THE DIAGNOSIS OF GLANDERS 121 
shown by their work that a suitable culture, when obtained, 
is liable, at unexpected intervals, to lose its responsiveness to 
the agglutinin. This can often be prevented by passing the 
organisms through a guinea pig. The organisms should be 
grown for from 48 to 72 hours on acid glycerin agar (5 % 
glycerin with a reaction of + 2.9 to phenolphthalein). In order 
to have a suitable culture on hand, subcultures should be made 
at short intervals. A culture about 72 hours old should be used 
in preparing the test fluid. 
Test fluid. The test fluid is prepared by washing the growth 
from the agar culture by the aid of a sterile wire loop into dis- 
tilled water containing 0.85 % sodium chloride and 0.5 % 
carbolic-acid crystals. This suspension is then placed in a 
thermostat at 60°C. for 2 hours, which kills the bacteria. 
A temperature higher than 65° C. or lower than 60° C. should 
be avoided. After heating, the suspension is thoroughly tritu- 
rated and filtered through sterile cotton. Thorough trituration 
of the emulsified growth is essential before filtering. The fil- 
trate thus prepared is diluted with the carbolized salt solution 
until it is of a faintly clouded appearance. The proper dilution 
of the filtrate can only be determined by experience. The 
test fluid gives the best results when made with freshly pre- 
pared carbolized salt solutions. 
Procuring the serum. The serum is easily obtained. At least 
10 cc. of blood should be drawn from the jugular vein, under 
aseptic precautions, into a small sterile bottle, and sent to the | 
laboratory. As soon as the clot forms, the supernatant serum ‘ 
is placed in a centrifuge and all the sediment thrown down, ' 
leaving the liquid perfectly clear. One cc. of the serum is then | 
' added to 39 cc. of a physiological salt solution, which makes 
a dilution of 1 to 40. It is desirable that the serum should be 
secured as soon as possible after the blood is drawn. If neces- 
sary to delay the test, the serum has given the best results if 
kept at about ro° C. until used. 
