jan. 2,1917 Diagnosis of Tuberculosis by Complement Fixation 
7 
EXPERIMENTAL WORK 
Preparation op the Antigens Employed 
Material used: Old tuberculin; saturated solution of ammonium chlorid; 
Schulke and Schleich parchment paper. 
To an amount of old tuberculin in an Erlenmeyer flask of convenient 
size sufficient saturated solution of ammonium chlorid is added to super¬ 
saturate the tuberculin. The mixture is stirred well with a glass rod and 
set aside for 48 hours. The fluid is carefully poured off from the pre¬ 
cipitate which has settled at the bottom of the flask. The liquid is centri¬ 
fuged at a high rate of speed and the sediment collected, which consists 
of a resinous sticky mass, dark brown in color, is readily dissolved in a 
small amount of salt solution, and is then transferred to the dialyzing 
(parchment paper) bag. The sediment contained in the Erlenmeyer 
flask is added to it and the material is then dialyzed for 8 consecutive 
days. The content of the bag is then tested for the presence of ammo¬ 
nium chlorid, and if no reaction for ammonia is obtained the substance 
is removed, filtered, and the filtrate evaporated to dryness over a water 
bath at 6o° C. 
The residue was scraped from the evaporating dish, and the chocolate- 
brown colored powder collected, placed in a bottle which was provided 
with a well-fitting stopper, and the precipitate kept in a cool place, 
protected from light. The amount of precipitate obtained corresponds 
to about 0.8 to 1 per cent. 
It should be emphasized that the preparation and handling of the 
precipitate must be carried out under precautions to prevent the inha¬ 
lation of the pulverized precipitate. Even slight amounts may cause 
disagreeable symptoms. These are manifested in severe headaches and 
chills, accompanied by high fever, the temperature ranging between 
102 0 and 104° F. for two or three consecutive days; pulmonary symp¬ 
toms resembling those of a lobular pneumonia and profuse night sweats; 
respiration sometimes difficult and painful; pains in the back are always 
present; the urine is dark-colored; micturition frequent and scanty. 
For protection it is advisable to cover the evaporating dish during the 
pulverization with several layers of cheesecloth moistened with a 5 per 
cent phenol solution. 
The material used for the preparation of defatted organisms was as 
follows: Surface growth of a bouillon culture of tubercle bacilli of both 
human and bovine strains; alcohol, 95 per cent; ether. 
The tubercle bacilli are macerated for seven days in 95 per cent ethyl 
alcohol in a closed vessel. The alcohol is then removed and the bacteria 
are macerated with 50 c. c. ethyl ether for six hours, shaking the mixture 
frequently. The ether is poured off and the bacilli are shaken two 
more times for one-half hour each time with 25 c. c. of ether. The 
bacilli are then collected, dried in vacuum over sulphuric acid, and 
