690 PLANT CHROMOSOMES 



Wratten B) when the chromosomes appear nearly black. With a 

 yellow screen (Wratten K3) they appear deep red. 



Mann {Science, Ix, 1924, p. 548) has a method of making per- 

 manent smears of pollen mother-cells, in which the contents of 

 the anther are distributed with a scalpel over a film of albumen 

 smeared on a slide. 



Webber {Univ. Calif. Pub. Bot., xiv, 1929, p. 345) uses a much 

 shortened schedule in which smears are fixed in acetic alcohol 

 and stained with alcoholic stains. All mordanting, staining, 

 washing, etc., is done in 60 per cent, or higher alcohols. Slides 

 stained with Delafield's haematoxylin are prepared in one and a 

 half hours, those with iron brazilin in four hours. Useful for 

 chromosome counts and morphology, cytokinesis, tetrad cells, 

 etc., but not for the finer details of prophase. The methods of 

 Mann {Science, xxxvi, 1912, p. 151) and Pickett {Science, xxxvi, 

 1912, p. 479) for preparing slides of unbroken jDollen mother-cells 

 have now little more than an historical interest. 



1373. Selling's Iron- Brazilin Method {Univ. Calif. Pub. 

 Botany, xiv, 1928, p. 293 ; The Use of the Microscope, p. 243). 

 Smears are made using a scalpel or a second slide held crosswise 

 over the first. Make one rapid slanting or curving sweep with the 

 scalpel or second slide and immediately invert the slide or slides 

 on the fixative. Fix in Belling's modified Navashin, grade into 

 70 per cent, alcohol and leave overnight. Fixation is rapid except 

 where sap from the anther wall or connective reaches the pollen 

 mother-cells. Mordant twenty-four hours in 1 per cent, ferric 

 ammonia alum in 70 per cent, alcohol. Wash briefly in 70 per 

 cent, alcohol and soak fifteen minutes to three hours in 70 per 

 cent, alcohol ; the shorter the time the more deeply the meta- 

 phase and cytoplasm stain. Stain two to twenty-four hours in 

 a solution of 0-5 grm. brazilin in 100 c.c. of 70 per cent, alcohol. 

 When using freshly made brazilin solutions, add 1 to 2 drops of 

 1 per cent, iron-alum solution per 50 c.c. Wash briefly in 70 per 

 cent, alcohol, and differentiate in iron alum in 70 per cent, alcohol 

 for one minute to three hours or more. Pachytene and chromo- 

 meres need only slight differentiation as only two to three hours' 

 staining is necessary for them. Stain metaphases for a longer 

 time ; they then need longer differentiation, of one or more 

 hours, especially if the chromosomes are small. Next wash 

 slides in 70 per cent, alcohol and transfer to 95 per cent, alcohol. 

 Pass successively through absolute alcohol, equal parts of absolute 

 and thin cedar oil, equal parts of xylol and thin cedar oil into 

 xylol. Mount in immersion cedar oil. Chromomeres and chromo- 

 somes should be brown to black, cytoplasm pink or colourless 

 and cell-walls unstained. Use green Wratten filters to heighten the 

 contrast, viz., 57A or 64 (blue-green) or a combination of 5Q and 

 64. The brazilin can be replaced by haematoxylin dissolved to 



