(6) Apply a saturated aqueous or alcoholic solution of Bismarck brown one-half to one minute, or a weak solution of fuchsin until the film become pink. The latter {40} probably gives a better contrast stain, but there is danger of overstaining. (7) Wash in water, dry, and mount. By this method Gram-staining bacteria are purple; Gram-decolorizing bacteria and nuclei of cells are brown or red. * * * * * [Illustration: FIG. 11.--Bacillus of influenza; cover-glass preparation of sputum from a case of influenza, showing the bacilli in leukocytes; highly magnified (Pfeiffer).] 2. Cells.--These include pus-corpuscles, epithelial cells, and red blood-corpuscles. (1) Pus-corpuscles are present in every sputum, and at times the sputum may consist of little else. They are the polymorphonuclear leukocytes of the blood, and appear as rounded cells with several nuclei or one very irregular nucleus (Fig. 8 and Plate II, Fig. 2). They are frequently filled with granules of coal-dust and are often much degenerated. Such coal-dust-laden leukocytes are especially abundant in anthracosis, where angular black particles, both intra- and extra-cellular, are often so numerous as to color the sputum (Plate II, Fig. 2, _B_). Occasionally mononuclear leukocytes are present. {41} _Eosinophilic leukocytes_ are quite constantly found in large numbers in the sputum of bronchial asthma near the time of the paroxysm, and constitute one of the most distinctive features of the sputum of this disease. They resemble ordinary pus-corpuscles, except that their cytoplasm is filled with coarse granules having a marked affinity for eosin. Large numbers of free granules, derived from disintegrated cells, are also found (Fig. 12). [Illustration: FIG. 12.--Sputum from a case of asthma showing leukocytes, some containing eosinophilic granules; free eosinophilic granules; and micrococci; stained with eosin and methylene-blue (Jakob).] Ordinary pus-cells are easily recognized in sputum stained by any of the methods already given. For eosinophilic cells, some method which includes eosin must be used. A simple method is to stain the dried and fixed film two or three minutes with saturated solution of eosin, and then one-half to one minute with Löffler's methylene-blue; nuclei and bacteria will be blue, eosinophilic granules bright red. (2) Epithelial cells may come from any part of the {42} respiratory tract. A few are always present. They have little diagnostic value, although a considerable excess would indicate a pathologic condition at the site of their origin. Any of the stains mentioned above will show them, and they can usually be identified in unstained sputum. In general three forms are found: