294 A MANUAL OF BACTERIOLOGY 



and undoubtedly occasionally a positive result may be obtained 

 by this longer incubation. 



Loffier's methylene blue gives much more characteristic prepara- 

 tions than Gram's method. 



Although eighteen to twenty hours is recommended for incubating 

 the cultures, a microscopical examination will sometimes reveal 

 the bacilli at a much earlier period. The writer has found them in 

 as short a time as six hours, but if bacilli are then not found the 

 tubes must be incubated for the longer period. 



Neisser's method of staining is as follows : 



(a) One gramme of methylene blue (Griibler's) is dissolved in 

 20 c.c. of 96 per cent, alcohol, which is then mixed with 950 c.c. 

 of distilled water and 50 c.c. of glacial acetic acid. 



(b) Two grammes of Bismarck brown are dissolved in one litre 

 of boiling distilled water and the solution is filtered. 



The preparations are stained in (a) for one to three seconds, 

 rinsed in water, and stained in (b) for three to five seconds, washed 

 in water, dried, and mounted. The bacilli are stained brown, and 

 contain two, rarely three, inky-blue dots. This is a valuable con- 

 firmatory stain for the diphtheria bacillus, but staining for a longer 

 time than that recommended by Neisser is advisable, viz. half a 

 minute in the blue and one minute in the brown. Tanner treats 

 with Gram's iodine solution for half a minute after the blue. The 

 staining solutions seem to keep well but occasionally fail to act, so 

 should be controlled on an undoubted diphtheria culture. 



Pugh's stain is also a very good one. It is a mixture containing 

 1 grm. of toluidine blue dissolved in 20 c.c. of absolute alcohol 

 and added to 1000 c.c. of distilled water and 20 c.c. of glacial 

 acetic acid. The mixture is applied for two minutes. The proto- 

 plasm of the bacilli is stained a pale blue and the polar bodies are 

 deeply stained and stand out in marked contrast ; by artificial 

 light they appear a reddish purple. 



In the majority of cases, after a little experience, the Klebs- 

 Loffler bacillus will be readily recognised if present. Occasionally, 

 however, bacilli may be present which resemble the Klebs -Loftier 

 very closely, and of which it is difficult to be certain. In such a 

 case the following points should be noted in attempting to arrive 

 at a decision : 



1. The character of the growth on the medium. 



2. The depth of staining with Loffler's blue, and the presence or 

 absence of segmentation or polar staining : the Klebs-Loffler 

 bacillus usually stains somewhat deeply, while the bacilli resembling 

 it stain but feebly. 



