39 
of air frequently reneAved. They passed filtered air across the surface 
of the culture. This procedure, once widely employed, has now been 
generally abandoned, it haA^ing been found that it is sufficient to haA^e 
a large surface of the culture exposed to air. It is now the uniA^ersal 
practice in almost all laboratories to groAV the toxine in shallow laA^ers 
of broth contained in flat-bottomed A’essels such as the Fernbach flask. 
Spronck^' believed that the presence of glucose in the bouillon pre- 
A^ents the bacillus of diphtheria from producing strong toxic sub- 
stances. He recommended, in order to eliminate the glucose, to use 
meat aged almost to the point of putrefaction. He also recommended 
that care should be taken to use a peptone freed from glucose and the 
addition to the bouillon of 0.5 per cent of sodium chlorid and a small 
quantity of carbonate of lime. 
Nicolle^ used fresh beef killed the same morning. He added 2 per 
cent peptone and 5 per cent salt. 
Park and Williams^ recommend the employment of a decidedly alka- 
line bouillon, containing 2 to d per cent peptone. They obtained the 
best results with a bouillon distinctly neutral to litmus and then an 
additional 7 c. c. of normal soda per liter. Such a l)ouillon has a dis- 
tinct alkaline reaction to litmus. 
MartiiP^ insists that the l^oullion should be made AAuth a fluid peptone 
obtained from the stomachs of hogs. 
Spronck,^ in his second process, reported success 1)A" groAving the 
culture in a decoction of A^east. 
In maiiA" laboratories a preference is gh^en for the simplest methods, 
using an ordinaiy bouillon distinctH alkaline, following the recom- 
mendations of Park and Williams. 
The methods used in this laboratoiy, as preAUouslA" described, are 
large!}" taken from the experimental AA^ork of Theobald Smith. 
Neutralization . — The reaction is taken with phenol-phthalin as an 
indicator as follows: 
To 5 c. c. of the bouillon add d5 c. c. of distilled water plus 1 c. c. 
of a 0.2 per cent solution of phenol-phthalin in 95 per cent alcohol. 
. . . N . . 
Heat to boiling and titrate Avhile nearly boiling AAuth a solution of 
«Spronck: Sur les conditions dont depend la production du poison dans les cul- 
tures diphteriques. Moyen simple de preparer une toxine tres acti\"e. yAnn. de 
rinst. Pasteur, IX, 1895, p. 758. 
^Xicolle: Preparation de la toxine diphterique. <7 Ann. de FInst. Pasteur, 10, 
1896, p. 333.' 
ePark & Williams: The production of diphtheria toxin. <Journ. exper. med., 
I, 1896, p. 164. 
Martin: Production de la toxine diphterique. <Ann. de FInst. Pasteur, 12, 
1898, p. 26. 
^Spronck: Preparation de la toxine diphterique; suppression de Femploi de la 
Auande. <Ann. de FInst.~ Pasteur, 12, 1898, p. 701. 
