METHODS OF DIAGNOSIS 459 



cholera anti-sera have little effect on at least most of the endo- 

 toxins, cannot 1x3 said to be shaken. It should be noted, how- 

 ever, that he disclaims having made the general statement, often 

 ascribed to him, that no antitoxins are formed to endotoxins. 



Anti-Cholera Inoculation. Haffkine's method for inoculation 

 against cholera exemplifies the above principles. It depends 

 upon (a) attenuation of the virus that is, the cholera organism, 

 and (b) exaltation of the virus. The virulence of the organism 

 is diminished by passing a current of sterile air over the surface 

 of the cultures, or by various other methods. The virulence is 

 exalted by the method of passage that is, by growing the 

 organism in the peritoneum in a series of guinea-pigs. By the 

 latter method the virulence after a time is increased twenty-fold 

 that is, the fatal dose has been reduced to a twentieth of the 

 original. Cultures treated in this way constitute the virus exalte. 

 Subcutaneous injection of the virus exalte produces a local 

 necrosis, and may be followed by the death of the animal, but if 

 the animal be treated first with the attenuated virus, the sub- 

 sequent injection of the virus exalte produces only a local oedema. 

 After inoculation first by attenuated and afterwards by exalted 

 virus, the guinea-pig has acquired a high degree of immunity, and 

 Haffkine believed that this immunity was effective in the case 

 of every method of inoculation that is, by the mouth as well as 

 by injection into the tissues. After trying his method on the 

 human subject and finding it free from risk, he extended it 

 in practice on a large scale in India in 1894. In the human 

 subject two or sometimes three inoculations were formerly made 

 with attenuated virus before the virus exalte" was used ; now, 

 however, a single injection of the latter is usually practised. 

 The results of preventive inoculation in India and also during 

 the recent epidemic in Russia have been such as to establish its 

 efficiency, both the case incidence and the mortality being 

 reduced. 



Methods of Diagnosis. In the first place, the stools ought 

 to be examinod microscopically. Dried film preparations should 

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

 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- 



