APPENDIX. 409 



treated, and the tissue should not be in pieces over 2 cm. in thickness ; 

 the fluid should be changed as soon as it becomes turbid, sometimes 

 within the first hour, and subsequently renewed as often as may be 

 necessary to maintain perfect transparency. Tissues are usually 

 allowed to remain in Muller's fluid for a considerable time, two weeks 

 being the minimum, while they may be permitted to lie much longer, 

 usually, without disadvantage ; it is advisable, however, to remove 

 specimens after six weeks, and preserve them in spirit. 



The tissue is transferred from the Muller's fluid to water, and thor- 

 oughly washed in the running stream from 4 to 6 hours, until all 

 excess of the fluid has been removed ; it is then placed in jo per cent, 

 alcohol and kept in the dark, the spirit being renewed whenever 

 strongly tinged by the removed fluid ; as long as discoloration occurs 

 an occasional change of alcohol is desirable. 



Where the interstitial methods of embedding are followed, no great 

 amount of hardening is necessary or even desirable, in which case 

 the tissues are best stored in 80 per cent, spirit, where they may lie 

 until needed. Portions of the nervous system which are subsequently 

 to be stained after the Weigert process may be fixed with advantage 

 in warm Muller 1 s fluid, being kept in an oven from 8 to 10 days at 

 a temperature of 35 C. 



b. Absolute Alcohol. For glands, skin, blood-vessels, etc., 

 absolute alcohol affords a rapid and admirable means of fixation, and 

 possesses the additional quality of simultaneously hardening the tissue, 

 a matter sometimes of great convenience, since the specimen may be 

 cut within 24 hours. Small pieces of tissue, so placed either by sus- 

 pension or support on cotton that they do not come in contact with the 

 bottom or the sides of the glass (to which they otherwise adhere), are 

 treated from 12 to 24 hours, the alcohol being invariably changed 

 at the end of the first three hours, whether cloudy or not. After 

 fixation the tissue is preserved in 80 per cent, spirit. It is to be noted 

 that the action of 95 per cent, alcohol is entirely different from that 

 of the absolute, with the weaker spirit the shrinkage being great and 

 the fixation imperfect ; it cannot, therefore, be substituted. 



c. Flemming's Solution. 



Chromic acid (one-per-cent. solution) 7.25 c.c. 



Osmic acid (two-per-cent. solution) 2.50 c.c. 



Glacial acetic acid, at least 25 c.c. 



Where the structure of the protoplasm or the nucleus is to be in- 

 vestigated, or where for any purpose an accurate picture of the cells 

 is desirable, Flemming's stronger solution (given above) will be found 

 the most trustworthy reagent at our command. Two drawbacks limit 



