226 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS 
The principles of immunization Calmette resorted to were: 
(1) Accustoming the animal to frequently repeated, gradually increased 
doses of diluted venom. 
(2) The administration of a fatal dose of venom, followed by immediate 
curative therapeutic treatment with chloride of calcium or gold. 
(3) The administration of repeated increasing doses of venom mixed 
with the chloride of calcium or gold. 
Of these three methods he preferred the first. The serums obtained from 
the immunized animals showed preventive as well as curative effects in ani- 
mal experiments. 
Somewhat later Calmette ' added to the above facts that the antitoxic 
property of his antivenin was equally effective in counteracting the action of 
all kinds of snake venoms, namely, those of vipers, Notechis scutatus and 
Pseudechis porphyriacus, as well as that of cobra, with which it was prepared. 
This statement, and also another, that the immunity can be developed by 
repeated injections of weak solutions of alkaline hypochlorites without the 
venom, have since undergone a complete modification through more careful 
and extended investigations by later workers on this subject, and to-day the 
question of specificity of antivenin may be considered as settled against this 
assertion. ‘To this point I shall return at length. 
As his study progressed Calmette ? at last found another method of immuni- 
zation, namely, by injecting animals with gradually increasing doses of venom 
modified by heat. He pointed out that the temperature of 75° C. inactivates 
all the local irritant and cedema-producing principles without affecting the 
activity and the immunizing quality of the venom. After 48 hours the ani- 
mals readily endure a fatal dose of venom and at the end of a month have 
quite a high immunity. At this time Calmette was still uncertain of the 
therapeutic value of the antivenin for the treatment of snake bite. 
He * next prepared the antivenin by immunizing two asses to venom (cobra) 
previously heated. ‘The first received 0.0022 gm. within a period of 97 days 
and another 0.0016 gm. within 76 days. The serum of the first was of such 
antitoxic value that 0.5 c.c. neutralized 0.001 gm. of the venom; 4 c.c. of the 
serum injected 4 hours before the venom protected against 2 minimal lethal 
doses. If one inoculated a rabbit with enough venom to kill a control rabbit 
in 3 hours, and an hour subsequently administered 4 to 5 c.c. of the serum, 
the animal recovered. The later the therapeutic serum is administered, 
however, the less certain is recovery to take place, so that Calmette in his 
experiments placed 14 hours as the limit of certain cure. He observed that 
the local action of the highly irritative venoms of Crotalus, Lachesis cerastes, 
and L. trigonocephalus is always present when these venoms are injected into 
the immunized animals. 

1Calmette. Propriétés du sérum des animaux immunisés contre le venin des serpents, thérapeutique 
de lenvenimation. Compt. rend. d. Acad. d. Sci., Paris, 1894, CXVIII, 720; Atti XI. Cong. 
Med. internaz., Roma, 1894, II, Patol. gen. ed Anat. patol., rog. 
2 Calmette. Contribution 4 l’étude du venin des serpents. Ann. |’ Inst. Pasteur, 1894, VIII, 275. 
3 Calmette. Ibid., 1895, IX, 225. 
