In this article we will discuss about the meaning and some methods of double staining.
Meaning of Staining:
Staining is essential because different tissues and other structures can be easily differentiated by staining. The plant materials, in which there is no differentiation of tissues such as members of algae, fungi and bryophyta, are stained by a single staining process.
But members of pteridophyta, gymnosperms and angiosperms are stained by the double staining method due to the presence of differentiation of tissues.
Members of different groups can be stained in general as follows:
Algae:
Algal members are generally stained with a few drops of safranin for a few minutes (time is variable for different members) and mounting is done in 10% glycerine. Aniline blue, fast-green, acetocarmine and haematoxylin are some of the other stains used for algal preparations.
Fungi:
Cotton blue is supposed to be one of the good fungal stains. After staining with cotton blue, mount the material in lactophenol if the former is not prepared in the lactophenol. Cotton blue serves as a staining as well as mounting medium if prepared in lactophenol. Aniline blue or haematoxylin also gives satisfactory staining results in certain cases.
Bryophyta:
Members are stained usually with safranin and mounted in 10% glycerine. Other good stains, to be used for members of bryophyta, are Delafield’s haematoxylin and fast-green.
Pteridophyta and Gymnosperms:
Representatives of these groups are stained by a double staining method.
Some Methods of Double Staining:
Under-mentioned are some of the commonly used methods of double staining:
1. Safranin-Fast Green Method:
Keep the material to be stained in safranin for three to five minutes and then wash it with water. See under the microscope that only thick-walled cells are stained. Excess of stain is destained by acid alcohol. Again wash the material very thoroughly with water so that even the traces of acid are removed.
Now stain the material with few drops of fast green for few seconds. Time for keeping the material in fast green varies from few seconds to one minute for different materials. Wash the material with glycerine and mount in a drop of glycerine.
With this method, all thick-walled cells get red stain and all thin-walled cells the green stain.
Entire process can be tabulated as follows:
2. Safranin-Aniline Blue Method:
Follow exactly the same procedure as mentioned above except that in place of fast green use aniline blue.
3. Haematoxylin-Safranin Method:
Keep the sections in Delafield haematoxylin for four to five minutes and remove the excess of stain with water. Wash with ammonia. Wash the material very thoroughly with water. Now stain with safranin for few minutes. Wash the sections with glycerine for removing excess of stain and mount in glycerine.
The entire method can be tabulated as follows:
