406 GLANDERS, ANTHRAX, PYOCYANEUS 
suspension of glanders bacilli to the foUowin^j; degrees: 1 to 500, 1 to 
1000, 1 to 2500, 1 to 5000, 1 to ToOO.i Glanders })acilli, virulent for 
guinea-pigs (obtained by passing glanders bacilli through a series of 
animals until the organism kills the animal within five days — intraperi- 
toneal injection), from glycerin agar slants are emulsified in physiologi- 
cal salt solution containing 0.5 per cent carbolic acid, thoroughly shaken, 
and filtered through a thin layer of absorbent cotton to remove clumps. 
Salt-phenol solution is added to the suspension until a moderately 
turbid suspension is obtained. Decreasing amounts of serum from 
the suspected animal are added to obtain the dilutions mentioned 
above. A normal serum and a known positive serum are diluted in 
the same manner to serve as controls. Incubation is continued at 
37° C. for seventy- two hours, because the reaction is usually slow in 
developing. Sterility must be maintained throughout. Strongly 
positive sera may give a definite clumping in twenty-four hours or 
less; the supernatant fluid becomes clear, and the organisms collect 
as a diffuse sediment at the bottom of the tube. A negative reaction 
is indicated by a turbid supernatant fluid. The reaction may be micro- 
scopically or macroscopically, the latter being preferable. 
Attempts have been made to shorten the reaction time by aiding 
sedimentation with the centrifuge. The various dilutions are incu- 
bated for a full hour at 37° C, allowing fifteen minutes for the tubes 
to reach 37° C. in the incubator; then they are whirled for fifteen 
minutes at a speed with a 24-inch radius not exceeding 1500 revolutions, 
placed in the ice-box and examined after three hours. The slowly 
developing reactions may not be definitely positive for twenty-four 
hours. 
A reaction in a dilution of 1 to 500 (horse, ass or mule) is the lowest 
limit to which a definite reaction may be attributed, and the result 
should be controlled with a mallein test. Dilutions of 1 to 750 or 
higher are usually safely regarded as diagnostic. In human cases a 
positive reaction in a dilution of 1 to 100 is diagnostic. 
The method of complement-fixation (see page 169 for details) is 
rapidly becoming a general method for the diagnosis of glanders.- 
Dissemination and Prophylaxis. — Glanders is transmitted by direct 
contact, by infection through cutaneous abrasions and cuts, and by 
feeding paraphernalia, watering troughs and buckets. In man cuta- 
neous infection is more common. 
BACILLUS ANTHRACIS. 
Bacillus anthracis was first seen by Davaine^ in 1863, in the blood of 
animals infected with anthrax. Koch'* confirmed Davaine's observa- 
• Povitzky: Jour. Immunol., 1918, 3, 463. 
2 Mohler and Eichhorn: Bull. No. 136, Bureau of Animal Industry, April 7, 1911. 
Meissner and Trapp: Centralbl. f. Bakteriol., orig., 1909, 52, 115. 
' Compt. rend. Acad. Sci., 1863, vol. 57. 
* Cohn's Beitr. z. Biol., der Pflanzen, 1876, 2, 277; Mitt. a. d. kais. Gesdth., 1881, 1, 49. 
