METHODS OF DIAGNOSIS 475 



If the organisms are very numerous, agar plates or plates of 

 Dieudonnd's medium (p. 46) may be inoculated at once and a 

 pure culture obtained from one of the colonies. 



If the spirilla occur in comparatively small numbers, the best 

 method is to inoculate peptone solution (1 per cent.) and incubate 

 for from six to eight hours. At the end of that time the spirilla 

 will be found on microscopic examination in enormous numbers 

 at the surface, and thereafter plate cultures can readily be made. 

 If the spirilla are very few in number, or if a suspected water is 

 to be examined for cholera organisms, the peptone solution 

 which has been inoculated should be examined at short intervals 

 till spirilla are found microscopically. A second flask of peptone 

 solution should then be inoculated, and possibly again a third 

 from the second, and then plates may be made. In such circum- 

 stances Dieudonne's medium has been found of much service. 

 For the separation of the organism Ottolenghi introduced a 

 medium composed of ox-bile to which 3 per cent, of a 10 per 

 cent, solution of sodium carbonate is added : it is sterilised in 

 the autoclave. It is used in the same way as peptone solution, 

 and the advantage claimed for it is, that it inhibits the growth 

 of most intestinal bacteria ; on the other hand, the cholera 

 organism appears to, grow r'ather less rapidly than in peptone 

 solution. 



When a spirillum has been obtained in pure condition by 

 these methods it should be tested, as regards agglutination 

 against a high titre anti-cholera serum. If it reacts positively 

 it may be accepted for practical purposes as the cholera organism. 

 Thereafter the cultural characters and the hsemolytic and patho- 

 genic properties may be tested. If it reacts negatively with 

 anti-cholera serum it may be one of the paracholera group, and 

 similar tests should be made. 



Dunbar introduced a method for rapid diagnosis which 

 depends on the properties of an anti-cholera serum. Two hang- 

 ing-drop preparations are made, each consisting of a small 

 portion of mucus from the suspected stool broken up in peptone 

 solution. To one a drop of a 50-fold dilution of normal serum 

 is added, to the other a drop of a 500-fold dilution of an active 

 cholera serum. If the spirilla present are cholera organisms, 

 they retain their motility in the first preparation, while they lose 

 it and then become agglutinated in the second. By this method 

 a diagnosis may sometimes be given in a few minutes. Others 

 have adopted the method of growing the suspected organism in 

 bouillon containing a small amount of anti-cholera serum ; in 

 the case of the cholera organism growth falls to the bottom as a 



