(3) Wash with water.
(4) Apply Gram's iodin solution one-half to two minutes.
(5) Wash in alcohol until the purple color ceases to come off.
(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 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.
| 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.
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).