PLANT CHROMOSOMES 691 



0-5 per cent, in 70 per cent, alcohol ; a longer differentiation is 

 needed and the cytoplasm is not so clear. 



See also Capinpin, Science, Ixxii, 10.30, p. .370. 



1374. Backman's Combined Fixing and Staining Method 

 (Stain. Tech., x, 19.35, p. 83). Substitute anthraquinone for picric 

 acid in Bouin formula and add alizarin red S : — 



Anthraquinone, sat. soln. . . 75 c.c. 



Formaldehyde .... 25 c.c. 



Acetic acid ..... 5 c.c. 



Alizarin red S .... 0-125 grm. 



Saponine ..... 0-150 grm. 



Immediately before use add 8 to 12 drops of normal solution of 

 metallic salt (e.ff. aluminium chloride, copper nitrate, ferric 

 chloride). Suitable concentration of salt solution found by 

 adding as many drops to the fixative as it will hold without 

 precipitation of hydroxide during maximum fixing time (three to 

 four hours). 



Smear and fix three to five minutes ; wash in tap-water two 

 to five minutes ; dehydrate to 95 per cent, alcohol. Differentiate 

 by flooding with 0-5 per cent. H2SO4 in 95 per cent, alcohol 

 saturated with picric acid ; this requires only a few seconds. 

 Wash in 95 per cent, alcohol. If slide to be used at once, intensify 

 stain by washing half to one minute in 95 per cent, alcohol con- 

 taining 4 per cent, by volume of cymene. Wash in absolute alcohol, 

 clear in xylol and mount. Baekman gives an alternative method 

 of differentiating and also a schedule for root-tips. 



1375. Internal (Spiral) Structure of Chromosomes at metaphase 

 and anaphase is frequently visible in well-fixed smears of pollen 

 mother-cells from plants with large chromosomes. Fixation with 

 2BD (La Cour) and S2 (Smith) is particularly good. Spirals are 

 seen particularly well in pollen mother-cells that have been 

 slightly pressed prior to fixation. Sakamuba {Bot. Mag., Tokyo, 

 Ixi, 1927, p. 59) fixes in hot water. 



KuwADA and Sakamura [Cijtologia, v, 1934, p. 244) squeeze 

 pollen mother-cells of Tradescantia out into a drop of 3 per cent, 

 cane sugar on a coverslip. The coverslip is exposed for a few 

 seconds, material downwards, on a cell containing a small piece 

 of cotton-wool soaked in ammonia and then fixed and stained 

 with iron aeeto-carmine. The spirals are spread out, or even 

 unravelled by prolonged treatment. La Cour [Stain Tech., 

 X, 1935, p. 57) makes smears on slides and carefully wets them by 

 putting them face downwards horizontally on to 3 per cent, 

 cane-sugar solution. The slides must also be removed horizontally 

 or much material washes off. The slides are then exposed to 

 ammonia vapour or the vapour of acetic acid or of mineral acids 

 (nitric or hydrochloric) for a few seconds. He then fixes the 

 smears in a Flemming fluid (medium Flemming slightly better 



