CALCIUM PHOSPHATE AND CALCIUM CARBONATE. 299 



8 hours or more in diluted aqueous solution of Gentian violet, 

 dehydrated in alcohol, cleared in benzol, and mounted in balsam. 



To stain the bone cells only, the periosteum was left on the 

 specimens, and this was later removed by dissection under a bi- 

 nocular microscope while the bones were in benzol. In this way 

 the matrix remained clear, while if the periosteum was removed 

 previous to staining, the matrix stained also. 



This method was used by me, but as the matrix was the main 

 object of study, most of my preparations were not stained, but 

 were mounted for observation clear, in three different ways : first, 

 without fixation, in a shallow cell filled with normal saline ; second, 

 in a shallow cell of glycerin ; and third, fixed in 95 per cent, alcohol, 

 then absolute alcohol, cleared in benzol, and mounted in balsam. 



A cover glass to which some of the precipitate of calcium car- 

 bonate was adherent in the form of both spherules and crystals 

 was passed through all the processes necessary to staining above 

 and mounted in balsam. There was no change to be seen in the 

 precipitate, so it can be inferred that calcium salts in the bony 

 matrix, if in this form, would not be altered by any of the processes. 



The specimens of bone used were parietal and ethmoidal bones 

 and nasal conchse of the mouse, frontoparietal bones of the frog, 

 and splinters and thin sections in various planes from the femur, 

 tibia, humerus, radius, ulna and scapula, of frog, mouse, guinea 

 pig, dog, and human foetus. The frog and mouse were young, so 

 that their bones were in process of growth, and if there is any 

 visible difference between new formed and old matrix it should be 

 evident here. Thin pieces of the long bones cut with a sharp knife 

 to take in areas of the total thickness of the shaft, or to show transi- 

 tion from the bony shaft to the cartilaginous epiphysis, were taken, 

 splinters were clipped off long bones, small pieces were crushed, 

 and all these were mounted as described above. The whole of a 

 concha, scapula or parietal, if small, was mounted as one specimen. 



In all cases the periosteum was carefully peeled off and was 

 mounted alongside of the bone so that examination of it for newly 

 formed matrix could also be made. 



