METHODS OF DIAGNOSIS 413 



be made and stained by any ordinary stains, though carbol-fuchsin 

 diluted four times with water is specially to be recommended. 

 Hanging-drop preparations, with or without the addition of a 

 weak watery solution of gentian- violet or other stain, should also 

 be made, by which method the motility of the organism can be 

 readily seen. By microscopic examination the presence of spirilla 

 will be ascertained, and an idea as to their number obtained. 

 In some cases the cholera spirilla are so numerous in the stools 

 that a picture is presented which is obtained in no other con- 

 dition, and a microscopic examination may be sufficient for 

 practical purposes. According to Koch, a diagnosis was made 

 in 50 per cent of the cases during the Hamburg epidemic by 

 microscopic examination alone. In the case of the first appear- 

 ance of a cholera-like disease, however, all the other tests 

 should be applied before a definite diagnosis of cholera is made. 

 Dunbar has recently introduced a method for rapid diagnosis 

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

 hang-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. 



If the organisms are very numerous, gelatin or agar plates 

 may be made at once and pure cultures obtained. 



If the spirilla occur in comparatively small numbers, the best 

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

 for from eight to twelve 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 the spirilla are found microscopically. A second flask of 

 peptone solution should then be inoculated, and possibly again 

 a third from the second. By this method, properly carried out, 

 a culture may be obtained which, though impure, contains a 

 large proportion of the spirilla, and then plate cultures may be 

 made. 



When a spirillum has been obtained in pure condition by 

 these methods, the appearance of the colonies in plates should 

 be specially noted, the test for the cholera-red reaction should 



