METHODS OF STUDYING H^MOLYSINS. 331 



course, the test is made in very narrow test-tubes. 1 The serum 

 to be tested is added to the various tubes in decreasing amounts. 

 The volume of fluid should be made the same in all the tubes by 

 the addition of salt solution, for the total amount of fluid present 

 may influence the course of haBmolysis. We usually keep the tubes 

 in a thermostat at 37 C. for two hours, frequently shaking them if 

 necessary. They are then kept in the refrigerator at 8 C. overnight, 

 which allows the intact blood-cells to settle. In the cases thus far 

 examined by us this method has always sufficed to produce the 

 maximum amount of haemolysis, though, of course, in a given case 

 it may have to be modified to suit the circumstances. 



It should be mentioned that in testing any substances for haBmolytic 

 action, the blood-cells must always be freed from serum by repeated 

 washing, for the serum may in some instances (e.g. with solanin) 

 give rise to a marked inhibitory action and so lead to errors. 



III. The Technique of Immunization. 



So far as the production of hcemolytic amboceptors by means of 

 immunization is concerned, only a few very general rules can be 

 given, for thus far sufficient systematic investigations have not been 

 made to determine the optimal conditions in any one direction. In 

 immunization one always selects such animals whose serum by itself 

 is not at all or but slightly hsemolytic for the blood employed, for 

 then the development of a hsemolysin is most readily determined 

 and the normal serum of this species always furnishes an ideal com- 

 plement. If animals are immunized whose serum by itself already 

 acts hsemolytically on the blood used, it is necessary to make an 

 exact preliminary determination of the haemolytic power of the 

 normal serum, and also to make a simultaneous control with normal 

 serum, when making the hsemolytic experiments. 



In some instances it may be necessary to subject the blood to a 

 preparatory treatment, for the purpose of removing the serum more 

 or less completely. This is done by means of the centrifuge and 

 is required especially in those cases in which intravenous injections 

 are made, or if large amounts of a blood are employed whose serum 



1 In certain cases the employment of very high columns of blood is indicated, 

 for in that case the development of zones (colorless feebly red strongly red) 

 permits of a very accurate estimation of the period of incubation of the poison, 

 or of the different vulnerability of the blood-cells. See also Madsen, I.e. 



