306 CYTOLOGICAL METHODS. 



teased pieces of fresh tissue immediately into cold Flemming's 

 solution (on ice) plus about 0-5 per cent, urea crystals. Flemming's 

 solution kept on ice registers about 4 to 5 C. Leave in cold 

 solution for twenty-four hours or longer. (3) If this fixation fails, 

 try the following : Allow small pieces of fresh tissue to remain in 

 the air for from ten to twenty minutes after removal from the 

 animal before placing them in the cold Flemming.* Then fix as 

 before. (4) Wash in water about twenty-four hours. (5) Dehydrate 

 by very gradual steps. (6) Clear from 95 per cent, alcohol in cedar 

 oil followed by xylol. Embed in paraffin. 



658. EZRA ALLEN'S Chromic Bouin and Urea. Used for work on 

 the spermatogenesis of rat, etc., and generally indicated for mammals. 



Picric acid, sat. sol. . . . . .75 c.c. 



Formol (pure) . . . . . . 25 ,, 



Glacial acetic . . . . . 5 



To freshly-made mixture raised to temperature of 38 C. add and 

 dissolve, first, 1-5 grms. of chromic acid crystals, and then 2 grms. 

 of urea crystals. Kill animal by decapitation, remove testis imme- 

 diately, snip into small pieces, fix at 38 or 40 C. for from one to 

 two hours. Fixative replaced by " drop method " with 70 per cent, 

 alcohol, picric acid washed out by addition to the alcohol of a sat. 

 sol. of lithium carbonate, a few drops at a time ; the alcohol is 

 replaced by anilin oil (freshly distilled), this by synthetic oil of 

 wintergreen, and this by paraffin of 52 melting point. The paraffin 

 is slowly added till the tissue is in a bath of high paraffin concen- 

 tration. It is then passed through several changes of pure paraffin 

 to remove oil (EZRA ALLEN, Jour. Morph., 1918 ; Anat. Record, 

 x, 1916). 



659. Amphibian Chromosomes. CHARLES PARMENTER (Jour. 

 Morph., 1919) uses Ezra Allen's chromic Bouin with success for 

 amblystoma. Ordinary Bouin with urea crystals, Hermann, and 

 Flemming were also good. 



660. Modified Bouin and Urea for Insects. Miss E. ELEANOR 

 CAROTHERS for orthopterous chromosomes uses the following fluid 

 which has been developed in the Zoology Laboratory, Pennsylvania. 

 It is a modification of Bouin' s formula : 



Picric acid, sat. sol. aq 75 c.c. 



Formalin (strong) 15 



* This, of course, is directly contrary to all the rules of fixing tech- 

 nique, but it might work satisfactorily. 



