682 PLANT CHROMOSOMES 



absolute alcohol, a further fifteen seconds in 1 per cent, aqueous 

 chromic acid, ten to fifteen seconds in absolute alcohol, differ- 

 entiates in clove oil and clears in xylol. 



After Carnoy fixation, Clausen {Ann. Bot., xliii, 1929, p. 741) 

 mordants slides one to two hours in 1 per cent, aqueous chromic 

 acid and then washes thirty minutes or more in water before 

 staining with gentian violet-iodine or Heidenhain's haematoxylin. 

 Alternatively, mordant material fixed in Bouin, Carnoy or 

 Ohlmacher in fresh or used Flemming fixative (Allen, Stain. 

 Tech., ii, 1927, p. 62). Or, mordant in 0-5 per cent, aqueous 

 osmic (Foley, Anat. Uec, Ixiii, 1929, p. 171). 



When gentian violet is used as a somatic chromosome stain, 

 JoHANSEN {Stain. Tech., vii, 1932, p. 17) adds 9-5 per cent, of 

 picric acid crystals to the dehydrating alcohols. A much better 

 differentiation completed in clove oil is possible. 



Smith {Stain. Tech., ix, 1934, p. 95) mordants ten to twenty 

 minutes in iodine solution, rinses in water, stains in gentian violet 

 five to twenty minutes and rinses in water. He then rinses in a 

 second iodine solution, then in 95 per cent, alcohol and floods 

 the slide quickly with a saturated solution of picric acid in absolute 

 alcohol, followed by immediate washing with absolute alcohol for 

 a few seconds. Finally, differentiate in clove oil and soak at 

 least ten minutes in xylol. The chromosomes are richly stained 

 by this method and are less transparent than when stained by 

 the ordinary method. Resting nuclei and prophase strands are 

 almost as sharply stained as fully formed chromosomes. The 

 cytoplasm is a very transparent yellow. 



Peto {Journ. Genet, xxviii, 1933, p. 113) states that the use of 

 distilled water and acid tap-water inhibits staining by the gentian- 

 violet-iodine method. Alvarez {Science, Ixxiv, 1931, p. 633) 

 finds that distilled water in equilibrium with the air absorbs 

 enough COg to lower its ^^H to 5-6 to 5-8. Peto finds that treat- 



N 

 ment with calcium hydroxide is satisfactory for producing a 



good stain. 



1365. Heidenhain's Haematoxylin. This stain is opaque and 

 should be avoided except for prophases of meiosis and for small 

 chromosomes when these are not too numerous. It is unsatisfac- 

 tory for side views of metaphase and other condensed stages. 



For plant material the classical method is as follows : mordant, 

 one to twelve hours in 4 per cent, iron alum ; wash five to fifteen 

 minutes in running (or frequently changed) water and stain one to 

 twelve hours in 0'5 per cent, haematoxylin. Rinse in water and 

 differentiate in 2 per cent, iron alum, observing the final stages 

 under the microscope. Wash thoroughly fifteen minutes to one 

 hour in water, dehydrate by gradual stages, clear in xylol and 



