STAINING METHODS. 31 



carbol-f uchsin solution (No. 3) the cover glass is placed for one or 

 two minutes in a solution containing: 



Sulphuric acid (twenty-five-per-cent solution), . . 100 cc. 



Methylene blue, ...... 2 cc. 



Wash, dry, and mount in cedar oil or balsam. 



METHODS OF STAINING SPORES. When preparations containing 

 the spores of bacilli are stained by any of the methods above given, 

 these remain unstained and appear as highly refractive bodies in the 

 interior of the rods or filaments in which they have been formed, or 

 scattered about in the field if they have been set free. Owing to 

 the contrast with the stained protoplasm of the rod or spore-bearing 

 filament, they are especially well seen in recent cultures ; while in 

 older cultures the bacilli often do not stain well, or are entirely dis- 

 integrated and spores only are to be seen. The discovery was made 

 at about the same time by Buchner (1884) and by Hueppe that 

 spores may be stained if they are first exposed to an elevated tem- 

 perature for some time. This may be accomplished by placing the 

 slide or cover glass, upon which the spore-containing culture has 

 been dried, in a hot-air oven at a temperature of 120 C. for an 

 hour; or a higher temperature (180 C.) may be employed for a 

 shorter time (fifteen minutes) ; or the cover glass may be passed 

 through the flame of an alcohol lamp or Bunsen burner eight or ten 

 times, instead of three times as is customary when the object in 

 view is simply to coagulate the albumen and fix the film upon the 

 cover glass. After such treatment the spores may be stained with 

 an aqueous solution of one of the basic aniline colors fuchsin, 

 methyl violet, etc. but the bacilli no longer take the stain so well. 



To obtain satisfactory double-stained preparations, showing 

 both spores and bacilli, a different method is employed. 



The film upon the cover glass is passed through the flame three 

 times, as heretofore directed ; it is then floated upon aniline-f uchsin 

 solution in a watch glass, and this is heated to near the boiling point 

 for an hour Neisser's method. The aniline-f uchsin solution is 

 prepared by shaking an excess of aniline oil in a test tube with dis- 

 tilled water, filtering the saturated solution into a watch glass, and 

 then adding a few drops of a saturated alcoholic solution of fuchsin. 

 After this prolonged action of the hot staining fluid the spores of 

 some bacilli are deeply stained, while others do not take the stain so 

 well. The cover glass is next washed in water and then placed in 

 a decolorizing solution containing twenty-five parts of hydrochloric 

 acid to seventy-five parts of alcohol. This removes the stain from 

 the bacilli, but, if not allowed to act too long, leaves the spores still 

 stained. The preparation is next stained in a saturated aqueous 



