xiii] MICROBES OF MALIGNANT ANTHRAX 307 



those which resemble diphtheria colonies are subjected to 

 microscopic examination in stained film specimens and to 

 subcultures. I have thus succeeded in finding a few diph- 

 theria colonies amongst crowds of colonies of cocci, whereas 

 serum tubes have failed to give a positive result. But by far 

 the best method is the Ascites Agar fluid set with slanting 

 surface, which was mentioned in a former chapter as Kan- 

 thack's serum Agar ; for by means of this medium the diph- 

 theria colonies can be demonstrated far more certainly than 

 with any other medium. I have seen this in cases in which 

 the diphtheria bacilli were scanty and much mixed up with 

 cocci, and yet a particle of the secretion on the membrane 

 rubbed over the slanting surface of Kanthack's serum 

 -Agar, and incubated at37°C., produced in twenty-four 

 hours a pure crop of diphtheria colonies. 



Recent cultures of the diphtheria bacillus on Agar, on 

 gelatine, on serum, and in broth prove virulent on guinea- 

 pigs, but this virulence decreases with the age of the culture. 

 A broth culture of which after forty-eight hours' incubation 

 at 37° C. o'25 to o'3 cc. is capable of killing in thirty to forty 

 hours one kilogramme body-weight of guinea-pigs is con- 

 sidered of normal virulence (Behring). Of gelatine subculture 

 made from a normal broth culture (three streaks on a slanting 

 surface six centimetres by two centimetres) incubated at 

 20-21° C. for seven to ten days, the growth being then 

 scraped down and suspended in sterile broth, qne-sixth of 

 the total growth is sufficient to kill one kilo, guinea-pig in 

 thirty to forty hours. In an Agar culture made in the same 

 way, incubated at 37° C, the same amount of virulence is 

 found during the first three or four days ; later the virulence 

 decreases, as does also often that of a broth culture after the 

 first six or seven days. 



Loffler has shown that with cultures of the diphtheria 



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