170 
Dourine 
organs, such as the liver and spleen, and are all more or less anticomple¬ 
mentary, more so in the presence of antigen than without it. Such 
action, of course, is non-specific and must be eliminated, otherwise it 
would be difficult or impossible to distinguish a specific from a non¬ 
specific reaction. Fortunately it can be eliminated, and the equilibrium 
of the serum fixed, by a proper and sufficient inactivation. It is more 
resistant to heat than is complement and is not wholly destroyed at 56° C. 
This is an important point, and one that appears to have been over¬ 
looked. I cannot help thinking that it is the explanation and the source 
of error of many of the apparent failures or discrepancies, especially 
that of non-specific fixation, which some serologists experience. A refer¬ 
ence to the literature on complement fixation methods shows a 
remarkable lack of uniformity in respect to the degree of heat and the 
length of time for the inactivation of suspected sera—fifteen to thirty 
minutes at degrees varying between 50 and 58° C. 
A few experiments with sets of ten or twenty different horse, mule 
and donkey sera, each set being heated for thirty minutes at different 
degrees between 50 and 62 C. and then tested in the haemolytic system, 
with and without antigen, will show the importance and necessity of a 
very careful inactivation and the temperature required (vide, p. 171). 
Method of inactivation recommended. A water bath, sufficiently 
large to hold 200 small test tubes, is heated to 60° C. The tubes, 
containing 2-0 c.c. serum in each (numbered for identification in water¬ 
proof india ink, labels being apt to become detached), are placed 
within the inner tank which is to contain sufficient water to mount to 
the level of the serum or to about half the height of the tubes. The 
cover of the tank should have two perforations for thermometer tubes 
which are inserted into control tubes within the tank, enabling the 
temperature to be read without removing the cover. Another ther¬ 
mometer passes directly into the outer tank. For the first few minutes 
the temperature will rapidly fall; it is brought up to 59° again—taking 
about ten minutes—and maintained at that point for a full half hour, 
for horse serum, and to 62° for one half hour for donkey or mule serum. 
There is no danger of destroying the specific antibodies of dourine 
sera by heating to the points given. Dourine sera can, in fact, be heated 
up to 65°, or to the point of coagulation, and still retain an active 
antibody content to give the test reaction, but the anticomplementary 
and non-specific factors in horse sera are wholly destroyed at 59°, and 
in donkey and mule sera at 62°. 
