618 VIBRIO 



V.P. test (see p 368). According to van Loghem (1938) the El Tor vibrio gives 

 usually a positive V.P. reaction. 



Hcemolysin Formation. — Many members of this group form a hsemolysin acting 

 on sheep, horse, or rabbit cells. This can be demonstrated either on plates or in 

 a broth culture. In general, the cholera vibrio does not produce a soluble hsemoly- 

 sin, while the El Tor and many non-cholera vibrios are able to do so. For diagnostic 

 purposes a standard technique is essential. Much confusion has in the past been 

 due to differences in method of studying haemolysis. Zimmermann (1932, 1933), 

 working with 5 per cent, defibrinated sheep blood broth cultures, obtained conflicting 

 results, but when he used a medium made up with peptone, asparagin, and 

 ammonium lactate containing 4 per cent, sheep blood, and read his results after 

 48 hours' incubation at 37° C, a clear differentiation between the non-haemolytic 

 cholera and the haemolytic El Tor and non-cholera vibrios was apparent. Van 

 Loghem (1932) states that sheep or goat's blood should be used ; guinea-pig and 

 rabbit red cells are too sensitive. He further points out that the cholera vibrio, 

 though not forming a true soluble hsemolysin, does digest blood pigment ; this 

 property, which has been shown by Bernard, Guillerm and Gallut (1937) to be 

 due to a ferment present in cultures but not in the bodies of the cholera vibrio, 

 is responsible for the greenish discoloration around individual colonies on blood 

 agar and for the complete clarification of the medium that occurs on further 

 incubation. The El Tor vibrio digests blood likewise, but in addition it produces 

 a soluble hsemolysin, the extraction of which has been recorded by Bernard, Guil- 

 lerm and Gallut (1939). The observations of these workers suggest that the 

 cholera vibrio forms a hsemolysin as well as a digesting ferment, but that it is 

 mainly intracellular and, unlike that of the El Tor vibrio, does not diffuse out 

 to any considerable extent into the medium. The identity of these two hsemolysins 

 is suggested by the finding of Vassiliadis (1937) that the injection of non-hsemolytic 

 cholera vibrios into animals gives rise to anti-heemolysins for the El Tor vibrio 

 as active as those prepared by injection of the El Tor vibrio itself. 



Doorenbos (1936) finds that haemolysis is very much more active in 8-hoar 

 than in 24-hour cultures. Many strains of true cholera vibrios that were non- 

 hsemolytic after 24 hours produced haemolysis of sheep or goat cells after 8 hours. 

 With so many factors influencing haemolysin production, it would clearly be 

 dangerous to place too much weight on this characteristic as a means of differentiat- 

 ing between the vibrios. 



The common routine method for testing haemolytic activity is to grow the 

 organism in broth for 3 days at 37° C, to add 1 ml. of the culture to 1 ml. of a 5 

 per cent, suspension of washed goat or sheep red cells, to incubate for 2 hours 

 at 37° C., and to read the results after the tubes have been left in the cold 

 overnight. 



Toxin Production. — Nicati and Rietsch (1884) injected dogs intravenously with the 

 filtrate of a broth culture of V. cholerce a week or more old. In their first series of experi- 

 ments there were vomiting, defsBcation, and general depression, with recovery in an hour. 

 In their second series there were dyspnoea, vomiting, and paresis of the extremities, followed 

 by recovery, or death in 12 hours. At necropsy in the fatal cases, ecchymoses were found 

 in the duodenum and larger haemorrhages in the stomach. Filtrates of young cultures were 

 innocuous. 



PfeifFer (1892) likewise experimented with filtrates. He found that even 4 ml. of a 

 20-days' glycerine broth filtrate, injected intraperitoneally into guinea-pigs, had no 



