DIAGNOSIS OF ASIATIC CHOLEEA. 355 



observe the appearance of the colonies. Usually at 

 about twenty-two hours the colonies of this organism 

 can easily be identified by one familiar with them. 



III. With another slimy flake start a culture in a tube 

 of peptone solution either the solution of Dunham or, 

 as Koch proposes, a solution of double the strength of 

 that of Dunham (Witte's peptone is to be used, as it 

 gives the best and most constant results). Place this at 

 37 to 38 C., and at the end of from six to eight hours 

 prepare cover-slips from the upper layers (without shak- 

 ing) and examine them microscopically ; if comma 

 bacilli were present in the original material, and are 

 capable of multiplication, they will be found in this 

 locality in almost pure culture. After doing this pre- 

 pare a second peptone culture from the upper layers of 

 the one just examined, also a set of gelatin plates, and 

 with what remains make the test for indol by the addi- 

 tion of ten drops of concentrated sulphuric acid for each 

 ten cubic centimetres of fluid contained in the tube. If 

 comma bacilli are growing in the tube the rose color 

 characteristic of the presence of indol should appear. 



By following this plan "a bacteriologist who is 

 familiar with the morphological and biological pecu- 

 liarities of this organism should make a more than 

 probable diagnosis at once by microscopic examination 

 alone, and a positive diagnosis in from twenty to, at the 

 most, twenty-four hours after beginning the examina- 

 tion." (Koch.) 



There are certain doubtful cases in which the organ- 

 isms are present in the intestinal canal in very small 

 numbers, and microscopic examination is not, therefore, 

 of so much assistance. In these cases plates of agar- 



