PLANT CHROMOSOMES 683 



mount in balsam. Use only good iron-alum crystals of pure 

 violet colour for making up the mordant and differentiator. 



More recent application of the method has tended towards a 

 shortening of the times as much as possible. Mordant, fifteen to 

 thirty minutes or even eight to ten minutes if the temperature is 

 high ; stain for five minutes or more according to the material. 



Of rapid methods, Cole's {Science, Ixiv, 1926, p. 452) is the best. 

 Prepare, as mordant, a solution of 20 c.c. 50 per cent, alcohol, 1 

 grm. ferric chloride and 2 c.c. glacial acetic acid, and as stock 

 stain 20 c.c. absolute alcohol, 0-2 grm. sodium hydrosulphite, 5 

 drops distilled water and 1 grm. light brown ha^matoxylin crystals. 

 The stock stain is very powerful and will keep without oxidation 

 for a long time. Use mordant and stain separately or combined. 

 To prepare the stain for use : (1) Add 5 drops of stock solution, 

 followed by 1 drop of anamonium hydroxide, to 5 c.c. of tap-water 

 in a dropping bottle ; the stain is ready for use in thirty seconds 

 and retains its potency for about four hours. Or, (2) use 95 per 

 cent, alcohol instead of tap-water ; the stain is ready for use 

 after twenty minutes and has a longer life than the aqueous solu- 

 tion. Or, (3) add 5 drops of stock solution to 10 drops of tap- 

 water and add 1 drop of ammonium hydroxide ; after thirty 

 seconds add 5 c.c. 95 per cent, alcohol and the stain is ready for 

 immediate use and lasts longer than (1). 



Method. Flood the slide with mordant for five minutes. Rinse 

 a few seconds in tap-water and flood with stain. Staining requires 

 ten minutes or less. Differentiate in 0-1 to 0-4 per cent, hydro- 

 chloric acid and when this is complete blue the slide in a jar of 

 water containing 1 or 2 drops of ammonium hydroxide. Counter- 

 stain in erythrosin, if desired. Dehydrate, clear and mount. The 

 fresh stain gives blue nuclei ; that an hour or more old gives blue- 

 black or black nuclei. To secure black nuclear stains use solu- 

 tions several hours old, or add 2 to 3 drops of ammonium 

 hydroxide in the preparation of the stain for use, or flood slides 

 with fresh or old stain and add 1 drop of ammonium hydroxide. 



See also Faure (C. R. Soc. Biol, xc, 1924, p. 87) ; Kaufmann 

 {Stain. Tech., ii, 1927, p. 88) ; Kornhauser {Stain Tech., i, 1926, 

 p. 78) ; Maheshwari {Journ. Ind. Bat. Soc, xii, 1933, p. 129). 



TuAN {Stain Tech., v, 1930, p. 135) recommends the use of a 

 saturated aqueous solution of picric acid to differentiate material 

 stained in Heidenhain's and Delafield's haematoxylin ; follow by 

 washing thirty minutes in water. A more transparent stain is 

 produced, no brownish or muddy colouration is obtained, and 

 since picric acid is milder in its destaining properties a more con- 

 trolled action is possible. 



1366. Mayer's Haemalum. Grind up 0-5 grm. haematein in a 

 glass mortar with 10 c.c. of 95 per cent, alcohol and add 500 c.c. of 

 5 per cent, aqueous solution of potassium alum (see Kornhauser, 



