BONE 



45 



BORAX CARMINE 



sections on a fine hone to remove preci- 

 pitated silver, dehydrate in alcohol, 

 clear in xylol and mount in balsam. 

 The lacunae and canaliculi appear black 

 in a yellowish brown background. To 

 impregnate thin sections with acid 

 fuchsin, dry them after extraction with 

 alcohol. Place them in watch glasses 

 in a 20% aq. sol. of acid fuchsin in a 

 desiccator connected with a suction 

 apparatus. Extract air for about an 

 hour and close the dessicator. After 

 24 hrs. the solution will have dried. 

 Rub off ppt. on a hone, pass through 

 xylol and mount in damar or balsam 

 (Shipley). Linings of lacunae and 

 canaliculi. (Schmorl's method modi- 

 fied by Shipley. ) Employ a fixative not 

 containing mercury. Decalcify in Miil- 

 ler's fluid, wash in running water, embed 

 in celloidin and section not over 10 mi- 

 crons. Stain in thionin solution al- 

 kalinised by 2 drops ammonia. Trans- 

 fer with glass needle to sat. aq. 

 phosphotungstic or phosphomolybdic 

 acid. Leave until blue, gray or green. 

 Place in water until sky-blue. Am- 

 monium hydroxide 1 cc. and aq. dest. 

 10 cc, 3-5 min. Several changes 90% 

 alcohol. 95% a)c. Clear in carbol- 

 xylol and mount in damar (or balsam). 

 This method is suggested for bones of 

 children. Processes of young osteoblasts 

 in growing bone. Shipley suggests fol- 

 lowing treatment of slices of bone of a 

 rickety animal. 4% aq. citric acid 

 20-30 min. in the dark. Rinse in aq. 

 dest. 1% aq. gold chloride in the dark, 

 20-30 min. 3% formic acid in the dark, 

 48 hrs. Rinse in aq. dest. and preserve 

 in pure glycerin. Make frozen sections, 

 mount in glycerin and ring with damar, 

 balsam, paraffin or cement. Keep spe- 

 cimens in dark when not in use. 



To determine relative age of deposi- 

 tion the following method has proved 

 useful in senile osteoporosis. Saw sec- 

 tions of bone not more than 0.5 cm. 

 thick and fix in 4% formalin 2-4 days. 

 Decalcify in 6% isotonic formalin, 40 

 cc, 85% formic acid, 60 cc, and sodium 

 citrate, 5 gm. changing every second 

 day for, say, a week, that is until they 

 become flexible and can be penetrated 

 by a fine needle. Embed in celloidin 

 (slow method). Prepare stain by dis- 

 solving 30 gm. potassium alum in 1 liter 

 hot water and by adding 1.5 gm. hema- 

 toxylin crystals. Cool and add 1 gm. 

 chloral hydrate. Ripening in sunlight 

 to rich dark color is hastened by addition 

 of crystal of potassium hydroxide. 

 Stain celloidin sections about 2 days 

 checking by microscopic e.xamination 

 until some areas are definite violet azur, 

 others lighter or colorless. Wash in tap 

 water 24 hrs. Stain in 100 cc. aq. dest. 



+ 2-3 drops 1% aq. eosin 1-2 days 

 (uncolored areas become dark rose 

 color). Dehydrate, clear in xylol and 

 mount in balsam. Old bone azur; new 

 bone bright rose (Belloni, L., Arch. 

 Ital. Anat. e Istol. Path., 1939, 10, 

 622) . See Madder staining of new bone, 

 Alizarin Red S staining of dentine, 

 various tests for Calcium, and Ossifica- 

 tion, Line Test for vitamin D potency. 

 Polarized light is excellent for the 

 demonstration of bone camellae. 



Bone Marrow. Microscopic examination of 

 bone marrow in vivo has not been 

 achieved because of the obvious techni- 

 cal difficulties involved. The best that 

 can be done is to study still living cells 

 removed from bone marrow unstained 

 or supravi tally stained. The methods 

 are essentially the same as for blood. 

 From humans samples can be obtained 

 by sternal puncture (Young, R. H. and 

 Osgood, E. E., Arch. Int. Med., 1935, 

 55, 186-203, and many others). Pri- 

 mitive cells of the erythrocytic and 

 leucocytic series can only be .identified 

 when hemoglobin and specific granules 

 respectively appear within them. Mi- 

 crochemical tests for Hemoglobin should 

 be more used. For the granules the 

 methods of Giemsa, Wright, Ehrlich 

 and others are the best available. 

 Special techniques have been described 

 for Megakaryocytes particularly in rela- 

 tion to platelet formation. To demon- 

 strate the vascular pattern special 

 methods are required (Doan, C. A., 

 Johns Hopkins Hosp. Bull., 1922, 33, 

 222-226). To reveal the nerve supply 

 is particularly difficult. Glaser (W., 

 Ztsch. f. Anat. u. Entw., 1928, 87, 741- 

 745) has described a fine network accom- 

 panying the vessels but Doan and Lang- 

 worthy (Downey, p. 1852) were less 

 successful . Sternal bone marrow during 

 first week of life (Shapiro, L. M., and 

 Bessen, F. A., Am. J. Med. Sci., 1941, 

 202, 341-354). Bone marrow of normal 

 adults (Plum, C. M., Acta Med. Scand., 

 1941, 107, 11-52. See chapters by Sabin 

 and Miller and by Doan in Downey's 

 Handbook of Hematology, New York, 

 Hoeber, 1938, 3, 1791-1961 for details. 

 A method for studying numerical and 

 topographic problems in the whole 

 femoral marrow of rats and guinea pigs, 

 with the use of undecalcified sections 

 (Mayer, E. and Ptuzicka, A. Q., Anat. 

 Rec, 1945, 93. 213-231). 



Borax Carmine (Grenacher). Make con. 

 sol. of carmine in borax (2-3% carmine 

 in 4% aq. borax) by boiling for 30 min. 

 Allow to stand 2-3 daj's with occasional 

 stirring. Dilute with equal volume 70% 

 ale, again allow to stand and filter. 

 Much used for staining tissues in bulk. 

 They are colored for days if necessary, 



