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 half 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.c. of pure toxins, of which 1 to 10 c.c. fatal to a 500- 

 gm. guinea-pig ; eighth day, 1 c.c. : fourteenth day, 1.5 c.c. : twentieth day, 

 2 c.c. ; twenty-eighth day, 3 c.c. ; thirty-third day, 5 c.c. ; thirty-eighth day, 

 8 c.c. ; forty- third day, 10 c.c. ; forty-seventh day, 20 c.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, 250 c.c. ; eighty-eighth clay, 

 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 oedema 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 



