168 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 
bacterial cell may be obtained in solution if the bacterial emulsion is 
shaken in a shaking machine with sterile, sharp quartz-sand for twenty- 
four hours: filtration through coarse Berkefeld filters removes 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 
XaCl to an impalpable powder, which is then gradually rubbed up in 
successive portions of water until a physiological salt solution is ob- 
tained (corresponding to 8.5 gm. 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 gm. of dried bacterial substance, groiuid 
in a mortar with 0.85 gm. NaCl crystals and gradually brought to a 
volume of 100 cc. with distilled water, would yield, theoretically, an 
antigen of 1 per cent strength, SmalP has found that bacteria grown 
upon solid media which are removed, dried and extracted with chloro- 
form make excellent antigens. Apparently the removal of some of the 
lipoidal substance by this method reduces the anticomplementary 
action of the preparation to a point where it is no longer a factor. The 
dried, extracted bacterial protein keeps for months if it is placed in a 
desiccator in the refrigerator. Bacterial antigens must be kept cold 
and in a dark place, preferably in sealed amber bottles. Deterioration 
gradually occurs and all bacterial antigens suspended or dissolved in 
liquids are relatively unstable. Melick^ has found that the less fastidi- 
ous pathogenic bacteria may be cultivated without loss of antigenic 
properties upon a modified Uschinsky medium. This has the advan- 
tage of freedom from protein constituents derived from the medium. 
Also he finds that freezing in liquid air and thawing, repeated many 
times gives a soluble antigen, free from anticomplementary substances, 
which is suitable for complement fixation. 
Standardization of Bacterial Ajitig ens. —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. 
1 Ann. Inst. Pasteur, 1906, 20, 149; Bull. Inst. Pasteur., 1914, 12, 145. 
2 Jour. Immunol., 1918, 3, 413. 
9 Jour. Med. Research, 1922, 43, 405. 
