39 
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of air frequently renewed, They^ passed filtered air across the surface 
of the culture. This procedure, once widely employed, has now been 
generally abandoned, it having been found that it is sufficient to have 
a large surface of the culture exposed to air. It is now the universal 
practice in almost all laboratories to grow the toxin e in shallow laj^ers 
of broth contained in flat-bottomed vessels such as the Fernbach flask. 
Spronck" believed that the presence of glucose in the bouillon pre- 
vents 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 emplo 3 "ment of a decidedly alka- 
line bouillon, containing 2 to T per cent peptone. The}^ 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 bouillon has a dis- 
tinct alkaline reaction to litmus. 
Martin insists that the boullion should be made with a fluid peptone 
obtained from the stomachs of hogs. 
Spronck,^ in his second process, reported success b}" growing the 
culture in a decoction of yeast. 
In inaii}^ laboratories a preference is given for the simplest methods, 
using an ordinal* v bouillon distinct!}" alkaline, following the recom- 
mendations of Park and Williams. 
The methods used in this laboratory, as previously described, are 
largely taken from the experimental work of Theobald Smith. 
Neutralization . — The reaction is taken with phenol-phthalin as an 
indicator as follows: 
To 5 c. c. of the bouillon add 45 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 while nearly boiling with a solution of 
«Spronck: Sur les conditions dont depend la production du poison dans les cul- 
tures diphteriques. Moyen simple de j^reparer une toxine tres active. C^Ann. de 
rinst. Pasteur, IX, 1895, p. 758. 
^Nicolle: Preparation de la toxine diphterique. <yAnn. de I’lnst. Pasteur, 10, 
1896, p. 3:I3. 
<^Park & Williams: The production of diphtheria toxin. CiJourn. exi)er. med., 
I, 1896, p. 164. 
Martin: Production de la toxine diphterique. <Aim. de I’lnst. Pasteur, 12, 
1898, p. 26. 
^Spronck: Preparation de la toxine diphtericiue; sui)pression de I’emploi de la 
viande. <Ann. de Plnst. Pasteur, 12, 1898, p. 701. 
