PLANT CHROMOSOMES 685 



1369. Belling's Iron Aceto-carmine Method (Amer. Nat., Iv, 

 1921, p. 573). Belling describes three procedures : (1) Heat 

 45 per cent, glacial acetic acid to boiling with excess powdered 

 carmine, cool and filter ; 1 grm. of carmine to 100 to 200 c.c. of 

 45 per cent, acetic acid is sufiicient. Do not boil more than half 

 a minute. Tease out anthers in a drop of the aceto-carmine solu- 

 tion with steel blades or needles until the colour changes toward 

 a bluish-red. Remove anther debris, cover with a large thin 

 coverslip, using a minimum of liquid. Seal the edges with 

 vaseline. The slide may improve in a day or two if no excess of 

 iron is present. (2) Add a trace of ferric hydroxide dissolved in 

 45 per cent, acetic acid until the liquid turns bluish-red, but there 

 is no visible precipitate. Add an equal amount of ordinary 

 aceto-carmine. Tease anthers with nickel instruments. If the 

 stain is too dark, add more aceto-carmine. The liquid may be 

 diluted with 45 per cent, acetic acid, never with water. Where 

 the stain is too heavy, McClintock {Genetics, xiv, 1929, p. 180) 

 extracts with dilute acetic acid, warming the whole slide gently 

 to hasten the action. (3) Anthers at the right stages are put into 

 a mixture of 1 part of glacial acetic acid to 9 parts absolute 

 alcohol, to which sufficient ferric hydroxide in 45 per cent, acetic 

 acid has been added to colour the liquid brown. After some days 

 or even weeks, the anthers are teased in ordinary aceto-carmine, 

 avoiding the use of steel instruments. 



Belling {Amer. Nat., Ivii, 1923, p. 92) found advantages in 

 using a water immersed aplanatic condenser with a water immer- 

 sion objective. Approximately monochromatic yellow-green 

 light, obtained Avith a colour filter (Wratten 57a or 58, or green B) 

 is useful. 



Belling later {Biol. Bull. Marine Biol. Lab., 1, 1926, p. 160) 

 recommends a solution of dammar in xylol or melted soft parafhn 

 wax as a seal for the edge. Chloral hydrate, followed by mount- 

 ing in glycerin, may be used for clearing pollen grains. 



The best seal is prepared by heating together equal parts by 

 weight of paraffin and gum mastic. Apply with a hot wire. 



The pollen mother-cells fixed and stained in iron aceto-carmine 

 pass tlu"ough three stages of hardening (Belling, Brit. Jour. Exp. 

 Biol., iii, 1926, p. 145). At first the cytoplasm is more or less 

 liquid, then it becomes a more or less stiff jelly and finally (after 

 some weeks) more and more brittle ; the chromosomes are always 

 harder than the cytoplasm. If the cells in the second stage 

 (reached in one to two days) are subjected to gradual pressure, the 

 cell contents may be squeezed flat within or without the cell-wall. 

 Slides keep better stored in the dark. 



Iron aceto-carmine is satisfactory with most material ; notable 

 exceptions are Oenothera and Rhododendron. 



McClintock {Proc. Nat. Acad. Sci., xvi, 1930, p. 791) studying 



