122 BACTERIOLYSINS AND HEMOLYSINS. 



As far as the Pfeiffer experiment is concerned the virulence titer in this case is 1/5 of a 

 loopful of an agar culture because this dose is fatal within twenty-four hours. In order, 

 however, to make sure of excluding all individual variations, which can and occasionally 

 do occur, it is advisable to use not the titer dose, but its fifth or tenth multiple, that is, in 

 this case, one loopful. 



Doses larger than one loopful should be avoided, so that if any particular 

 strain of typhoid bacilli is not sufficiently virulent, necessitating the use of 

 larger doses, the virulence must first of all be increased. This is done by 

 passing the organism through animals such as guinea-pigs. 



The method is as follows: A very large dose of the culture, for example 



To Increase the surface of an entire agar tube, is injected intraperitoneally. Every 



the Virulence, animal succumbs to this enormous dose. The bacteria-laden exudate 



from the abdominal cavity, which, of course, must be removed under 

 sterile precautions is then inoculated into a second guinea-pig and when it dies, into a 

 third, and so on. As a rule, after passing through one or two animals the bacterial strain 

 (which must be grown pure from the cadaver) becomes more virulent, as can be proven 

 by titration. Very often the virulence is increased exclusively for the species of animal 

 used and occasionally this is associated with a decrease in virulence for other species. 

 After a series of passages through animals, the strain reaches its maximum strength 

 beyond which it cannot be increased. The degree of virulence varies with the type of 

 bacteria. Typhoid and cholera usually reach only a moderate virulence (i/io to 1/20 

 loopful); the bacteria of the hog cholera group can acquire a distinctly higher virulence; 

 for instance, B. paratyphosus, i/ioo to i/iooo of a loopful, while the streptococcus and 

 pneumococcus reach the highest figures, i/ioooo to i/ioooooo of a loopful. 



For the Pfeiffer's experiment with cholera or typhoid, the most suitable strains are 

 those of such a virulence that 1/5 to i/io of a loopful injected intraperitoneally kills in 

 twenty-four hours. 



The serum to be investigated is freed of its serum by heating in 



Technique of a water-bath for one-half hour at 56 C. Then a series of 



Pfeiffer's dilutions are made in bouillon (not in salt solution) for instance 



Experiments, i/io, i/ioo, i/iooo, etc. A c.c. of each dilution is put into a 



test-tube (a sterile pipette should be used) and rubbed up with 



a standard loopful of an 18- to 24-hour agar culture of typhoid bacteria. 



Finally the contents of each test-tube are injected intraperitoneally into a 



guinea-pig of 250 grams weight. 



Inasmuch as small amounts are apt to be lost when aspirating the fluid 

 with the syringe as well as when pouring the bacterial emulsion into a watch 

 glass, it is better to rub up two loops of the culture in 2 c.c. of bouillon 

 instead of i loop in i c.c., and then withdraw only i c.c. for use in the 

 experiment. 



The following controls should be prepared: 



i. Dilutions of the serum of a normal person (or animal of the same 

 type) f typhoid culture. 



