2. Stain in Pranter’s solution (orcein D 0.1 grm., hydrochloric acid 2.0 cc., 70 per cent alcohol 100 cc.) for 8-24 hours.
3. Wash rapidly in 70 per cent alcohol.
4. Wash in water.
5. Stain in polychrome methylene-blue 10 minutes to several hours.
6. Wash in distilled water.
7. Differentiate in glycerin ether until no more clouds of color come away and section is light blue.
8. Wash in distilled water.
9. 70 per cent alcohol for a few seconds; absolute 5-10 minutes; xylol; balsam.
Protoplasm is gray-brown; bacteria dark-blue; background colorless. Zieler’s method is especially good for the staining of the glanders, typhoid and chancroid bacilli and the gonococcus.
For Unna’s methylene-blue eosin and the modifications of the Romanowsky method see Pages [260] and [290] respectively. Pappenheim’s methyl-green-pyronin method is also recommended for the staining in sections of Gram-negative bacteria.