324 PUBLIC HEALTH BACTERIOLOGY 



(d) After eight to ten hours examine any colonies, and if 

 pure cultures, plant out as follows : (i) Peptone and salt 

 solution : in twenty-four hours at 37 C, turbid, and gives 

 cholera-red ; (ii) Gelatin plates : characteristic colonies 

 with irregular margins ; (hi) Gelatin stab : typical funnel- 

 shaped liquefaction ; (iv) Agar slope : growth in twenty- 

 four hours at 37 C. must give with anti-cholera serum, 

 agglutination and Pfeiffer's test ; (v) A portion of colony 

 should be examined for typical microscopical appearances. 



Make films and hanging drops direct from stools. 

 Dunbar diagnoses from two hanging drops, one having 

 added to it an equal quantity of 1-50 normal serum, the 

 other an equal quantity of 1-500 anti-cholera serum. 

 Cholera organisms retain their motility in the first instance, 

 but lose it and agglutinate in the second. The hanging 

 drop is mounted from peptone water in which a piece of 

 mucus has been broken up. 



2. From Water. Keep 10 per cent peptone water 

 sterilized. Take 900 c.c. of suspected water, and add 

 100 c.c. of strong peptone water. Divide into ten flasks, 

 each containing 100 c.c. Incubate at 37 C. In eight to 

 twelve hours make a film and hanging-drop preparation 

 from the surface of each flask. From those flasks showing 

 most similar forms, make subcultures, proceeding as 

 above. If a spirillum conforms to all the above tests, it is 

 probably the true cholera vibrio, but it must be remem- 

 bered that a certain number of spirilla, although agglutina- 

 ting to some extent with cholera serum, are sharply 

 differentiated by being multiciliated and hemolytic. 



SPIRILLA OTHER THAN THE 



CHOLERA SPIRILLUM. 



(Often present in water but not necessarily pathogenic). 



Sp. Metchnikovi Is found in a disease resembling fowl 

 cholera in the faeces and blood. It is practically identical 

 with the Sp. cholerae, having a single polar flagellum. 

 Culturally, it fluidifies gelatin twice as rapidly, and grows 

 slightly more luxuriantly. It is sharply distinguished, 

 however, by being very pathogenic to pigeons (doves), 



