96 LABORATORY BACTERIOLOGY 



145. Work for this Exercise — Examine and describe the 

 cultures made in Exercise XLVI. 



Examine the agar and bouillon cultures microscopically in 

 (i) hanging-drop preparations and (2) stained cover-glass 

 preparations. Stain with alkaline methylene-blue and with 

 carbol fuchsin. The preparation should be stained for fully 

 5 minutes with the alkaline methylene-blue. Also stain a 

 preparation after Gram's method (38). 



Examine carefully a fresh culture made directly from a diph- 

 theritic throat, including stained cover-glass preparations.^ 

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

 is not always possible to obtain these cultures at this particular 

 time, in which case the examination will be postponed until 

 they are available.) 



146. 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 c.c. of 96% alcohol, which is then mixed with 950 c.c. of 

 distilled water and 50 c.c. 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 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 diphtherice is to be differentiated from the 

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

 decayed teeth, and which is said to resemble in its morphology the Klebs- 

 Loeffler bacillus very closely. It is also to be distinguished from the 

 Xerosis bacillus isolated by Neisser. For detailed descriptions of these 

 organisms see text-books. 



