BACTERIUM DIPHTHERIA 103 



150. Work for this exercise. Examine and describe the 

 cultures made in Exercise XLII. 



Examine the serum, glycerin agar, and bouillon cultures 

 microscopically in stained cover-glass preparations. Stain with 

 alkaline methylene blue. The preparation should be stained 

 for fully 5 minutes and decolorized for a few seconds with 

 alcohol. Examine the bouillon culture in a hanging-drop 

 preparation. 



Examine carefully a fresh culture made directly from a diph- 

 theritic throat, including stained cover-glass preparations. 1 

 Stain with alkaline methylene blue and by Neisser's method. 

 (It is not always possible to obtain these cultures at this par- 

 ticular time, in which case the examination will be postponed 

 until they are available.) 



151. Neisser's method of staining diphtheria bacteria. Neisser 

 has recently recommended the following method of staining, 

 in which 2 solutions are employed, viz. : 



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

 20 cc. of 96% alcohol, which is then mixed with 950 cc. of 

 distilled water and 50 cc. of glacial acetic acid. 



(b) Two grams of vesuvin are dissolved in i litre of boiling 

 distilled water and filtered. 



The cover-glass preparations are stained in (a) for from i 

 to 3 seconds, washed in water, and then stained in (b) for from 

 3 to 5 seconds, again washed in water, dried, and mounted. 

 Stained in this manner the bacilli are brown, and contain 2, 

 or rarely 3, but never more, blue corpuscles. The corpuscles 

 are oval, not round, in shape, and their diameter appears 

 greater than that of the bacilli in which they are situated. 



1 Clinically, Bacterium diphtheria is to be differentiated from the 

 pseudo-diphtheriae organism and from a bacillus which has been found in 

 decayed teeth, and which is said to resemble very closely in its mor- 

 phology the Klebs-LoefHer bacillus. It is also to be distinguished from 

 the Xerosis bacillus isolated by Neisser. For detailed descriptions of 

 these organisms, see text-books. 



