METHODS OF DIAGNOSIS 427 



microscopical examination alone depends much upon the experi- 

 ence of the observer. In some cases the bacilli are present in 

 characteristic form in such numbers as to leave no doubt in 

 the matter. In other cases a few only may be found, mixed 

 with large numbers of other organisms, and sometimes their 

 characters are not sufficiently distinct to render a definite opinion 

 possible. The bacillus may be frequently obtained by means 

 of cultures, when the result of microscopical examination is 

 inconclusive. As already said, however, microscopical examina- 

 tion alone is more reliable after the observer has had experience 

 in examining cases of diphtheria and making cultures from them. 



(b) By making Cultures. For this purpose a piece of the 

 membrane should be separated by forceps from the pharynx or 

 other part when that is possible. It should be then washed 

 well in a tube containing sterile water, most of the surface im- 

 purities being removed in this way. A fragment is then fixed in 

 a platinum loop by means of sterile forceps, and a series of 

 stroke cultures is made on the surface of any of the media 

 mentioned (p. 414), the same portion of the membrane being 

 always brought into contact with the surface. The tubes are 

 then incubated at 37 C., and, in the case of the serum 

 media and blood-agar, the circular colonies of the diphtheria 

 bacillus are well formed within twenty- four hours. A small 

 portion of a colony is then removed by means of a platinum 

 needle, stained, and examined in the usual way, Neisser's stain 

 being also applied. When the material has been taken from 

 the throat, an organism with all the morphological and cultural 

 characters of the diphtheria bacillus may for all practical 

 purposes be accepted as the diphtheria bacillus. 



In cases where a suspicion arises that the organism found 

 is a pseudo-diphtheria bacillus, bouillon containing a trace 

 of glucose should be inoculated and incubated at 37 C. 

 The reaction should be tested after one and after two days' 

 growth. If it remains alkaline, the diphtheria bacillus may be 

 excluded. If an acid reaction results, then all the microscopical 

 and cultural characters must be carefully observed, and the 

 virulence of the bacillus may be ascertained by inoculating a 

 guinea-pig, say with 1 c.c. of a broth culture of two days' growth. 

 (See also pp. 417, 423.) A fatal result with characteristic 

 appearances may be taken as positive evidence ; but if the animal 

 survive there is still theoretically the possibility that the 

 organism is an attenuated diphtheria bacillus (p. 421). 



