PLANT CHROMOSOMES 689 



Crush anthers on clean shdes with a clean flat-honed scalpel, and 

 spread their contents over the centre of the slide with quick 

 strokes. Immediately invert the slide in the fixing fluid, bringing 

 it down in a horizontal position, so that the whole smear face is 

 wet simultaneously. The time from first crushing to the fixing 

 must not exceed three to five seconds. If the slide is brought 

 down obliquely, much of the material washes off. Fix in a Petri 

 dish with a thin glass rod in the bottom, sufficient liquid being 

 used just to cover the rod. Square porcelain dishes (3^ X 3| in.), 

 with two small ridges near opposite ends are better. Fix not less 

 than fifteen minutes, wash in water and remove the larger pieces 

 of anther walls and other debris. Bleach in dilute aqueous 

 hydrogen peroxide, rinse and place four to twelve hours in 2 per 

 cent, iron ammonia alum. Wash fifteen minutes or more in 

 running water, stain four to twelve hours in 0-5 per cent, aqueous 

 hsematoxylin, followed by a rinse in water and differentiation in 

 iron-alum solution under the microscope. Finally, wash for one 

 hour. Dehydrate through alcohols by 10 per cent, stages, two to 

 three minutes in each. The chief difficulty is to get a brilliant 

 stain, some slides being distinctly muddy. 



By ICaufmann's {Stain Tech., ii, 1927, p. 88) method fewer 

 muddy slides are obtained. Mordant forty-five minutes to one 

 hour in 2 per cent. alum. Wash in running water ten to fifteen 

 minutes. Stain twenty^ to thirty minutes in 0-25 to 0-5 per cent, 

 haematoxylin ; as soon as the slides are deep purplish-black 

 transfer to water. Differentiation in 0-5 per cent, ammonio-ferric 

 alum can then more often be completed prior to the appearance 

 of a muddy colour than is possible after prolonged staining. 

 Kaufmann found no advantage in allowing the htematoxylin to 

 ripen. He also fixed smears in a picric acid-acetic acid-formalde- 

 hyde combination and found organic adjuvants such as lactose, 

 maltose and urea in 1-5 per cent, concentration improved 

 penetration. 



Newton (J. Linn. Soc. (Bot.), Ixvii, 1927, p. 339) and 

 Darlington {J. Genet, xvi, 192G, p. 237) used gentian violet-iodine 

 as a stain, following fixation in Flemming. 



Sax {Stain Tech., vi, 1931, p. 117) fixes smears in Navashin's or 

 modified Flemming solution for one to two hours. Wash in 10 to 

 30 per cent, alcohol for fifteen to thirty seconds and stain in 

 1 per cent, aqueous crystal violet one to five minutes. Rinse in 

 water and pass through 30 and 50 per cent, alcohols, fifteen to 

 twenty seconds in each. Transfer to 80 per cent, alcohol (contain- 

 ing 1 per cent, iodine and 1 per cent, potassium iodide) for thirty 

 seconds. Destain with absolute alcohol, followed by clove oil, 

 xylol and balsam. 



Observation of preparations stained with gentian violet (or 

 crystal violet) is facilitated by using a green screen {e.g. 



