METHODS OF DIAGNOSIS 369 



normal saline should be added. The films are then dried in the 

 usual way and stained with any ordinary basic stain, though 

 methylene-blue is on the whole to be preferred, used either as 

 a saturated watery solution or in the form of Loffler's solution. 

 After staining for two or three 'minutes the films are washed in 

 water, dried, and mounted. As a rule no decolorising is 

 necessary, as the blue does not overstain. Neisser's stain (p. 108) 

 may also be used with advantage. Any secretion from the 

 pharynx or other part is to be treated in the same way. The 

 value of microscopical examination alone depends much upon 

 the experience 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 quantities of other organisms, and sometimes 

 their characters are not sufficiently distinct to render a definite 

 opinion possible. We have frequently obtained the bacillus by 

 means of cultures, when the result of microscopical examination 

 of the same piece of membrane was non-conclusive. As already 

 said, however, microscopical examination 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 are made on the surface of any of the media 

 mentioned (p. 357), the same portion of the membrane being 

 always brought into contact with the surface. The tubes are 

 then placed in the incubator 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 

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