ASIATIC CHOLERA AND THE CHOLERA ORGANISM 585 



Egg-White Medium. 



A. White of egg and water a.a. 

 Sodium carbonate cryst. 12 per cent. 



Mix in equal parts, steam in Arnold sterilizer for 20 minutes. 



B. Meat pepton 3 per cent agar, neutral to litmus. 



30 parts of A are added to 70 parts of B. 



Another modification recommended by them is as follows: 



Whole-Egg Medium. 



A. Whole egg and water a.a. 



Sodium carbonate 12 to 13.5 per cent. 



Mix in equal parts, steam for 20 minutes. 



B. Meat free agar, viz., pepton, salt, and 3 per cent agar. 



30 parts of A are mixed with 70 parts of B while the agar is boiling hot as 

 above. 



The medium is poured on the plates in a thick layer and allowed 

 to stand open for 20 to 30 minutes and then the inoculation is carried 

 out by surface streaking. 



Isolation. Isolation of the cholera vibrio from the feces, while 

 easy in many cases, is occasionally attended with some difficulty 

 owing to the large number of other bacteria present. The most 

 satisfactory method of procedure is to inoculate a set of gelatin 

 plates, another of agar plates, and a number of Dunham's pepton- 

 broth tubes, with small quantities of the suspicious material. When 

 the spirilla are numerous they can frequently be fished directly from sus- 

 picious colonies in the plates and isolated for further identification. 

 When less numerous, they can usually be found in relatively increased 

 numbers after eight or ten hours at 37.5 C., in the topmost layers of 

 the Dunham broth, which is an almost selectively favorable medium for 

 these organisms. They collect at the surface where free oxygen is 

 readily obtained. From the pepton broth, plate dilutions can then be 

 prepared and colonies fished. 1 Once isolated, the spirilla are identified 

 by their morphology, by the appearance of their colonies, by their 

 manner of growth upon gelatin stabs, by the cholera-red reaction, 

 and, finally, by agglutinative and bacteriolytic tests in immune sera. 

 Owing to the existence of other spirilla morphologically and cultu- 

 rally similar, the serum reactions are the only absolutely positive dif- 

 ferential criteria. 



1 Abel und Claussen, Cent, f . Bakt, xvii, 1895. 



