KAHN TEST 173 
brittle. It is then ground into powder form by means of a mortar or 
a coffee grinder which is used for no other purpose. Powdered beef 
heart is obtainable on the market (Digestive Ferments Co., Detroit). 
A large number of hearts enter into the preparation of each lot of this 
product and a higher degree of uniformity is obtained than is possible 
when the powder is prepared on a smaller scale. It should be pointed 
out that the powdered muscle should be stored in the cold. Ageing at 
room temperature may render it unfit for antigen preparation. 
Ether Extraction.— Twenty-^ve grams of powdered beef heart are 
placed in a 250 cc. Erlenmyer flask and 100 cc. ether (anesthesia) added. 
The flask is shaken from time to time for an interval of ten minutes 
after which the ether is filtered oft' through qualitative filter paper. 
The filtration process is hastened by applying gentle pressure to the 
beef heart with a spatula. Filtration is completed when such pressure 
does not cause drops of ether to pass through the funnel. The moist 
beef heart is then returned to the original 250 cc. extraction flask. 
This may be done by first transferring the beef heart from the filter 
paper to a sheet of white paper and breaking the material with a spatula 
into pieces small enough for the mouth of the flask. Seventy-five 
cubic centimeters of ether are added and the mixture again extracted 
for a ten-minute interval and filtered as above. After the second filtra- 
tion with ether, the beef heart is transferrefl to the flask for a third time 
and 75 cc. ether again added. The mixture is extracted for a ten- 
minute interval and again filtered as described above. The moist beef 
heart is transferred for the fourth and last time to the flask and 75 cc. 
ether added, the mixture extracted for a ten-minute period and then 
filtered. When gentle pressure with a spatula no longer causes drops 
of ether to pass through the funnel— the end-point employed in each 
of the four ether filtrations— the beef heart is transferred to a sheet of 
white paper and dried either at room temperature or at 37° C. The 
drying usuall}- requires from ten to fifteen minutes. When no ether 
odor is detectable, the beef heart is ready for extraction with alcohol. 
Alcohol Extraction.— An alcoholic extract of the beef-heart powder 
after its extraction with ether is prepared as follows : The dry powdered 
muscle is weighed and placed in the 250 cc. Erlenmeyer flask used for 
ether extraction— after the flask has been freed from ether odor. There 
will be approximately 23 gm. remaining of the 25 gm. used in the ether 
extraction. Five cubic centimeters of 95 per cent alcohol are added 
per gram of powder. The flask is shaken for ten minutes and extraction 
allowed to continue at room temperature, in the dark, for three days 
without shaking. At the end of this period, the mixture is shaken for 
five minutes and filtered. The filtrate is kept in the dark at room 
temperature, as stock antigen solution. 
C hoi esterolization. — Cholesterol is added to the alcoholic extract in 
the proportion of 6 mg. per cc. of extract. The cholesterol is dissolved 
b\' rotating the flask in a water-bath at 37° C. WTien all the cholesterol 
