78 LOCAL ILSSUE REACniVLrY 



ol recovery. It may l)e concliidecl, however, that tlie process ol 

 precipitation w iili animoniiim sul})hate offers the (jpj)ortiinity of 

 ofjtaining' stabile active principles of skin-j5rej)aratory and react- 

 ing potencies in a dry form (Sliwart/man, h)2()(:) . 



PURIFIED AND I'KF.SKRVl.D S 1 RF 1' I()C;( )C:CAL RKACTING FACTORS 



Attempts ^vere also made by me to purify the streptococcal 

 reacting factors and to obtain them in stable form. In these ex- 

 periments, some of the steps of the method of concentration of 

 streptococcal scarlet fever toxin recently described by Shiiin 

 (1930) , were employed Avith certain modifications, as follows: 



Ten and one-half liters of i per cent dextrose broth of pH 7.6 

 in Florence flasks (Y^ full) were inoculated \vith Streptococcus 

 hemolyticus scarJatinae, strain 4014. The cidtures ^vere incubated 

 for twenty-lour hours at 37.5° c. After incubation, the flasks were 

 chilled in the refrigerator, the contents transferred to suitable 

 sterile containers and diluted with 31.5 liters of 95 per cent alco- 

 hol containing 0.03 \)gx tent glacial acetic acid. The mixtures 

 Avere allo\ved to stand in the refrigerator for one hour and ^vere 

 then filtered under negative pressure through a Buchner funnel, 

 hard filter paper being used. The j^recipitate was taken off the 

 paper and dissolved in 0.85 per cent sodiiun chloride solution 

 previously adjusted to pH 8.0 and containing 0.5 })er cent phenol. 

 The solution of the precipitate ^^•as dried in a Freas vacuum o\en 

 at 37.5° c. The dried precipitate ^vas ground into a fine powder 

 and preserved in a desiccator over phosphorus pentoxide at room 

 temperature. The total yield in dry powder was 42 gms. Just be- 

 fore use, a ^veighed amount of the po^vder was suspended in dis- 

 tilled ^vater. The powder made a fairly good suspension. 



Inasmuch as the streptococcus yields skin-preparatory factors 

 of lo^v and irregular potency, the rabbits ^vere jjrepared by intra- 

 dermal injection of B. typhosus "agar washings"" filtrate and then 

 given intravenous injections of suspensions of various amounts of 

 the streptococcus powder. The powder was able t(^ elicit severe 

 reactions at the prepared skin sites. The potency of the material 

 could be easily titrated. The procedure employed for the prepara- 

 tion of the powder apparently brought al)out some loss of the 

 reacting potency of the cultures. In fact, the live cultures from 

 which the material was derived, in the amount of 3 c.c. per kilo 

 of body \\eight injected intravenously, gave about 66 per cent 

 positi\e reactions, ^vhereas ^vith the powder it 'tvas necessary to 



