316 PROTECTIVE INOCULATIONS. 
of an antitoxin suitable for use in the treatment of diphtheria in man. 
The horse has been found the most suitable animal for this purpose, 
on account of his slight susceptibility and the ease with which a high 
degree of immunity can be established; and because of the large 
amount of blood that may be drawn without injury to the animal. 
Roux prepares his toxin by cultivating the diphtheria bacillus in a 
slightly alkaline bouillon made from beef and containing two per 
cent of peptone and 0.5 per cent of sodium chloride. This medium 
is placed in flat-bottomed flasks, and should not be more than balf an 
inch in depth. Two glass tubes pass into the flask, which serve for 
inlet and outlet tubes to be used in passing a current of air over the 
cultures. This is commenced when the growth is fairly started, at 
the end of twenty-four hours, and the air should be moist to prevent 
the evaporation of the culture. In Roux’s laboratory a flask is used 
which has a tube attached to one side, about an inch from the bottom, 
and which is known as a Fernback flask. A flocculent deposit falls 
to the bottom and gradually accumulates for about a month. This 
consists of bacilli which have for the most part lost their vitality and 
are undergoing degeneration. At the end of thirty days, during 
which time they are kept in an incubating oven at a temperature of 
37° C., the cultures are passed through a Pasteur-Chamberland filter, 
and 0.5 per cent of carbolic acid may be added in order to preserve 
them. This filtrate is so toxic that a dose of 0.1 cubic centimetre 
will kill a guinea-pig weighing five hundred grammes in less than 
forty-eight hours. A healthy horse is selected and receives at first a 
dose of 0.5 cubic centimetre of the filtered culture (or of the clear 
‘fluid obtained from a culture by decantation, and containing 0.5 per 
cent of carbolic acid). The dose is gradually increased at intervals 
of a few days, and is followed each time by some febrile reaction and 
tumefaction at the point of inoculation. When the reaction is exces- 
sive, a little Gram’s solution is added to the following dose. The 
usual plan of treatment is stated by Kinyoun as follows: 
‘‘First day, 1 to 2 ¢.c. of pure toxins, of which 1 to 10 c.c. fatal to a 500- 
gm. guinea-pig ; eighth day, 1c¢.c.; fourteenth day, 1.5 c.c. ;: twentieth day, 
2c.c.; twenty-eighth day, 3 c.c. ; thirty-third day, 5 ¢.c. ; thirty-eighth day, 
8 c.c.; forty-third day, 10 c.c. ; forty-seventh day, 20 ¢.c.; fifty-first day, 30 
c.c.; fifty-sixth day, 50 c.c.; sixty-second day, 50 c.c. ; sixty-eighth day, 60 
c.c. ; seventy-fourth day, 100 c.c. ; eightieth day, 250c.c.; eighty-eighth day, 
250 c.c. 
‘‘ When the first injections are given there is quite a marked local and gen- 
eral reaction to the poison; there is an cedema at the point of the injection, 
which is followed by a distinct inflammatory process—hard in the centre and 
soft and cedematous at its periphery. The general reaction is manifested by 
a rise in the temperature, 1° to 2° C., loss of appetite, and occasionally cramps. 
The reaction must be taken as the guide in the future dosage, and a sufficient 
