METHODS OF DIAGNOSIS. 375 



the blue does not overstain. Neisser's stain (p. 363) 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 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 quantities of other organisms, and sometimes their charac- 

 ters 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. 361), the same portion of the membrane being al- 

 ways brought into contact with the surface. The tubes are then 

 placed in the incubator at 37° C, and, in the case of the serum me- 

 dia and blood agar, the circular colonies of the diphtheria bacillus 

 are visible in twenty-four hours. A small portion of a colony is 

 then removed by means of a platinum needle, stained, and ex- 

 amined in the usual way, the characteristic appearance of the 

 organism being readily recognised. 



In cases where a suspicion arises that the organism found is 

 the pseudo-diphtheria bacillus, bouillon containing a trace of 

 glucose should be inoculated and incubated at 37" C. The re- 

 action 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 I c.c. of a broth culture of two days' growth. (See also 

 PP- 370, 371. 372.) 



