APPENDIX. 



3. Transfer to 70 per cent, alcohol ; keep in dark ; change alcohol 

 whenever it becomes deeply tinged, until it remains colorless. 



4. Stain in excess of borax-carmine 24-48 hours. 



5. Transfer directly, without washing, from stain to acid alcohol 

 24-48 hours. 



6. Wash well in 70 per cent, alcohol, several times renewed 24 

 hours. 



7. Transfer to 80 per cent, alcohol 24 hours. 



8. Transfer to 95 per cent, alcohol 24-48 hours. 



9. Dehydrate in absolute alcohol 24-48 hours. 



10. Transfer to pure chloroform until tissue sinks 6-8 hours, 

 u. Transfer to saturated solution of paraffin in chloroform 6 

 hours. 



12. Transfer to pure melted paraffin, kept at constant temperature 

 of about 50 C. , until all chloroform is driven off 6-8 hours. 



13. Transfer to fresh melted pure paraffin of consistence for em- 

 bedding 10-15 minutes. 



14. Embed tissue in mould ; cool rapidly. 



15. Section in microtome, first suitably trimming block for cutting. 



1 6. Fix sections to slides by gum or collodion-clove oil. 



17. Remove paraffin by benzole, succeeded by turpentine. 



1 8. Drain off excess of turpentine, apply balsam, and cover. 



19. Place freshly-mounted slides in horizontal position. 



20. Clean up and permanently label when thoroughly -dry ; store 

 in suitable cabinet. 



While the duration of the several manipulations as indicated in 

 the above summary represents the time usually required by ordinary 

 objects, yet the individual character of the tissue must be considered 

 in each case, as density exerts much influence on the rapidity with 

 which the fluids penetrate. 



When it is desirable to stain the tissue after sections have been 

 cut, the above manipulations must be modified ; steps 4, 5, and 6 in 

 such case are omitted and the tissue is at once dehydrated. Re- 

 moval of the paraffin from the fixed sections on the slides (17) by 

 benzole is at once succeeded by the following manipulations : 



a. Transfer to 95 per cent, to remove the benzole 5-10 minutes. 



b. Transfer to clean 95 per cent, alcohol to insure complete absence 

 of benzole 5 minutes. 



c. Transfer to 80 per cent, alcohol 5 minutes. 



d. Transfer to 70 per cent, alcohol 5 minutes. 



e. Stain in borax-carmine solution 10-15 minutes. 



f. Differentiate in acid alcohol (10 per cent.) 6-10 minutes. 



g. Wash in 70 per cent, alcohol, renewed 10-15 minutes. 

 h. Transfer to 80 per cent, alcohol 15 minutes. 



