PROTECTIVE INOCULATIONS. 319 
lon cultures for the preparation of mallein. The potatoes are to be 
washed, before sterilization, in a five-per-cent bicarbonate of soda 
solution, “until the wash-water remains clear.” They are then 
cooked for an hour and twenty minutes. After planting upon the 
surface glanders bacilli from a previous culture they are placed in an 
incubator at 36° to 36.5° C., with provision to prevent them from be- 
coming dry. At the end of two weeks the growth is removed with a 
platinum spatula and added to nine parts of water, in which it is well 
mixed by rubbing. It is then allowed to stand for twenty-four hours, 
after which it is sterilized for fifteen minutes at 110° C. (a lower tem- 
perature would no doubt answer quite as well). After cooling it is 
passed through a Chamberlain filter by means of a pressure of six 
atmospheres. The filtrate is then carefully evaporated over a water- 
bath to one-fourth its volume, and to this concentrated extract gly- 
cerin is added in the proportion of one part to two. The mixture is 
again sterilized in the autoclave at 110° C. When injected into 
healthy horses in the dose of two cubic centimetres this mallein does 
not cause an elevation of temperature exceeding 0.5° to 0.8°. But 
one cubic centimetre injected into a horse having glanders causes its 
temperature to mount to 40° C., and at the point of inoculation a con- 
siderable swelling is developed which lasts from four to six days— 
in healthy horses a swelling the size of a man’s fist is developed at 
the point of inoculation, which disappears within twenty-four hours. 
In Pasteur’s laboratory, according to Nocard (1892), mallein is 
prepared as follows: The glanders bacillus is first made so virulent 
by successive inoculations in susceptible animals that it will kill a 
rabbit or a white mouse in a fewhours. This virulent bacillus is cul- 
tivated in glycerin-peptone-flesh-infusion (five per cent of glycerin and 
five per cent of peptone). The cultures are kept in the incubating 
oven for four weeks at a temperature of 31° C., and then sterilized in 
the autoclave at 110° C. They are then filtered through paper and 
evaporated, in vacuo, over sulphuric acid, at-a low temperature, to one- 
tenth of the original volume. The result is a syrup-like, dark-brown; 
strong-smelling liquid, which is about one-half glycerin. This can 
be preserved in a cool and dark place for a long time. When it is to 
be used nine parts of a 0.5-per-cent solution of carbolic acid are 
added to one part of the glycerin extract. The concentrated extract, 
when injected into a healthy horse in the dose of one-half to one cubic 
centimetre, causes a local swelling which disappears after two or three 
days. The temperature of the body is elevated from 1.5° to 2° C. as 
a result of the injection, and there are chilliness, loss of appetite, and 
debility. When the diluted mallein is injected in healthy animals in 
