SUGAR IN THE NUTRIENT MEDIUM. 159 



virulence, the inoculation must be made upon a sensi- 

 tive animal, and the proper channel of infection must 

 be selected. 



The virulence of bacteria varies like all their other 

 functions (production of coloring matters, fermenta- 

 tion, etc.), and is best retained by constant inocula- 

 tion from one sensitive animal to another. This is 

 also done in many varieties by tolerably frequent 

 transmission (about once a month) from one artificial 

 nutrient medium to another, preferably with an occa- 

 sional intermediate inoculation of an animal. On 

 the other hand, the virulence usually suffers when, on 

 account of rare inoculations, the cultures remain for a 

 long time in contact with their accumulating meta- 

 bolic products. 



Attenuation of the virulence is easily effected : 



(a) By making the cultures at somewhat too high 

 a temperature. For example, at 42.5 anthrax is en- 

 tirely deprived of virulence in three to four weeks, at 

 47 in a few hours, at 50-53 in a few minutes. By 

 the proper regulation of the action of heat Jhe bacil- 

 lus anthracis may be attenuated to such a degree that 

 it will kill only mice, or mice and guinea-pigs, or 

 these animals and rabbits. 



Spores may also be "attenuated" by dry heat or 

 brief, careful disinfection with steam. 



(b) By cultures upon an unsuitable nutrient me- 

 dium. The addition of phenol (1 : 600), potassium 

 bichromate (0.04-0.02 per cent) was employed suc- 

 cessfully to attenuate bacillus anthracis, iodine tri- 

 chloride to attenuate the bacilli of diphtheria. 



(c) By the action of sunlight, compressed oxy- 

 gen, etc. 



