APPENDIX. 



its use : its very limited power of penetration, which necessitates the 

 tissue being cut in layers not over 2-3 mm. thick, and the consider- 

 able expense attending the use of large quantities of the fluid. The 

 mixture should be made up each time just before using, and cannot 

 be employed a second time. The living tissue is placed within the 

 solution in a glass-stoppered bottle, and allowed to remain, without 

 changing, 24 hours ; then transferred to running water 1-2 hours, 

 after which it is placed in 70 per cent, alcohol, and, after several 

 changes, preserved in 80 per cent, spirit. 



d. Picro-Sulphuric Acid (Kleinenberg's) Solution. 



Picric acid, saturated watery solution 200 c.c. 



Sulphuric acid, pure 4 c.c. 



resulting in dense precipitate ; after one hour filter, and dilute with 

 three volumes (600 c.c.) of distilled water. 



This solution is an admirable and trustworthy reagent for embryos 

 and other delicate structures, its principal objection being the time 

 required to remove the yellow tinge of the picric acid. The embryos 

 are placed directly, without washing, into the fluid, where they remain 

 5 hours if very large the time may be extended to 10-12 hours, 

 with a renewal of the fluid ; they are then transferred to 70 per cent, 

 alcohol, which is repeatedly changed until discoloration no longer 

 takes place ; preserve in 80 per cent, alcohol. 



2. Preservation of Tissues. In connection with fixation, the 

 subsequent preservation of tissues in 70 per cent, spirit has been indi- 

 cated ; when, however, the condition of the specimen, as when ob- 

 tained some time after death, or other considerations, render fixation 

 useless, it becomes necessary to preserve the tissue from further change. 

 To this end Miiller's fluid may also be advantageously employed, 

 observing the precautions already pointed out, followed after some 

 weeks by alcohol. In many cases, however, when fixation is no 

 longer possible, alcohol offers the most convenient method of preser- 

 vation, possessing as it does the merits of simplicity and of rendering 

 the tissue receptive to all forms of staining. 



In the employment of alcohol for hardening, the tissue should be 

 passed through a series of gradually-increasing strength ; beginning 

 with 60 per cent, spirit for 2-3 days, with renewals when turbid, the 

 tissue is placed successively, at intervals of 3-4 days, into 70 per 

 cent., 85 per cent., and 95 per cent, alcohol, finally, after sufficient 

 hardening, to be preserved indefinitely in 80 per cent, spirit. 



In those cases where bone or calcareous matters are present, fixation 

 and hardening must be followed by de calcification and softening ; 



