46 BACTERIOLOGY FOR NURSES 



FORMULA OF SOME OF THE MOST FREQUENTLY USED STAINS 

 Loeffler Methylene Blue. 



Saturated alcoholic solution of methylene blue ... 30 c.c. 

 1-10,000 solution of caustic potash in distilled water . 100 c.c. 



Films may be stained with the above preparation for from two to 

 five minutes without being overstained. It is a useful stain for 

 structural differentiation and is generally employed in routine 

 examination for the diphtheria bacillus. 

 Neisser's Stain. Two solutions are used : 



(a) 5 per cent alcoholic solution of methylene blue . 20 c.c. 



Glacial acetic acid 50 c.c. 



Distilled water 950 c.c. 



(6) Bismarck brown 2 gm. 



Distilled water 1000 c.c. 



Films are stained in (a) for three to five seconds, washed in water 

 and stained in (6) for five seconds, dried and examined. 



The stain was originally introduced by Neisser as an aid in the 

 identification of the diphtheria bacillus. If the film is made 

 from a twenty-four hour culture grown on serum medium the 

 bacilli frequently show when stained by this method deep blue 

 granules with surrounding protoplasm of a faint brown. 



Ziehl-Neelsen's Carbol Fuchsin Stain. 



Basic fuchsin 1 gm. 



Absolute alcohol 10 c.c. 



5 per cent solution of carbolic acid 100 c.c. 



After fixing the film may be placed in the stain, heated until steam 

 rises and allowed to remain there for five minutes, or it may be 

 allowed to remain in the cold stain from twelve to twenty-four 

 hours. The excess of stain is then washed off with water and the 

 film placed in a decolorizing solution ; 3 per cent hydrochloric acid 

 in 80 per cent alcohol gives good results as a decolorizing agent. 

 After a few seconds remove the film and wash in water ; if only the 

 faintest pink color persist decolorization has been sufficient; if 



