CONTRIBUTION TO THE STUDY OF SERUM-THERAPY IN DIPHTHERIA. 661 
facts to enable us to judge the method properly. Now we are 
enabled to declare that our results confirm, in all essential 
points, those that have been published by Behring and his col¬ 
laborators. 
i. Preparation of the Diphtheritic Toxine .—The animals 
which furnish the antitoxic serum are immunized against diph¬ 
theria, that is to say, they are accustomed to the diphtheritic 
toxine. 
The toxine is produced by cultivating the virulent diph¬ 
theritic bacillus in broth, in the presence of air. Under usual 
conditions, the cultures' must be maintained fora number of 
months at a temperature of 37c. (98.6 F.) in order to allow the 
poison to accumulate. A more rapid procedure has been 
recommended by Roux and Yersin ; it consists in making the 
cultures in a current of damp air. 
Flat-bottomed flasks are used, provided with a lateral tube 
(Fernbachs flasks), containing alkaline broth, peptonized at 2 
per cent, so that the liquid layer will be of but slight thickness. 
After sterilization in the autoclave, they are impregnated with 
fresh and very virulent diphtheritic bacilli, and the incubator is 
heated to 37c. When the development has well begun, the 
bent tube of each flask is connected by means of a rubber tube 
with a pipe issuing from a copper tube which is itself connected 
with a water blast pump. By means of screw closers placed on 
the lubber tubes, the amount of air entering the neck of each 
flask may be regulated as it arrives after bubbling up through 
a washing jar. This arrangement is better than that which 
connects all the flasks together and supplies them all with air 
from the same current. After three weeks, or four at the most, 
the culture is sufficiently rich in toxine to be employed. At the 
bottom of the flasks we see a heavy deposit of bacilli, while the 
surface is covered with a veil formed by younger microbes. At 
this time the reaction is strongly alkaline. 
All diphtheritic bacilli, even when they appear to be equally 
virulent for guinea-pigs, do not give the same amount of toxine 
in the cultures. A trial of bacilli from various sources will 
