METHODS OF EXAMINATION 283 



and reliable method is to grow the bacillus in bouillon containing a small 

 proportion of the serum to be tested. In this way he has obtained a 

 distinct sedimenting reaction with a serum which did not agglutinate at 

 all distinctly in the ordinary method. Further observations are still 

 required to determine the precise value of the test. 



Mallein and its Preparation. Mallein is obtained from cultures of the 

 glanders bacillus grown for a suitable length of time, and, like tuber- 

 culin, is really a mixture comprising (1) substances in the bodies of the 

 bacilli and (2) their soluble products, not destroyed by heat, along with 

 substances derived from the medium of growth. It was at first obtained 

 from cultures on solid media by extracting with glycerin or water, but is 

 now usually prepared from cultures in glycerin bouillon. Such a culture, 

 after being allowed to grow for three or four weeks, is sterilised by heat 

 either in the autoclave at 115 C. or by steaming at 100 C. on successive 

 days. It is then filtered through a Chamberland filter. The filtrate 

 constitutes fluid mallein. Usually a little carbolic acid ('5 per cent) is 

 added to prevent it from decomposing. Of such mallein 1 c.c. is usually 

 the dose for a horse (M'Fadyean). Foth has prepared a dry form of 

 malleiu by throwing the filtrate of a broth culture, evaporated to one- 

 tenth of its bulk, into twenty-five or thirty times its volume of alcohol. 

 A white precipitate is formed, which is dried over calcium chloride and 

 then under an air-pump. A dose of this dry mallein is '05 to "07 grm. 



The use of Mallein as a means of Diagnosis. In using mallein for the 

 diagnosis of glanders, the temperature of the animal ought to be observed 

 for some hours beforehand, and, after subcutaneous injection of a suitable 

 dose, it is taken at definite intervals, according to M'Fadyean at the 

 sixth, tenth, fourteenth, and eighteenth hours afterwards, and on the 

 next day. Here both the local reaction and the temperature are of 

 importance. In a glandered animal, at the site of inoculation there is a 

 somewhat painful local swelling, which reaches a diameter of five inches 

 at least, the maximum size not being attained until twenty-four hours 

 afterwards. The temperature rises 1'5 to 2 C., or more, the maximum 

 generally occurring in from eight to sixteen hours. If the temperature 

 never rises as much as 1 '5, the reaction is considered doubtful. In the 

 negative reaction given by an animal free from glanders, the rise of 

 temperature does not usually exceed 1, the local swelling reaches the 

 diameter of three inches at most, and has much diminished at the end 

 of twenty- four hours. In the case of dry mallein, local reaction is less 

 marked. Veterinary authorities are practically unanimous as to the 

 great value of the mallein test as a means of diagnosis. 



Methods of Examination. Microscopic examination in a 

 case of suspected glanders will at most reveal the presence of 

 bacilli corresponding in their characters to the glanders bacillus. 

 An absolute diagnosis cannot be made by this method. Cultures 

 may be obtained by making successive strokes on blood serum or 

 on glycerin agar (preferably the former), and incubating at 37 C. 

 The colonies of the glanders bacillus do not appear till two days 

 after. This method often fails unless a considerable number of 

 the glanders bacilli are present. Another method is to dilute the 

 secretion or pus with sterile water, to varying degrees, and then 

 to smear the surface of potato with the mixture, the potatoes 



