ACQUIRED IMMUNITY 71 



substance thoroughly dissolves all but the acid-fast bacteria when 

 used in concentrations of 2.5 per cent. Since it is alkaline it is 

 necessary to neutralize it with hydrochloric or sulphuric acid before 

 use. 



For the preparation of antigen from pneumococci Neufeld 37 has 

 utilized the solvent action upon these organisms of bile. He adds 

 the bile and broth cultures just as this is done in the diagnostic 

 "bile test" (0.1-0.2 c. c. of fresh bile to a broth culture; sodium 

 taurocholate solution can also be used). Many bacteria can also be 

 broken up by emulsifying them in 17 per cent, salt solution and 

 allowing them to stand for some time in this medium and then 

 diluting this to 0.85 per cent. 



B. Extraction by Mechanical Methods 



One of the most useful methods for obtaining extracts of bacteria 

 within a relatively short time is that which Besredka 38 has applied 

 mainly for the preparation of typhoid (endotoxin), 24-hour agar 

 cultures washed up in very small quantities of physiological salt 

 solution, killed by heat at 60-65 C. and dried in vacuo. The dried 

 mass is mixed with a measured quantity of dry salt and the mix- 

 ture thoroughly triturated in a mortar for a considerable time. 

 While triturating distilled water is added in small quantities until 

 the fluid represents a 0.85 per cent, salt solution. This is allowed 

 to stand for anywhere from a few hours to a week, and the bacteria 

 are then removed by centrifugalization. This method has been modi- 

 fied by many observers and gives good results whenever thorough 

 trituration is practiced. It is also probable that the exposure to the 

 hypertonic salt solution in the earlier stages of the trituration may aid 

 considerably in breaking up the bacteria. 



Trituration after freezing is a method which has yielded excel- 

 lent results in the hands of Macfadyen and others. This requires a 

 rather complicated piece -of machinery originally described by Mac- 

 fadyen and Rowland. The principle of this is one of mechanical 

 trituration in a steel cylinder which is surrounded by an ice-brine 

 mixture so that the bacteria and sand may be kept frozen during the 

 process. 



Appleton, 1913, p. 1020) gives its composition as follows: "Antiformin 

 consists of equal parts of liquor sodae chlorinate of the British Pharmacopoeia 

 and a 15 per cent, solution of caustic soda. The formula for liquor sodce 

 chlorinate is as follows: 



Sodium carbonate 600 



Chlorinated lime 400 



Distilled water 4,000" 



"Neufeld. Zeitschr. f. Hyg., Vol. 34, 1900. 



38 Besredka. Ann. de I'Inst. Past., 19-20, 1905, 1906. 



