164 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 



the sand and broken bacterial cells, and the filtrate is preserved with 

 0.5 per cent, phenol. Besredka prepares a bacterial antigen from dried 

 bacterial cells, which are obtained by drying bacteria scraped from 

 agar slants or other solid media over sulphuric acid or calcium chloride. 

 The dried organisms are ground in agate mortars with crystals of 

 NaCl to an impalpable powder, which is then gradually rubbed up 

 in successive portions of water until a physiological salt solution is 

 obtained (corresponding to 8.5 grams NaCl in a liter of distilled water). 



It has been found that much of the antigenic substance of bacteria 

 is precipitated by an excess of alcohol; a considerable excess of alcohol 

 is added to a suspension of bacteria, or to an emulsion of the cell 

 substance prepared according to Besredka's process, outlined above. 

 The precipitate from the alcoholic solution is separated by filtration, 

 dried, and ground to an impalpable powder with NaCl crystals. 

 The powder is gradually brought into solution by the addition of 

 water in successive amounts until isotonicity is reached. An attempt 

 is made to create a definite concentration of antigen by starting with 

 a known quantity of dried bacteria and a corresponding amount of 

 NaCl crystals. Thus, 1 gram of dried bacterial substance, ground 

 in a mortar with 0.85 gram NaCl crystals and gradually brought to 

 a volume of 100 c.c. with distilled water, would yield, theoretically, 

 an antigen of 1 per cent, strength. Bacterial antigens must be kept 

 cold and in a dark place, preferably in sealed amber bottles. Deter- 

 ioration gradually occurs and all bacterial antigens suspended or 

 dissolved in liquids are relatively unstable. 



Standardization of Bacterial Antigens. The standardization of bac- 

 terial antigen differs in no respect from that of a syphilitic antigen. 

 The anticomplementary titer and the antigenic titer are determined, 

 the latter by titration with a specific immune serum. 



The Diagnosis of Glanders by the Method of Complement-fixation 

 The antigen is prepared from glycerin-agar cultures 1 of several strains 

 of B. mallei incubated at 37 C. for forty-eight hours. The organisms 

 are autolyzed in distilled water for several hours at a relatively high 

 temperature (70 to 80 C.), then freed from suspended particles by 

 filtration through coarse Berkefeld filters. The filtrate is stored in 

 amber bottles in the ice-box after the addition of 0.5 per cent, phenol. 



The anticomplementary titer is determined from a series of tubes 

 containing constant amounts of complement and graduated amounts 



1 Reaction 1.5 per cent, acid to phenolphthalein. 



