DIPHTHEROID ORGANISMS 31 



of Hodgkin's disease we ought to be able to show a definite relationship 

 between serums from patients and organisms isolated from these and 

 other cases. By the delicate method of complement fixation we may 

 detect similar antigenic substances and establish the identity, if such 

 there be, of the several strains isolated. 



AGGLUTINATION 



Four serums, obtained from 4 authentic cases of Hodgin's disease were 

 studied. Eleven strains of bacteria were tested, of which 8 were isolated from 

 glands in Hodgin's disease and the remaining 3 from a case of leukemia, 

 ascitic fluid in cirrhosis and blood. 



Forty-eight-hour agar cultures were suspended in salt solution and emul- 

 sions made up to approximately the same density. These were shaken with 

 glass beads for thirty minutes and then centrifugated for 5 minutes at low 

 speed to throw down coarser particles. Equal volumes of the different serums 

 (diluted with NaCl solution) were mixed with the bacterial suspensions in a 

 hanging drop. The preparations were well sealed with vaselin and incubated at 

 room temperature for 1 hour, then at 38 C. for another hour, when the results 

 were read. 



The results were entirely negative and confirm the results of Fox. 



COMPLEMENT FIXATION 



In these experiments the aim was to show by tests for cross fixation what 

 relationship, if any, existed between the different types of bacteria isolated 

 from cases of Hodgkin's disease. The cultures, as in the preceding, were 

 obtained from Drs. Bunting, Yates and Rosenow and from cases studied by 

 me. These strains have been described fully in Part I. 



Four strains were used in the production of immune serums from rabbits. 

 These cultures are designated 2, 8, 13 and 57. No. 2 was isolated by Dr. C. H. 

 Bunting from a patient who had a very acute form of Hodgkin's disease ; it 

 fermented dextrose and saccharose with acid. No. 8 was sent to me by Dr. 

 E. C. Rosenow, and labeled B. hodgkinii; it produced acid in dextrose, maltose, 

 saccharose, and dextrin. No. 13 was obtained from the same source; it fer- 

 mented dextrose and maltose. No. 57 was isolated by me from a case of acute 

 Hodgkin's disease. This organism fermented none of the carbohydrates and 

 was identical in this respect with a strain (No. 1) obtained from Dr. Bunting. 



Healthy rabbits, weighing about 1,600 gm., were systematically treated with 

 each of these cultures heated to 58 C. for 30 minutes. Injections were made 

 with increasing doses every week for a period of 6 weeks and the serums drawn 

 10 days after the last injection. 



Antigens were prepared by grinding to a fine powder the flocculent precipi- 

 tate obtained from a mixture of bacteria with an excess of absolute alcohol. 

 The powder was dried in vacuo and then ground in a mortar with solid NaCl. 

 Subsequently sterile distilled water was added to isotonicity and the anti- 

 complementary unit determined. 



Complement was obtained from normal guinea-pigs and titrated in the 

 usual way. Sensitizer was prepared with thoroughly washed sheep blood cells 

 injected into rabbits and the unit determined, after which a 5% suspension 

 of sheep cells was sensitized with 2 units of the amboceptor. Readings were 

 taken after the tubes were incubated for 15 minutes in a 37 C. water bath, and 

 8 hours in the ice-chest. 



