PLANT CHROMOSOMES 675 



chromosome threads should be more or less generally disposed through- 

 out the nuclear cavity at resting and early prophase stages, and their 

 doubleness should be recognisable from the earliest stages. The 

 nucleolus should show a non-bubbly structure ; it should not be 

 surrounded by a clear space, which is due to the shrinkage away from 

 it of other nuclear contents. Spiral thread structures, carrying granules, 

 should be recognisable within metaphase and anaphase chromosomes. 

 The cytoplasm should not shrink. ^ 



Pollen mother-cells that cannot be directly exposed to the 

 fixative will generally shrink. In the case of very small anthers 

 and sporangia little can be done to improve the results. Embryo- 

 sacs and deeply immersed megaspore mother-cells of Gymno- 

 spermas and Angiospermae are also difficult. Every effort should 

 be made to secure direct access of the fixative to the cells that are 

 to be studied. 



Fragmentation of long chromosomes, reported in the past by 

 many plant cytologists, is probably an artifact due to the imper- 

 fections in the sectioning process, e.g. brittle paraffin, dull or 

 saw-edged knife, etc. Chromatin extrusion from resting and 

 early proj^hase nuclei is probably due in many cases to pressure 

 on the tissue at fixation or cutting or some intermediate stage. 



FIXATIVES 



1356. Flemming-type fluids are most satisfactory for detailed 

 studies. Fix root-tips, anthers and ovaries twelve to twenty-four 

 hours, smears two to four hours or less. Wash in water, pre- 

 ferably tepid. Bleach in hydrogen peroxide in aqueous or alco- 

 holic solution. Use 1 part of hydrogen peroxide with 4 parts 

 80 per cent, alcohol, either cold or tepid (stand the jar on thin 

 cardboard on the oven top). Gwynne-Vaughan and Barnes 

 {The Fungi, p. 336) use nascent chlorine in 60 per cent, alcohol. 

 Place 1 drop of concentrated hydrochloric acid on a few crystals 

 of potassium chloride and add the alcohol when a green colour 

 indicates the evolution of chlorine. Afterwards wash the slides 

 thoroughly in alcohol. 



McCluxg {Anat. Rec., xlv, 1918, p. 265) recommends that 

 Flemming fixations should be carried out at 0° C. to keep the 

 tissues unchanged until fixed. 



Try medium or strong Flemming solution, Benda with lower 

 acetic acid content, or better, one of the following modifications. 

 It is frequently desirable to use a higher percentage of osmic acid 

 and chromic acid when fixing anthers. 



The principal modifications are : — 



Bonn. 10 per cent, aqueous chromic acid, 0-33 c.c. ; 10 per 

 cent, aqueous acetic acid, 30 c.c. ; 2 per cent, aqueous osmic 

 acid in 2 per cent, chromic acid, 0-62 c.c. ; water, 6-27 c.c. 



Newton and Darlington's (J. Genet., xxi, 1919, p. 1) formula 



