A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.