INCREASE OF VIRULENCE OF BACTERIA 135 



As far as the Pfeiffer experiment is concerned the titer of virulence 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 par- 

 ticular 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 a 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, 1/10,000 to 1/1,000,000 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 complement by 

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

 Pfeiffer's series of dilutions are made in bouillon (not in salt solution) 

 Experiments. f or ms t a nce i/io, i/ioo, i/iooo, etc. A c.cm. of each dilu- 

 tion is put into a test-tube (a sterile pipette should be used) 

 and rubbed up with a standard loopful of an 1 8 to 24 hour agar culture 

 of typhoid bacteria. Finally the contents of each test-tube are injected 

 intraperitoneally in a guinea-pig of 250 gms. 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.cm. of 

 bouillon instead of i loop in i c.cm., and then withdraw only i c.cm. 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) + typhoid culture. 



