662 
E. ROUX, L. MARTIN AND A. CHAILLOU. 
show those that make the largest amount of toxine. No bac¬ 
teriologist will be astonished to hear that the strength of the 
toxine is not always the same in cultures made, in all appear¬ 
ances, under identical conditions. Hence it is well to make a 
large provision of toxine before beginning a series of experi¬ 
ments, so that these may be thoroughly comparable one with 
another. 
The finished cultures are filtered upon a Chamberlain tube 
and the clear fluid kept in well filled flasks, corked and kept 
in the dark at the ordinary temperature. Thus prepared, the 
toxine commonly kills a 500 grm. guinea-pig in 48 hours, with 
a dose of a tenth of a c.c. After a long time it loses its activ¬ 
ity, but very slowly if kept in the manner indicated above. 
11. Immunization of Animals. —Dr. Carl Frankel was the 
first who immunized guinea-pigs against diphtheria by carefully 
inoculating them by injection, with toxine that was modified by 
heating it to 70.c (158° F.) Then Behring recommended the 
mixture of toxine with trichloride of iodine. He now prefers to 
inject very small doses of pure toxine at sufficient intervals to 
allow the animals to remain healthy. Brieger and Wasserman 
obtain the result by injecting a culture of diphtheritic bacilli in 
thymus broth, after heating it to 65-70c. for a quarter of an 
hour. All these processes are successful, but it should be well 
understood that the thorough immunization of small animals, 
such as rabbitts and guinea-pigs, is always a lengthy and deli¬ 
cate operation. 
The method which we prefer is that which employs the 
iodized toxines used by Roux and Vaillard in their researches 
upon tetanus. Diphtheritic toxine combined with iodine is 
much less dangerous than pure toxine. Just before using the 
toxine we add one-third of its volume of Gram’s solution, and 
then the mixture is injected under the skin. A medium sized 
rabbit can stand from the first half a cc. of this liquid ; after a 
few days the injection is repeated and this is kept up for several 
weeks, after which we may increase the amount of iodized 
toxine or lessen the proportion of iodine. Later still pure 
