THE CHOLERA SPIRILLUM AND ALLIED VARIETIES 403 



importance. When present in great amount such agglutinins can be 

 used for identifying doubtful spirilla. 



Variations of the Cholera Spirillum. From the great majority of 

 all cases of epidemic cholera examined, cholera spirilla agreeing in 

 all essential characteristics have been obtained, usually in great 

 numbers and often in almost pure culture. In their agglutination 

 with a specific serum they are also alike. Some cultures agglutinate 

 with more difficulty than others, so that the same serum may agglutinate 

 different cultures in dilutions varying from 1 : 1000 up to 1 : 10,000. 

 Such a serum would not agglutinate cholera-like spirilla above a 1 : 50 

 dilution. Especially among isolated cases of cholera-like diseases 

 spirilla are met with which do not agree in agglutination charac- 

 teristics. 



Biological Diagnosis of the Cholera Vibrio. Plan of Procedure. A. 

 Dejecta (fluid) or intestinal contents of a cholera patient or cholera 

 suspect. 



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

 ing 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 and 0.5 

 per cent. NaCl in distilled water) and place them at once in the incu- 

 bator. Note the time. 



8. Examine hanging drop for form, size, and motility (and arrange- 

 ment). 



9. Make stained cover-glass preparations and examine. 



10. Then try indol reaction with the same tubes. 



11. While these tubes are incubating use material from the suspected 

 colonies on the plates (1 and 5) for hanging-drop cultures. 



12. Meanwhile make stained cover-glass preparations from other 

 colonies of suspected cholera on the plates (1 and 5). 



13. Make gelatin-tube cultures from colonies on plates (1 and 5). 



14. Make gelatin-tube cultures daily for five or six days, to study 

 shape of growth along the line of puncture to preserve the culture. 



1 These direct microscopic examinations of the intestinal contents are, as a rule, very unsatis- 

 factory, 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 mem- 

 brane, 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. 



