DIAGNOSIS OF DIPHTHERIA 351 



when lying side by side do not seem quite to touch, while the 

 bacilli which resemble the Klebs-Lomer and show a parallel 

 grouping frequently lie much closer together than the Klebs- 

 Lomer bacillus ever does. 



9. The reaction with Neisser's or Pugh's stain (the culture must 

 be a young serum one) : the pseudo -bacillus and other bacilli do 

 not give the diphtheritic reaction (polar staining). 



10. The final test of virulence may be applied. For this pur- 

 pose the organism must be isolated in pure culture by plate culti- 

 vations. Two guinea-pigs, of 250-300 grm. weight, are each 

 inoculated with 2 c.c. of a forty-eight hours' broth culture, one 

 receiving at the same time 1 c.c. of diphtheria antitoxin. If the 

 guinea-pig inoculated with culture only dies, while the one receiv- 

 ing culture and antitoxin lives, this is complete proof that the 

 organism is the diphtheria bacillus ; if both live no inference 

 can be made except that the organism is non-virulent ; if both 

 die it shows that the organism is virulent, but that it is not 

 neutralised by antitoxin, and is therefore not the diphtheria 

 bacillus. In cases in which bacilli persist, the test of viru- 

 lence is frequently applied. If the organism proves to be non- 

 virulent, presumably the patient is non-infective. Such a pre- 

 sumption, in the author's opinion, however, is not necessarily 

 true. 



11. Agglutination tests are unsatisfactory and not of service. 

 It occasionally happens that a conclusion cannot be arrived at 



without an extended investigation. 



If serum tubes are not available an egg may be used. It is 

 boiled hard, the shell chipped away from one end with a knife 

 sterilised by heating, and the inoculation made on the exposed 

 white ; the egg is then placed, inoculated end down, in a wine- 

 glass of such a size that it rests on the rim and does not touch the 

 bottom. A few drops of water may with advantage be put at the 

 bottom of the glass to keep the egg-white moist. The preparation 

 is kept in a warm place for twenty-four to forty-eight hours and 

 then examined. Antitoxin itself may be used as a culture medium, 

 provided it contains no antiseptic (this is now rarely the case). A 

 test-tube is sterilised by heating, or with boiling water or steam 

 from a kettle, antitoxin to the depth of about an inch is poured in, 

 and is coagulated by holding the tube very obliquely in boiling 

 water or steam. After coagulation and cooling the medium is 



