834 PATHOGENIC MICROORGANISMS 



To seventy parts of ordinary 3 per cent agar, neutralized to litmus, there 

 are added thirty parts of a sterile mixture of equal parts of defibrinated 

 beef blood and normal sodium hydrate. 



The latter is sterilized by steam before being added to the agar. This 

 pure alkali agar is poured out in plates and allowed to dry several days at 

 37 or five minutes at 60. 



The material to be examined is smeared upon the surface of these 

 plates with a glass rod. If the blood-alkali mixture is prepared 

 beforehand and allowed to stand for four or five weeks, the plates 

 may be used immediately after pouring (Teague). 



The principle of this medium is that cholera will grow in the 

 presence of an amount of alkali which inhibits other fecal bacteria. 



For other cholera media see the section on media in the first part 

 of this book. 



The rational basis for the isolation of cholera spirilla from fecal 

 or other material is found in two chief properties of the spirilla. 

 These have been described to us by Teague as follows: (1) It grows 

 on media of an alkalinity that retards or completely inhibits the 

 growth of most of the fecal bacteria. (2) It comes to the surface 

 of fluid media, rich in oxygen, to a greater extent than do the 

 fecal bacteria. 



The best results in the practical isolation of the cholera spirilla 

 from stools are obtained by making use of both of these 

 properties from the beginning. A portion of the stool is seeded 

 directly into alkalin-pepton water. The broth used should be 

 distinctly alkalin, titrated to 0.5, or 1.0, with phenolphthalein. 

 After six to twelve hours, a loopful from the surface of these pepton 

 water tubes is plated upon plates of Dieudonne's medium, and is 

 also transferred to a second series of alkalin-pepton water tubes. 

 Once isolated, the spirilla are identified by their morphology and 

 motility, by the appearance of their colonies, by their manner of 

 growth upon gelatin stabs, by the cholera-red reaction, and, finally, 

 by agglutinative tests in immune sera. Owing to the existence of 

 other spirilla morphologically and culturally similar, the serum reac- 

 tions are the only absolutely positive differential criteria. 



For isolation of the bacteria from water, it is, of course, necessary 

 to use comparatively large quantities. Fliigge and Bitter advise 

 the distribution of about a liter of water in ten or twelve Erlen- 

 meyer flasks. To each of these they add 10 c.c. of sterile pepton-salt 

 solution (pepton ten per cent, NaCl five per cent). After eighteen 



