﻿REPORT ON CHOLERA IN MANILA. 417 



saline solution. 4 The method of its preparation has been described 

 in detail in former articles from this laboratory. It will be sufficient 

 to state here that an organism known to possess high immunizing and 

 peptonizing powers and one of maximum virulence is selected and grown 

 upon the surface of large test tubes containing 1 per cent alkaline agar. 

 After twenty hours, the growth of one-half of the number of inoculated 

 culture tubes is suspended in 0.85 per cent saline solution, 1 cubic 

 centimeter being employed for approximately every 30 to 35 milligrams 

 of bacteria. The suspension is heated for one hour at 60° C. and then 

 placed at a temperature of 37° for from three to four days. At the end 

 of this time its sterility is tested, it is filtered through a Berkefeld 

 candle and the filtrate saved. The remaining half of the twenty-hour 

 agar cultures is suspended in sterile distilled water, 1 cubic centimeter 

 to each 30 to 35 milligrams. This suspension of the living organisms 

 is then placed on an electrical shaking machine and throughly shaken 

 for from three to four days. At the end of this time cultures are taken 

 to ascertain if the growth is pure and the suspension is then also filtered 

 through a Berkefeld candle. The two filtrates are subsequently mixed 

 in equal proportions and carbolic acid added to 0.5 per cent. The pro- 

 phylactic is finally bottled in glass flasks, the smaller ones being sealed 

 in the flame. Two cubic centimeters of this mixture represents an adult 

 dose. After the sterility of each sample of the vaccine has been tested 

 by animal inoculation and by anaerobic cultures, it is standardized 

 according to the number of units of immunity to which it gives rise 

 after inoculation ; one unit of immunity equaling the amount of immune 

 serum which will protect a guinea pig of 250 grams weight against the 

 intraperitoneal inoculation of ten times the fatal dose of living cholera 

 organisms. If 1 cubic centimeter of the vaccine, when injected intra- 

 venously into a rabbit, does not give rise to at least 10,000 units of 



4 The process by means of which this prophylactic is prepared may be regarded 

 as the outcome of the experimental work performed by Koch, Neisser and Shiga, 

 Wassermann, Brieger, and myself. The idea of using the immunizing substances 

 (free receptors) extracted from Bacillus typhosus and Bacillus dysenteriae for 

 producing immune sera originated in Ehrlich's laboratory with Neisser and 

 Shiga G in 1903, and that of using the extracted free receptors of the cholera 

 spirillum for prophylaxis against cholera in Wassermann's laboratory a few 

 months later (during the same year) where 1° was able to carry on the first 

 extensive and conclusive experiments in regard to its value. Later Shiga " 

 advocated and used the method for preparing a prophylactic against typhoid 

 fever. 



Brieger and Mayer 8 called attention to the advantage of extracting the soluble 

 substances of the organisms by shaking in distilled water. 



3 Deutsche med. Wchnsch. (1903), 4, 61. 



"Am. Med. (1903), 6, 272. 



7 Berl. klin. Wchnsch. (1904), 4, 79. 



8 Deutsche Med. Wchnsch. (1904), 30, 980. 



