SPIRILLUM CHOLERJE ASIATICS. 587 



Plan of Procedure for the Biological Diagnosis of the 

 Cholera Vibrio. A. Dejecta (fluid) or intestinal con- 

 tents of a cholera patient or cholera suspect. 



1. Use one drop (one platinum loop) for gelatin plate- 

 cultures, making two dilutions. Do this in duplicate 

 or triplicate. Cultivate at 22 C. 



2. Inoculate a couple of bouillon tubes and a couple 

 of Dunham's 1 per cent, peptone solution with one drop 

 each, and place them in the incubator (37 to 38 C.) 

 for six to eight hours. 



3. Examine a drop of the dejecta in the hanging drop. 



4. Examine a drop of the dejecta in stained cover- 

 glass preparation. 1 



5. Make gelatin plates from one drop taken from 

 the surface of each of the bouillon and peptone solution 

 tubes and cultivate at 22 C. 



6. As soon as the plates (see 1 and 5) are sufficiently 

 developed (thirty-six to forty-eight hours) fish the 

 suspected cholera colonies and use the material for the 

 following procedures : 



7. Inoculate six or eight peptone tubes (1 per cent, 

 peptone, 0.5 per cent. NaCl in distilled water) and 

 place them at once in the incubator. Note the time. 



8. Examine hanging drop for form, size, and motility 

 (and arrangement). 



9. Make stained cover-glass preparations and exam- 

 ine. 



1 These direct microscopical examinations of the intestinal contents are, as 

 a rule, very unsatisfactory, at least in those in which the symptoms are not 

 marked. In a few the spirals will make up from 50 to 100 per cent, of the 

 bacteria present. In most of the cases during the last epidemic in New York 

 Dunham found abundance of columnar epithelium from the intestinal mucous 

 membrane, numerous straight, thick bacilli, and only a few curved bacilli or 

 segments of spirals too few to identify. Plate cultures from these showed 

 from 20 to 80 per cent, of all the colonies developing to be cholera spirilla. 



