Double staining:

Safranin :
Stain Lignified tissue such xylem and secondary tissue/ wall.
Light green:
Ferrous sulphate; stain Non Lignified tissue.

Safranin and Light green staining Procedure:-

• Keep section for 5 minutes each in 30%,50% alcohol.
• Keep section for 20-30 minutes in 1% Safranin.
• Dehydrated the after staining the section in 70 %,80 %,90 %
alcohol each for 5 minutes.
• Then Light green stain apply and keep it for 10 to 15 second.
• Wash it in 90/95% alcohol ( যতখন না সমস্ত Stain বেরিয়ে
যাযে।)
• Then wash it in absolute alcohol for 5 minutes.
• Pass through alcohol Xylol (1:1) mixture for 5 minutes.
• Clear this section in Xylol for 5 minutes again.
• Mount the specimen with Canada balsam [Oleoresin of the
balsam fir tree (Abies balsamea)] or DPX ( Distyrene –
Plasticizer- Xylene used as a synthetic resin mounting media)