The spores of different bacilli differ greatly in their resistance to decolourising reagents; even the spores of the same species of organisms vary according to their age. Young spores are more easily decolourised than those more mature. Sulphuric acid, 1 per cent. aqueous solution, and hydrochloric acid, 0.5 per cent. alcoholic (90 per cent.) solution, are useful decolourising reagents. ~3. Moeller's Method.~-- 1. Prepare and fix films in the usual manner. 2. Immerse in absolute alcohol for two minutes, then in chloroform for two minutes; wash in water. This dissolves out any fat or crystals that might otherwise retain the "spore" stain. 3. Immerse in chromic acid, 5 per cent. aqueous solution, for one minute; wash in water. 4. Pour Ziehl's carbolic fuchsin on the film, warm as in previous methods, and allow it to act for ten minutes. 5. Wash in water. 6. Decolourise in sulphuric acid, 5 per cent. aqueous solution, for five seconds. 7. Wash in water. 8. Counterstain with Kuehne's carbolic methylene-blue for one or two minutes. 9. Wash in water. 10. Dry and mount. (Spores red, bacilli blue.) ~4. Abbott's Method.~-- 1. Prepare and fix films in the usual manner. 2. Pour Loeffler's alkaline methylene-blue on the film; warm cautiously over the flame till steam rises and allow the hot steam to act for one to five minutes. 3. Wash thoroughly in water. 4. Decolourise in nitric acid, 2 per cent. alcoholic (alcohol 80 per cent.) solution. 5. Wash thoroughly in water. 6. Counterstain in eosin, 1 per cent. aqueous solution. 7. Wash. 8. Dry and mount. (Spores blue, bacilli red.) DIFFERENTIAL METHODS OF STAINING. ~Gram's Method.~--This method depends upon the fact that the protoplasm of some bacteria permits aniline gentian violet and Lugol's iodine solution, when applied consecutively, to enter into a chemical combination which results in the formation of a new blue-black pigment, only very sparingly soluble in absolute alcohol. Such organisms are said to "stain by Gram," or to be "Gram positive." 1. Prepare a cover-slip film and fix in the usual way. 2. Stain in aniline gentian violet three to five minutes. Filter as much aniline water on to the cover-slip as it will hold; then add the smallest quantity of alcoholic solution of gentian violet which suffices to saturate the aniline water and form a "bronze scum" upon its surface--if too much of the alcoholic gentian violet is added the alcohol present redissolves this scum. To prepare aniline water, pour 4 or 5 c.c. aniline oil into a stoppered bottle and add distilled water, 100 c.c. Shake vigourously and filter immediately before use. The excess of oil sinks to the bottom of the bottle and may be used again. 3. Wash in water.