60 Methods in Plant Histology 



aqueous safranin in water, thus making a 1 per cent solution of 

 safranin in 50 per cent alcohol. Use a 1 per cent aqueous solution of 

 gentian- violet, and a 1 per cent aqueous solution of orange G. 



Transfer paraffin sections to the stain from 95 per cent alcohol 

 after the turpentine or xylol used in dissolving away the paraffin has 

 been rinsed off. Stain 3 to 24 hours. If the period be too short, 

 the washing out is so rapid that it is difficult to stop the differentiation 

 at the proper point and, besides, the red is likely to be less brilliant. 

 Rinse in 50 per cent alcohol until the stain is properly differentiated. 

 Leave the slide in the 50 per cent alcohol until the stain is washed 

 out from the spindle and cytoplasm, but stop the washing out 

 before the chromosomes begin to lose their bright red color. If the 

 washing out takes place too slowly, treat with slightly acidulated 

 alcohol (1 drop of HC1 to 50 c.c. of 50 per cent alcohol) for a few 

 seconds. The acid must be removed by washing for 15 to 30 seconds 

 in alcohol which has not been acidulated. 



Then dip the slide 5 or 6 times into water and stain in gentian- 

 violet. The time required is so variable that definite instructions 

 are impossible. The gentian-violet should stain the spindle, but 

 not the chromosomes. If the stain be too prolonged, it may be 

 impossible to get it out from the chromosomes and still leave it bright 

 in the spindle. If the period be too short, the stain will wash out 

 from the spindle. For mitotic figures in the germinating spores of the 

 liverwort, Pellia, 30 minutes is not too long. In this case, the stain 

 washes out easily from the chromosomes without the use of acid, and 

 the spindle takes a rich violet which is not easily washed out. In 

 embryo-sacs of Lilium try 10 minutes. In pollen mother cells try 

 5 to 10 minutes. For root-tips try 2 to 10 minutes. Chromatin in 

 the early prophases and in telophases will stain with the violet, and 

 the violet will not wash out, but in phases in which fully formed 

 chromosomes are visible the violet can be washed out if the period has 

 not been too long. 



Remove the slide from the gentian-violet and dip it 5 or 6 times 

 in water and then stain 30 seconds to 1 minute in orange G. The 

 orange stains cytoplasm and at the same time washes out gentian- 

 violet. 



