134 Immunity 



hemolytic action. From this time on the literature 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 5 per cent, suspension of the blood-corpuscles in an iso tonic 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 5 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 cor- 

 puscles, 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 corpuscles 

 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 at 3 7C., 

 the other after twenty-four hours in the ice-box, both observations being made on 

 the same series of tubes. Hemolysis is shown by the appearance of a beautiful 

 clear red color of the formerly cloudy greenish suspension. One must notice the 

 difference between partial and complete hemolysis, different additions of the 

 hemolytic substance being required for these results. 



Cytolysis. The phenomena of hemolysis corresponds to those 

 by which many other cells, vegetable and animal, are destroyed and 

 dissolved through the activity of immunity products. Delezene* 

 first produced a leukolytic or leukocyte-destroying serum by in- 

 jecting animals with the leukocytes of a heterologous species; 

 Metalnikofl,t by injecting the spermatozoa of one animal into 

 another of another species, produced a spermatoxic or spermalytic 

 serum; von Dlingern,t a serum capable of dissolving the ciliated 

 epithelium scraped from the trachea of an ox by injecting the 

 dissociated epithelial cells into an animal, Delezene found that 



*"Compt. rendu de 1'Acad. de Sciences de Paris," 1900. 

 "Ann. de 1'Inst. Pasteur," 1899. 

 ^Munchener med. Wochenschrift," 1899. 



"Compt. rendu de PAcad. de Sciences de Paris," 1900, cxxx, pp. 938 and 

 1488. 



