206 BACTERIA PATHOGENIC TO MAN 



of carbolic acid. After forty-eight hours the dead bacilli have settled 

 on the bottom of the jar and the clear fluid above is syphoned off or it 

 is filtered through ordinary sterile filter paper and stored in full bot- 

 tles in a cold place until needed. Its strength is then tested by giving 

 a series of guinea-pigs carefully measured amounts. Less than 0.01 

 c.c., when injected hypodermically, should kill a 250-gram guinea- 



pig- 



The horses used should be young, vigorous, of fair size, and abso- 

 lutely healthy. Vicious habits, such as kicking, etc., make no differ- 

 ence, of course, except to those who handle the animals. The horses 

 are severally injected with an amount of toxin sufficient to kill five 

 thousand guinea-pigs of 250 grams' weight (about 20 c.c. of strong 

 toxin). After from three to five days, so soon as the fever reaction 

 has subsided, a second subcutaneous injection of a slightly larger dose 

 is given. With the first three injections of toxin 10,000 units of 

 antitoxin are given. If antitoxin is not mixed with the first doses 

 of toxin only one-tenth of the doses advised is to be given. At 

 intervals of from five to eight days increasing injections of pure 

 toxin are made, until at the end of two months from ten to twenty 

 times the original amount is given. * There is absolutely no way of 

 judging which horses will produce the highest grades of antitoxin. 

 Very roughly, those horses which are extremely sensitive and those 

 which react hardly at all are the poorest, but even here there are excep- 

 tions. The only way, therefore, is at the end of six weeks or two months 

 to bleed the horses and test their serum. If only high-grade serum is 

 wanted all horses that give less than 150 units per c.c. are discarded. 

 If moderate grades only are desired, all that yield 100 units may be 

 retained. The retained horses receive steadily increasing doses, the 

 rapidity of the increase and the interval of time between the doses 

 (three days to one week) depending somewhat on the reaction follow- 

 ing the injection, an elevation of temperature of more than 3 F. being- 

 undesirable. At the end of three months the antitoxic serum of all the 

 horses should contain over 300 units, and in about 10 per cent, as much 

 as 800 units in each cubic centimetre. Very few horses ever give above 

 1000 units, and none so far has given as much as 2000 units per c.c. 

 The very best horses if pushed to their limit continue to furnish blood 

 containing the maximum amount of antitoxin for several months, and 

 then, in spite of increasing injections of toxin, begin to furnish blood of 

 gradually decreasing strength. If every nine months an interval of three 

 months' freedom from inoculations is given, the best horses furnish high- 

 grade serum during their periods of treatment for from two to four years. 



In order to obtain the serum the blood is withdrawn from the jugular 

 vein by means of a sharp-pointed cannula, which is plunged through 

 the vein wall, a slit having been made in the skin. The blood is carried 

 by a sterile rubber tube into large Erlenmeyer flasks and allowed to 

 clot, the flasks, however, being placed in a slanting position before 

 clotting has commenced. The serum is drawn off after four days by 

 means of sterile glass and rubber tubing, and is stored in large flasks. 



