cent of 

 bouillon was added, did it result in good growth. For the prepara- 

 tion of toxins, the amount of bouillon present must be increased to 10 

 per cent. Addition of peptone, on the contrary resulted in a slight 

 growth and toxin formation. Toxin is not supposed to be a synthetic 

 product of bacterial cells ; but a product of metabolism which makes 

 its appearance only in the presence of certain amino acids and 

 vitamine. 



Agulhon and Legroux (164) investigated the influence of vitamine 

 on B. influenzae (B. Pfeiffer). The growth-accelerating influence of 

 blood, serum and ascitic fluid, could not have been due to protein 

 addition but to the influence of vitamine. The vitamine extract of 

 defibrinated blood was obtained in two ways. By the first, blood 

 was precipitated by about four volumes of absolute alcohol; the 

 precipitate was extracted with the same volume of water, centrifuged 

 and filtered through a Chamberlain filter. By the second procedure, 

 blood was extracted with sodium chloride solution, and the extract 

 heated to 80C. for 15 minutes. From 5 to 10 per cent of these 

 extracts, added to the usual medium, showed a great influence on 

 the growth of B. Pfeiffer. Even a one per cent solution showed a 

 definite effect. It might well be conceived that the vitamines in 

 the cells are present in combination and that alcohol or heating sets 

 them free. They seemed to be insoluble in alcohol, for a cold 

 alcoholic extract was inactive, while one prepared at 80C. was only 

 slightly active. Acetone extracts, prepared either hot or cold, were 

 inactive. Warming to 80C. in the presence of alcohol or acetone, 

 with subsequent drying at a lower temperature did not bring about 

 decomposition, since extraction of this powder with the water yielded 

 an active substance. The watery vitamine extract partially lost its 

 activity after heating to 90C. for 15 minutes. It appears to be more 

 stable when the heating is done in the presence of gelose, although in 

 this case too, it is partially or completely inactivated, depending upon 

 whether the temperature is raised only to 100or to 120C. When 

 a totally defibrinated blood was used, it was observed that the vita- 

 mines were found in the corpuscles, since the watery extract of the 

 serum gave completely negative results, while the washed blood 

 corpuscles gave positive results. Hemoglobin and blood corpuscle 



