The Cytotoxins 145 



The technic of hemolytic studies 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. 



The Preparation of Blood-corpuscles. For the study of hemo- 

 lysis and hemo-agglutination it is necesary to prepare a 5 per cent, 

 suspension of the blood-corpuscles in an isotonic salt (NaCl) solution. 

 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 thor- 

 oughly shaken to distribute the corpuscles. This slightly greenish 

 fluid is the 5 per cent, solution of corpuscles. It is, of course, not per- 

 manent, and easily spoils if bacteria enter. It also gradually deterior- 

 ates through changes in the corpuscles, 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 requirement 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 lo-c.c. doses of defibrinated blood of 

 the animal for whose corpuscles the serum is to be made hemolytic, 

 the doses being given intraperitoneally or intravenously, 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 c.c. 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 37 C., 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 dif- 

 ference between partial and complete hemolysis, different additions 

 of the hemolytic substance being required for these results. 



Cytolysins. It was soon found that the phenomena of 

 hemolysis corresponded to those by which many other cells, 

 vegetable and animal, were destroyed and dissolved through 

 the activity of immunity products. To such as were applic- 

 able to bacteria, the term bacteriolysis was applied ; to such 

 as were applicable to animal cells, the term cytolysis was 

 applied ; epitheliolysis, endotheliolysis, hemolysis, spermato- 



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