94 LOCAL TLSSUE REACTIVIIY 



aiTiinoniimi sulplKik' wcic injected subcutancously. Unwaslicd Ji. lyj)li()SHs 

 heat-killed vaccines were injected intravenously. The length ol time during 

 whicli the innnuni/alion ^vas continued before each bleeding is indicated in 

 Tables in and i\. Llie doses were increased by 25 per cent each week. 

 Previous to the bleediirg on the eighth month, some of the horses {e.g., No. 

 2 and No. 4) were receiving the following rather large doses: 400 c.c. of 

 vaccine, intravenously, 1,200 c.c. of filtrate, subcutaneously, a suspension of 

 9 gms. of the precipitated toxic substances (a yield from 1000 c.c. of tryptic 

 digest broth culture filtrate) , subcutaneously, and about 400 c.c. of the vac- 

 cine intravenously. In later work doses considerably smaller were found 

 sufficient. 



Rabbit serum R.201 was a mixture of sera of 5 rabbits immunized with 

 material similar to that employed for horses. The injections were carried out 

 for four to six weeks, 25 c.c. of the filtrate was the largest subcutaneous dose 

 and 20 c.c. of the vaccine was the largest intravenous dose. 



Normal rabbit and heterologous horse sera (meningococcus and erysipelas 

 therapeutic sera) Avcre also employed. 



Preliminary titrations estalilished the fact that a dose as small 

 as 0.25 c.c. of the fikrate j^reviously dikited to 1:100 was capable 

 of preparino tlie skin of some of the rabbits to the phenomenon 

 providing sufficiently large intravenous doses ^vere subsequently 

 used. Because of the high dilution, however, there occurred in- 

 dividual fluctuations. In order to obtain a high degree of reac- 

 tivity necessary for the work about to be described, rabbits ^vere 

 prepared Avitli many multiples of the above indicated minimal 

 skin-preparatory dose. Intravenous doses of 0.01 c.c. and 0.5 c.c. 

 per kilo of body ^veight, were found capable of producing con- 

 sistent reactions in ral)bits prepared Avith filtrate A7 undiluted 

 and diluted 1:4, respectively. 



For the basic neutralization experiments several multiples of 

 the above indicated minimal doses of reacting factors were em 

 ployed in order to be assured of a high percentage of positive 

 reactions in rabbits. Furthermore, at least 5 ralibits ^vere em- 

 ployed for eacli neutralization experiment. 



The filtrate (A7) stored in the refrigerator, was employed 

 for these experiments. The Hltrate Avas first used approximately 

 two Aveeks after its preparation and tiiis use was continued for 

 two montlis afterwards. Since tliere were observed changes in the 

 jiotency of different l)atches of filtrates from time to time, 4 con- 

 trol titrations of the reacting factors were carried out during the 

 two months. In addition, Aviienever a serum neutralization experi- 

 ment Avas made, a control group of 4 to 6 rabfjits ^vas injected 

 with the fiftrate alone. Tliese control experiments definitely 



