XIX] AND BIOLOGICAL METHODS 361 



2. Wash thoroughly in 70 per cent, alcohol, with several changes during 

 24 hours or longer. 



3. Pass into Soap Solution (soft soap in 70 per cent, alcohol) for 24 hours, 

 or longer. This softens the chitin. 



4. Wash again as in 2. 



5. Pass into 90 per cent, alcohol ; 6 hours. 



6. Pass into absolute alcohol; 6 hours. 



7. Pass into absolute alcohol + cedar oil (equal parts); 24 hours. 



8. Pass into pure cedar oil ; 24 hours. 



9. Introduce fine parings of soft paraffin- wax (38 M.P.) into the cedar 

 oil, until it has dissolved as much as possible. 



10. Remove specimen to melted paraffin-wax (38 M.P.) at about 40, for 

 2 hours, on top of bath. 



11. Remove to melted paraffin-wax (52 M.P.) for 2 or 3 hours, within 

 bath. 



12. Embed in paraffin-wax (52 M.P.) in the usual way. 



From this, the block may be cut out, trimmed up and sectioned in the usual 

 way. The times given must be varied according to the size and character of 

 the object. Care must be taken (a) to soften the chitin sufficiently, (b) not 

 to leave the object too long in the higher grades of alcohol, as this causes 

 hardening, (c) to extract all the cedar oil at stage 10 [I usually have two dishes 

 of soft paraffin, and give 1 hour or more in each], (d) to keep the object in the 

 higher paraffin only long enough to allow of complete penetration, and not 

 "cooking." 



The sections having been prepared, any of the usual methods of staining 

 may be employed. I find the combination of Ehrlich's Haematoxylin with 

 Eosin to be unequalled for general use. Another very good combination is 

 Heidenhain's Iron Haematoxylin with Orange G. 



B. Double Embedding : After carrying out processes 1-6 as in A, 

 above, proceed as follows: 



7. Pass into absolute alcohol + ether (equal parts) ; 24 hours. 



8. PJace in covered crystal-dish in \ per cent, celloidin solution 

 (dissolve the celloidin in equal parts of ether and alcohol) ; 24 hours. 



9. Change to 2 per cent, celloidin solution; 24 hours. 



10. Change to 5 per cent, celloidin solution; 24 hours. 



11. Harden in chloroform vapour under a bell-jar, until the celloidin 

 block is of the consistency of cheese; 2-3 hours. 



12. Shape and trim up block, with object correctly orientated; transfer 

 to liquid chloroform for further hardening (half-an-hour or more). 



13. Place hardened block in liquid chloroform with parings of paraffin- 

 wax (58 M.P.) on top of bath (about 40 C.). Leave overnight. (The chloro- 

 form dissolve* the wax, and does not wholly evaporate.) 



14. Repeat processes 11, 12 as in A, above, treating the celloidin block 

 as the object to be embedded. Section in the usual way. 



Method B must be used for all the more delicate work, such as embryology, 



