STAINS AND STAINING 67 



Wash in water for a couple of minutes and then stain in gentian 

 violet. 



The time required is so variable that definite instructions are im- 

 possible. 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 Lilimn try 10 

 minutes. In pollen mother-cells try 5-10 minutes. For root-tips try 

 2-10 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. If the aqueous gentian violet 

 or crystal violet fails to stain the spindle, try the anilin oil solutions. 



Remove the shde from the gentian violet and dip it 5 or 6 times in 

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

 orange stains cytoplasm and at the same time washes out gentian 

 violet. 



Transfer from the orange G to 95 per cent alcohol, dipping the shde 

 a few times in this merely to save the absolute alcohol. Dehydrate in 

 absolute alcohol 3-30 seconds. 



Clear in clove oil, as already described in the paragraph on gentian 

 violet. Transfer to xylol and mount in balsam. 



Safranin and gentian violet are often used without the orange. In 

 this case, transfer from the gentian violet directly to 95 per cent alco- 

 hol, and proceed as before. 



An objection to both these methods is that the gradual series of al- 

 cohols cannot be used, because the gentian violet washes out so rapid- 

 ly. If one should try a filament of Spirogyra with either of these 

 methods, it would hardly be recognizable when it reaches the balsam; 

 but with thin sections, especially when well fastened to the shde, con- 

 ditions are different and there does not seem to be much damage. 

 With any aqueous solution of gentian violet or crystal violet, the violet 

 is less likely to be lost, if permanganate of potash is used before the 

 stain, or Gram's iodine after it. 



