QUALITATIVE BACTERIOLOGICAL ANALYSIS. 253 



give any trace of this reaction. As a result of his comparisons 

 of the water- vibrios with true cholera spirilla, he concluded that 

 the two groups could not be distinguished by cultural reactions? 

 and that a diagnosis of cholera could only be made when the 

 bacteriological examination was confirmed by epidemiological 

 facts and the appearance of disease. 



METHODS PROPOSED FOR THE ISOLATION OF THE CHOLERA 

 SPIRILLUM FROM WATER. 



The .simplest method is to convert a considerable bulk of the 

 specimen of water into a 1 per cent, peptone and 0*5 per cent, 

 salt solution. This is easily done by keeping a stock solution con- 

 taining 10 per cent, peptone and 5 per cent, salt ; 10 c.c. of the 

 stock solution are added to 90 c.c. of the water placed in a 

 sterile flask, which is then incubated at 37 C. After twelve to 

 twenty-four hours a faint pellicle forms on the surface if cholera 

 spirilla are present. Loopfuls must then be removed from the 

 surface and plated out in gelatine and rubbed over the surface 

 of agar solidified in Petri dishes. If pure cultures of spirilla 

 are obtained the colonies must be fished and planted out on the 

 following media : 



(1) Peptone and Salt Solution. After twenty-four hours 

 incubation at 37 C. the tube will become turbid, and on the 

 addition of pure sulphuric acid the cholera-red reaction will be 

 obtained. 



(2) Gelatine Plates. The characteristic colonies with irregular 

 margins will appear. 



(3) Gelatine-stab. The typical air-bubble liquefaction will be 

 obtained. 



(4) Agar-slope. The growth which appears in twenty-four 

 hours must be tested for agglutination and for Pfeiffer's reaction 

 with anti-cholera serum. 



A portion of the colony should be examined in a hanging- 

 drop for the characteristic appearance and motility of Koch's 

 vibrio. If a spirillum conforms to all the tests it may be classed 

 with the true cholera spirilla. 



Metchmkoff' recommended the following method: A series of 

 flasks are prepared, and into each the following solution is 

 poured, i.e., water 50 c.c., peptone 2 grammes, salt 



