322 MANUAL OF BACTERIOLOGY. 



The diagnosis should be confirmed by the use of culture- 

 methods. Using the small, semi-solid particles from the 

 intestinal discharges, gelatin plates in the usual three dilu- 

 tions (see page 97) should be made and kept at a tempera- 

 ture of 20 to 22 C. At the end of twenty-four hours or 

 less the colonies of the spirillum of cholera should have 

 been developed and should present the picture characteristic 

 for these colonies in gelatin plates (Fig. 92), which enables 

 them to be differentiated from colonies of other bacteria. 

 From one of these colonies, preparations may be made for 

 microscopic examination, and a set of tubes may be inocu- 

 lated. The most characteristic growth will be from stick- 

 cultures in gelatin. The growth in Dunham's peptone 

 solution may be tested for the development of indol and 

 nitrites. 



At the time that the first smear preparations and gelatin 

 plates are prepared, tubes of peptone solution should be 

 inoculated directly from the intestinal contents, and kept 

 in the incubator (Schottelius). After development has oc- 

 curred, the production of indol may be tested by the addi- 

 tion of sulphuric acid. These tubes are especially valuable 

 when unfavorable material or when material containing 

 small numbers of the spirilla is used. In the incubator the 

 spirilla may be expected to multiply in the peptone solution 

 rapidly, and to appear upon the surface of the liquid in 

 large numbers, even forming a visible film in six hours. 

 Smears may be made from the surface part of these tubes, 

 stained, and examined with a microscope. From the same 

 material gelatin plates should be prepared, and examined 

 as soon as the colonies develop. 



\Yhen cultures are obtained, their effects may be tested 

 upon guinea-pigs by injecting them into the peritoneum. 

 The reaction described by Pfeiffer as resulting from the 

 injection of cholera spirilla into the peritoneum of immune 



