688 PLANT CHROMOSOMES 



them in aceto-carmine under a cover-glass and heats them to 

 boihng point. The aceto-carmine is then replaced with glacial 

 acetic acid, by an irrigation method with the aid of filter-paper, 

 and the acetic acid with Venetian turpentine. 



The clu'omosomes in young, vigorous root-tips can be examined 

 by the method of Whitaker {Stain Tech., ix, 1934, p. 107). Tips 

 are fixed in aceto-alcohol (1 part glacial acetic acid, 2 parts 

 absolute alcohol) for twenty-four to forty-eight hours. The 

 material can be stored indefinitely by transference to 80 per cent, 

 alcohol. To prepare the smears, place the root-tips on a slide 

 and slice them as thinly as possible with a sharp razor blade. 

 Then smear the slices on the slide, immediately flood with aceto- 

 carmine and add a coverslip. . Using absorbent paper and 

 exerting considerable pressure, the excess aceto-carmine can be 

 removed and the material flattened at the same time. Finally, 

 warm the slide to a jooint slightly below boiling ; this aids con- 

 trast. Sealed with gum mastic and paraffin, such preparations 

 keep well for five to ten days. 



Warmke's Permanent Root-tip Smear Method (Stain Tech., x, 

 1935, p. 101). Fix twelve or more hours in 1 part glacial acetic 

 acid and 3 parts absolute alcohol. Then place for five to ten 

 minutes in a solution of 1 part 95 per cent, alcohol and 1 part 

 concentrated HCl to dissolve the pectic substances of the middle 

 lamella. Harden again with Carnoy for five or more minutes. 

 Place a small piece of the root-tip (0-5 nnn.) on a slide in a small 

 drop of iron aceto-carmine. Press material with scalpel tip and 

 cover. Press gently with flat blunt end of pencil and then heat 

 three to four times in spirit flame ; do not boil. Seal edge of 

 preparation. Preserve good slides by McClintock's method. 

 Pollen grains also satisfactory this way ; the heavy exine is split 

 by the HCl, leaving the naked protoplast adhering to the slide. 



Probably other carmine stains could be used successfully 

 following aceto-alcohol fixation. Fyg {Zeits. wiss. Mikr., xlv, 

 1928, p. 442) gives formulae for the i^reparation of carmine stains 

 using chrome alum, aluminium sulphate, copper alum, sodium 

 bicarbonate, ferric oxide and ammonium sulphate as mordants. 

 The first three give nuclei a violet or blue-black colour. See also 

 ScHMELZER (Zeits. wiss. Mik., li, 1934, p. 66). 



Barrett (Stain Tech., vii, 1932, p. 63) uses Heidenhain's 

 haematoxylin according to the following formula, for studying 

 chromosomes in temporary smears of pollen mother-cells : 1 part 

 0-5 per cent, haematoxylin and 4 per cent, iron alum (equal parts 

 of each), 1 part of 95 per cent, alcohol and 2 parts glacial acetic 

 acid. 



1372. Permanent smears of plant material were first made by 

 Taylor (Bot. Gaz., Ixxviii, 1924, p. 236) who used Flemming 

 (Taylor's modification) followed by Heidenhain's haematoxylin. 



