Double staining or counter staining

Safranin and fast green method

Procedure

Primary staining:
  • Take the stem material and cut numerous fine sections with the help of a sharp blade.
  • Dip the cut sections in a big-sized watch glass and pour a few drops of safranin stain (prepared in 50 % alcohol).
  • Leave it for about two minutes by gentle agitation with the help of a fine brush.
  • Put the watch glass on a bench and gently move it in a circular direction so that all the sections should aggregate in the center.
  • Now decant the excess stains and repeat this process until washing water becomes colorless.

Destaining:
  • Decant the excess stains and wash it in 50 % alcohol until sections stop losing color.


Double staining
R.L.S. of Pinus stem showing differential staining of safranin (red) and fast green (green)

Secondary staining/counter staining:
  • Pour one drop of fast green (prepared in 90 % alcohol) in the watch glass containing sections stained in safranin.
  • Gently agitate the section and decant the stains in 15 seconds.
  • Wash multiple times in water to remove excess of stains.
  • Mount in glycerin or water on a glass slide and observe under a compound microscope.

Explanation

  • Safranin upon staining gives red color. Hard and secondary tissues such as, xylem vessels, tracheids, sclerenchyma, fibers and secondary wall catch the safranin stain firmly and appear red in color.
  • After destaining in 50 % alcohol, all the soft tissues become colorless.
  • When counter stained in secondary stain (Fast green), soft tissues like, xylem and phloem parenchyma and ground tissues catch the fast green and appear green in color.


Method of stain preparation

Safranin

Safranin is basic stain of azin group. Its solubility in water is 5.45% and in alcohol solubility is 3.41%. This is the most commonly used stain used by bthe botanists in morphology anatomy and cytology. It stains lignified, cutinized, suberized and chitinized structures as well as chromosomes, nucleoli and centrosomes.
Stock solution of safranin is prepared by dissolving 0.5 g of Safranin O in 95% ethyl alcohol. Stock solution may be diluted with equal amount of water at the time of use.

Content first created on 06-09-2022
last updated on 29-09-2022