APPENDIX. 



this is most conveniently and quickly accomplished by placing the 

 fixed and partially -hardened tissue in a large quantity of dilute nitric 

 acid, varying in strength from 3 to 9 per cent. The fluid should be 

 changed daily for three days, subsequently every second day. The 

 completion of the decalcification may usually be determined by judi- 

 ciously passing a fine needle into the tissue. After suspending the 

 acid solution, whose too prolonged action may result very disastrously 

 for the softer parts, the tissue is thoroughly washed for some hours in 

 running water, and then placed in alcohols of gradually-increasing 

 strength to complete the hardening. 



3. Staining. Since the introduction by Gerlach, now some forty 

 years ago, of a means of differentially coloring tissues, the list of 

 staining methods has gradually been extended, until their description 

 at the present time would cover pages ; notwithstanding the multipli- 

 cation of formulae and their claimed advantages for particular purposes, 

 all ordinary investigations may be satisfactorily carried on with the aid 

 of a very limited selection. Among the important stains, carmine 

 and heematoxylin stand pre-eminent on account of their general 

 applicability and their certainty. The relative merits of carmine and 

 haematoxylin are well defined by their respective advantages. 



Carmine is, as usually now employed, a pure nuclear stain, pos- 

 sessing great penetrating properties, and hence being well adapted 

 for staining tissues and small animals in toto, a matter of much 

 importance in many lines of work requiring serial sections ; further, 

 carmine is permanent, remaining bright and unfaded after years of 

 exposure, does not overstain, and produces preparations admirably 

 adapted to the needs of the improved methods of photomicrog- 

 raphy. 



Haematoxylin, on the other hand, is more than a nuclear stain, 

 yielding, when successfully employed, beautifully crisp pictures of 

 cellular structure seldom, if ever, equalled by carmine; its applicability 

 in its usual formulae, however, is limited to staining sections, since its 

 powers of penetration are feeble. This latter defect may be overcome 

 by employing the stain in the form of Delafield' s h&matoxylin, given 

 below, which answers admirably for bulk-staining. The liability to 

 fade, the possibility of overstaining, and the necessity of using water 

 for differentiation are among the disadvantages of haematoxylin as 

 usually employed. 



The student is strongly advised to adopt carmine as his staple stain, 

 reserving haematoxylin as a valuable, and sometimes indispensable, 

 supplementary means of bringing out parts of cells not satisfactorily 

 displayed in carmine preparations. 



In the order of procedure given above, staining follows the preser- 



