542 APPLICATION OF METHODS OF BACTERIOLOGY 



in from eighteen to twenty-two hours. In general, the 

 steps to be taken and points to be borne in mind are as 

 follows: 



1. Microscopic Examination. — ^From one of the small slimy 

 particles seen in the semi-fluid evacuations, obtained as 

 soon as possible after their passage, prepare a cover-slip 

 preparation in the ordinary way and stain it. If, upon 

 microscopic examination, only curved rods, or curved rods 

 greatly in excess of all other forms, are present, the diagnosis 

 of Asiatic cholera is more than likely correct; and particularly 

 is this true if these organisms are arranged in irregular linear 

 groups with the long axes of all the rods pointing in nearly 

 the same direction. 



2. Plate Cultures. — From another slimy flake prepare a set 

 of gelatin plates. Keep them at a temperature of from 

 20° to 22° C, and after sixteen, twenty-two, and thirty-six 

 hours observe the appearance of the colonies. Usually after 

 about twenty-two hours the colonies of this organism can 

 easily be identified by one familiar with them. 



3. Peptone Cultures. — 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). Keep this 

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

 hours prepare cover-slips from the upper layers (without 

 shaking) 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 the microscopic examination prepare 

 a second peptone culture from the upper layers of the one 

 just examined, also a set of gelatin plates, and with what 



