PLANT CHROMOSOMES 681 



per cent, alcohol, though it is better to add 5 to 10 per cent, of 

 glycerin if it is to be left for a long period. Do not leave longer 

 than twelve hours in absolute alcohol, and in the case of larger 

 buds use two or three changes. Chloroform is preferable to xylol 

 for clearing and infiltration, as it does not harden so much and 

 evaporates more quickly. The time in the oven at a high tempera- 

 ture is thereby shortened. When in pure chloroform, add a small 

 piece of wax and place the phial on the paraffin oven on a piece of 

 cardboard. Leave here three to five days, with the stopper in 

 the phial ; add a small piece of wax each day, but never more 

 than will go into solution. Place inside the oven for two hours. 

 Pour into an open dish or watch-glass and allow the chloroform to 

 evaporate off ; this usually requires four to five hours. Then 

 imbed. 



A'^-butyl alcohol can be used on root-tips and buds (Zirkle, 

 Science, Ixxi, p. 103 ; see § 1257) and is advantageous since 

 material can be taken up into wax in a shorter time and the 

 material is not rendered brittle. In La Cour's (Journ. Roy. Micr. 

 Soc, li, 1931, p. 119) modification material is taken up to pure 

 n-butyl alcohol by Zirkle's method and then placed successively 

 for one hour in each of 25, 50, 75 per cent, chloroform in 

 /«-butyl alcohol, and then into pure chloroform with wax. 



Use a harder wax for cutting in the summer (54° to 56° C. m.p. 

 if necessary) and a softer wax for cutting in winter (50° C. m.p. 

 is usually satisfactory). Sections should be cut of suthcient 

 thickness to preclude a high proportion of cut cells ; 15 to 20 /x is 

 sufficient for most plants, but 30 )Lt or thicker is required for those 

 with long chromosomes. 



1364. Stains. Newton's Gentian Violet (or Crystal Violet) 

 Iodine Method (Newton, Journ. Linn. Soc, Bot., Ixvii, 1927, 

 p. 31.6 ; HusKiNS, Journ. Genet., xviii, 1927, p. 315 ; Newton 

 and Darlington, Journ. Genet., xxi, 1929, p. 1). Bring slides 

 down to water and steep in gentian-violet solution (1 per cent, 

 boiled and filtered) for three to ten minutes according to the age 

 of the stain. Rinse in water and mordant thirty to forty-five 

 seconds in 80 per cent, alcohol containing 1 per cent, iodine and 

 1 per cent, potassium iodide. Rinse two seconds in 95 per 

 cent, alcohol. Rinse two to five seconds in absolute alcohol. 

 Differentiate in clove oil ; clear in three changes of xylol, with 

 at least fifteen minutes in the last change before mounting in 

 xylol-balsam. Manton {Ann. Bot., xlvi, 1932, p. 509) also 

 mordants with iodine before staining. 



For material difficult to stain, and after certain fixatives 

 (Carnoy, Bouin) La Cour {Journ. Micr, Soc, li, 1931, p. 119), 

 after staining in gentian violet, mordants in iodine solution two 

 minutes, rinses two seconds in absolute alcohol, fifteen seconds in 

 1 per cent, aqueous solution of chromic acid, five seconds in 



