The Cytotoxins 137 



and by Flexner and Noguchi,* and upon the efifects upon corpuscles 

 of warm-blooded animals, of the poisonous serum of certain eels 

 by MossOjt Camus and Gley,t and Kossel.§ The serious considera- 

 tion of the subject was,however, deferred until Belfanti and Carbone|| 

 showed that if horses were injected with red corpuscles of rabbits, 

 the serum thereafter obtained from the horses would be toxic for 

 rabbits; Bordet** had shown that the serum of guinea-pigs injected 

 several times with 3 to 5 cc. of the defibrinated blood of rabbits 

 acquired the property of rapidly dissolving the red corpuscles of the 

 rabbit in a test-tube, and EhrHch and Morgenrothff had shown the 

 mechanism of the hemolytic action. From this time on the litera- 

 ture of hemolysis rapidly grew and the subject- assumed a more 

 and more important place in the domain of chemico-physiological 

 research. 



The technic of hemolysis is comparatively simple, and it is intended 

 in this chapter to do no more than offer the student a simple method 

 of performing experiments which he can modify to suit his own 

 purposes. 



For the study of hemolysis and hemo-agglutination it is necessary to prepare 

 a s per cent, suspension of the blood-corpuscles in an isotonic salt (NaCl) solu- 

 tion. To do this the blood of the animal is permitted to flow into a sterile tube 

 and is immediately stirred with a small stick or a platinum wire until completely 

 defibrinated. Some salt solution (0.85-0.9 per cent.) is then added and the 

 mixture shaken. It is then placed in a sterile centrifuge tube and rotated until 

 the corpuscles are packed in a mass at the bottom. The supernatant fluid is 

 poured off, replaced by an equal volume of salt solution, and shaken until the 

 corpuscles are again thoroughly distributed. It is then again centrifugated and 

 the fluid again poured off, after which 95 parts (by volume as compared with 

 the corpuscular mass) of the salt solution are added and the fluid thoroughly 

 shaken to distribute the corpuscles. This slightly greenish-red fluid is the s per 

 cent, solution of corpuscles. It is, of course, not permanent, and easily spoils 

 if bacteria enter. It also gradually deteriorates through changes in the corpus- 

 cles, so that it is not usually useful after the third day, even when kept on ice. 



The hemolytic substance to be investigated must be isotonic with the corpus- 

 cles and therefore must be dissolved in, or diluted with, the same salt solution as 

 that used for making the corpuscular suspension. Neglect to observe this re- 

 quirement may lead to error by diminishing the tonicity of the solution and 

 inducing spontaneous or hypotonic disintegration of the corpuscles. 



To secure a specifically hemolytic serum one injects an animal — say a rabbit 

 or guinea-pig — with increasing doses of the washed blood corpuscles of the animal 

 for whose corpuscles the serum is to be made hemolytic, the doses being given 

 intraperitoneally about six times, at intervals of a week. The animal is then 

 bled, the blood permitted to coagulate, the serum separated and filtered, if 

 necessary. 



The contact of the corpuscles and the hemolytic substance is best conducted 

 in small test-tubes holding about 2 cc. of the mixed fluids. It is usually best to 

 work with a constant volume of the blood-corpuscle suspension and varying 

 quantities or concentrations of the hemolytic substances. Two observations 

 are to be made, one after thirty minutes' sojourn in the thermostat at37°C., 



* "Journal of Exp. Med.," 1901-1905, vr, p. 277. 



t "Archiv f. Exp. Path, and Pharmak.," xxv, pp. in and 145. 



i "Compt. rendu de la Soc. de Biol, de Paris," 1898, p. 129. 



§ "Berliner klin. Wochenschrift.," 1898. 



11 "Jour, de la R. Acad. d.'Med. de Torino," 1898, No. 8. 

 ** "Ann de I'Inst. Pasteur," 1898, xn, 688. 

 ft "Berliner klin. Wochenschrift," 1899. 



