THE CONNECTIVE TISSUES. 



133 



is changed daily, and the bone remains immersed until it is soft enough 

 to be cut. This stage is reached when a needle can be introduced with 

 no resistance. 



(<) An aqueous solution of nitric acidic strengths of 3% to 10^, ac- 

 cording to the delicacy of the specimen, and of a specific gravity of 1.4. 

 Instead of water, 70% alcohol may be used as a solvent for the acid. 

 Thoma has recommended for this purpose a solution consisting of i 

 vol. nitric acid of a specific gravity of 1.3, and 5 vols. alcohol. This 

 fluid is changed daily and decalcifies small objects in a few days. The 

 specimens are then washed several times in 70% alcohol to remove 

 as much as possible of the acid. 95% alcohol, with the addition 

 of a little precipitated calcium carbonate, has been recommended for 

 washing sections that have been treated by Thoma' s method. After from 

 eight to fourteen days the specimens are again washed with clear 95% 

 alcohol. 



(c) The process of decalcification recommended by v. Ebner (75) is 

 of considerable value, as it also reveals the fibrillar structure of the 

 bone lamellae. A cold saturated solution of sodium chlorid is diluted 

 with 2 vols. of water, and 2 % of hydrochloric acid added. This fluid 

 decalcifies very slowly, and must either be changed daily or a small 

 quantity of hydrochloric acid occasionally added. As soon as the speci- 

 men is thoroughly decalcified, it is washed with a half-saturated solution 

 of sodium chlorid. A little ammonia is now added from time to time 

 until the reaction of the fluid and bone is neutral. 



(</) Very small pieces that contain very little lime-salts, as, for in- 

 stance, bones in an embryonal condition where calcification has only just 

 begun, can be deprived of their lime-salts by means of acid fixing solu- 

 tions like Flemming's fluid, chromic acid, picric acid, etc. 



(^) Bone should be first fixed in some one of the fixing fluids and 

 then decalcified. 



Schmorl's Method for Demonstrating the Bone Corpuscles 

 and their Processes in Decalcified Preparations. The tissues are 

 fixed in Mtiller's fluid or in Miiller's fluid with formalin, decalcified in 

 V. Ebner's fluid, and imbedded in celloidin. The sections are stained 

 in either of the following thionin solutions: concentrated 50% alcoholic 

 thionin solution, 10 c.c.; i per cent, carbolic acid water, 90 c.c.; or 

 concentrated 50% alcoholic thionin solution, 10 c.c.; distilled water, 

 100 c.c.; liquor ammonias, 10 drops. Bring sections from water into the 

 stain, in which they remain from five to ten minutes or longer. Rinse 

 sections in water, and place them in a saturated aqueous solution of pic- 

 ric acid for one to two minutes or longer. Rinse in water and wash in 

 70% alcohol until no more stain is given off. Dehydrate in alcohol, 

 clear in xylol, and mount in balsam. The bone corpuscles and processes 

 are stained brownish-black, the ground substance yellow, the cells red- 

 violet. 



SchmorT s method for staining the boundary-sheaths of the bone cor- 

 puscle: Harden, decalcify, imbed, and stain as in the preceding 

 method. After staining wash in water for two minutes or longer ; rinse 

 in alcohol for one-half minute, and again rinse in water and place the 

 sections in a saturated aqueous solution of phosphomolybdic or phospho- 

 tungstic acid for three minutes or longer ; wash in water which needs to 

 be changed frequently for ten minutes. The sections are now placed for 

 three to five minutes in a 10% aqueous solution of liquor ammonise, after 



