70 TOXINES AND ANTITOXINES. 



the cultivation. A culture forty-two days old killed a rabbit in 

 five to six hours with toxic symptoms analogous to those of a 

 severe attack of diphtheria, whilst six days was necessary with 

 an equal dose (35 c.c.) of the same cultivation of seven days' 

 growth. The poison was also characterised by the same pheno- 

 mena and the same degree of infectiousness as in the case of 

 inoculation with living cultures. The conclusions of Roux and 

 YERSIN were fully confirmed by KOLISKO and PALTAUF/ who 

 produced the symptoms of poisoning by means of the filtrates of 

 a bouillon cultivation of fourteen days' growth. 



Roux had already noticed that the poison was destroyed by 

 heat, and concluded that it was akin to the enzymes. As such 

 it was, in his opinion, a secretion product of the diphtheria 

 bacilli excreted by them into the surrounding media. Yet this 

 view was apparently quite out of keeping with the fact that 

 young and vitally active cultivations produced relatively little 

 toxine, whilst older cultivations gave richer yields. 



Hence, GAMALEIA 2 concluded that diphtheria virus was not 

 a secretion product of the bacilli but a constituent of their cell 

 contents, which was not discharged by healthy bacilli, and that 

 it was only when the cultivation became old and many of the 

 bacilli died that the poison was extracted from their decomposed 

 cells. This view, however, has frequently been convincingly 

 opposed. H. KossEL, 3 in particular, proved that the toxine was 

 a secretion product and not a decomposition product, by the fact 

 that, on the one hand, he observed a very plentiful formation 

 of poison, even in quite young cultivations (two days old) when 

 grown by suitable methods, and that these began to grow weaker 

 after as little as five days, whilst, on the other hand, he showed 

 that but little toxine was present in the cells of the bacilli 

 themselves. 



He cultivated diphtheria bacilli upon as large a surface as possible, 

 making the inoculations from the surface skin of a cultivation of one day's 

 growth. After some days the bacilli formed a tough skin from which he 

 poured off the bouillon. The cells of the bacilli were then repeatedly 

 washed with distilled water and subjected to centrifugal force until the 

 washings no longer gave the biuret reaction, after which they were 

 extracted with water rendered faintly alkaline. This extract was only 



1 Kolisko and Paltauf, "Zum Wesen des Croup und der Diphtheric," 

 Wien. klin. Woch., 1889, No. 2. 



2 Gamaleia, "Les poisons bacte"riens, " Arch, de M6d. Exper., 1892. 



3 H. Kossel, "Zur Kenntnis d. Diphtheriegiftes," CentralU. f. Bakt., 

 xix., 977, 1898. 



