132 THE TISSUES. 



the whole has been done with sufficient rapidity the air will have been 

 caught in the open spaces of the bone before the Canada balsam has had 

 a chance to enter these spaces. 



Other substances may be used to demonstrate the spaces in 

 bone. Ranvier (75) recommends the following method: A few c.c. 

 of a concentrated alcoholic solution of anilin blue (which is soluble in 

 alcohol and not soluble in water and sodium chlorid solution) are placed 

 in an evaporating dish containing the dry bone. The solution is very 

 carefully evaporated, as the alcohol may otherwise ignite. The specimen, 

 which will soon be covered on both surfaces by a blue powder, is taken out 

 and ground upon a rough glass plate until thoroughly clean. While being 

 polished the bone should be kept moist by a solution of sodium chlorid. 

 On heating in the evaporating dish, the air is driven from the spaces and 

 replaced by the anilin blue. As already stated, anilin blue is insoluble 

 in sodium chlorid solution, and it therefore remains unaffected by the 

 latter during the process of grinding and cleaning. Hence it remains in 

 the lacunse and canaliculi of the bone, which then appear blue. The 

 specimen may either be mounted in glycerin-sodium chlorid and the edge 

 of the cover-glass sealed with varnish, or the section may be washed for 

 a short time in water (in order to remove the sodium chlorid), dried, 

 and finally mounted in Canada balsam as directed. 



A method adapted to the study of the hard and soft parts together 

 is that first used by von Koch in studying corals. The specimen 

 is first fixed, and if it be a long bone, the marrow cavity should first be 

 opened to permit the fixing agent to come in contact with all parts of the 

 tissue. After fixing, the bone is stained and then placed in absolute 

 alcohol, and when completely dehydrated the pieces are placed in chloro- 

 form, then in a thin solution of Canada balsam in chloroform, and finally 

 put into an oven kept at a temperature of about 50 C. for from three to 

 four months. By this means the pieces are completely penetrated by 

 the Canada balsam, and as the latter becomes very hard on cooling, the 

 sections may be afterward ground without difficulty. Long as this pro- 

 cedure may seem, it is still the one which enables us to see the soft 

 and hard parts of bone in a relationship the least changed by manipu- 

 lation. 



In bone, as also in cartilage, there sometimes occur amorphous 

 as well as crystalline deposits of lime-salts. Upon the addition of acetic 

 acid the carbonate of calcium gives off bubbles ; upon the addition of 

 sulphuric acid, short, thin needles will be formed crystals of gypsum. 

 Hematoxylin stains the lime-salts blue, with the exception of the oxalate 

 of lime. Alkaline solution of purpurin stains calcium carbonate red. 

 Caustic potash does not affect lime. 



In order to study the organic constituents of bone, it must 

 first be decalcified and thus rendered suitable for sectioning /. e. , the 

 lime-salts must first be removed, and that without destroying the cellular 

 elements of the bone. The process of decalcification consists in substi- 

 tuting the acids of the decalcifying fluids for those of the bone salts. 

 As a consequence^ new combinations are formed, soluble in water or in 

 an excess of the decalcifying acids themselves. 



The decalcifying fluids most frequently used are : 

 (a) Hydrochloric acid (i% aqueous solution), used in quantities 

 amounting to about fifty times the volume of the specimen. The solution 



