5. Stain sections, well spread out, for 5-10 minutes in a solution of 2 cc. of a saturated solution of thionin in 50 per cent alcohol and 10 cc. of water, to which 1-2 drops of ammonia are added.
6. Wash in water.
7. Stain ½-1 minute in hot saturated and cold filtered watery-solution of picric acid.
8. Wash in water.
9. Differentiate in 70 per cent alcohol until the color ceases to come away in blue-green clouds, 5-10 minutes or more.
10. Dehydrate in 96 per cent alcohol.
11. Clear in phenol xylol; xylol; balsam.
Lacunæ and canaliculi dark brown to black; bone cells red; ground substance yellow or brownish yellow. Calcified areas take a darker yellow than non-calcified. This method consists in an impregnation with a fine precipitate rather than a staining. Should the precipitate be too heavy in portions of the section it may be removed by thorough washing between 9 and 10.
Schmorl’s Thionin and Phosphotungstic or Phosphomolybdic Acid Method.
1. Fix thin pieces of fresh bone in formol, then in Müller’s for 6-8 weeks, or 3-4 weeks in the incubator. Fixation is best at 37°C.