The following points highlight the four main methods of pollen preparation. The methods are: 1. Fischer’s Method of Pollen Preparation 2. Wodehouse’s Method of Pollen Preparation 3. Erdtman’s Method of Pollen Preparation 4. Nair’s Method of Pollen Preparation.
Methods of Pollen Preparation:
- Fischer’s Method of Pollen Preparation
- Wodehouse’s Method of Pollen Preparation
- Erdtman’s Method of Pollen Preparation
- Nair’s Method of Pollen Preparation
Method # 1. Fischer’s Method of Pollen Preparation:
Under this method, the pollen grains are placed on a clean slide, washed with xylol or benzene and stained by a weak solution of fuchsin stain. They are finally mounted in glycerine gelly and studied.
Method # 2. Wodehouse’s Method of Pollen Preparation:
R.P. Wodehouse also suggested a method of pollen preparation in 1935. In this method the pollen grains are treated with absolute alcohol which removes oily and resinuous substances from the surface of the grains. The grains are then stained with methylene green and mounted in glycerine gelly.
Method # 3. Erdtman’s Method of Pollen Preparation:
G. Erdtman (1952, 1964) suggested a widely accepted standard method of pollen preparation and this method is called “acetolysis method”. In this method, pollen grains are treated with a mixture of 9 parts acetic anhydride and 1 part concentrated sulphuric acid.
Method # 4. Nair’s Method of Pollen Preparation:
P.K.K. Nair (1960) suggested a modification of the “acetolysis method’ of Erdtman. This method helps in the comparative study of acetolysed and unacetolysed pollen grains and also helps in understanding the effect of acetolysis on pollen grains.
A brief schedule of Nair’s method is undermentioned:
(a) Pre-treatment and Staining:
1. Fix the pollen material (fresh or dry anthers, flowers or spiles, etc.) in 70% alcohol in glass vials for at least 24 hours if dry and at least for one hour if fresh.
2. Transfer the material with alcohol in a centrifuge tube and crush the material with a glass rod.
3. Pass the dispersion through a brass metal sieve having at least 48 meshes per sq. inch and collect it in a centrifuge tube ‘A’.
4. From centrifuge tube ‘A’, transfer one half of the dispersion to another centrifuge tube ‘B’.
5. Centrifuge the contents of tube ‘A’, decant the alcohol and add about 2 drops of safranin (5% in water). Wait for 5-10 minutes.
6. Wash the stained sediment with 70% alcohol, centrifuge it, decant off the water, wash with water at least twice or thrice by centrifuging till it becomes colourless.
7. Add 2 ml of dilute glycerine (50% in water) and keep this centrifuge tube ‘A’ in the rack.
8. Centrifuge the tube marked ‘B’ and decant off the alcohol.
9. Add about 5 ml glacial acetic acid, centrifuge and decant off the acetic acid.
10. Add about 6 ml of acetolysis mixture (acetic anhydride and conc. sulphuric acid in a ratio of 9 : 1) over the pollen grains in tube ‘B’, place it in a water bath and heat the water from 70°C to 100°C. When the water starts boiling, stop the flame and leave this centrifuge tube ‘B’ in hot water for 3-5 minutes.
11. Centrifuge tube ‘B’ and decant off the waste acetolysis mixture.
12. Add about 10 ml of glacial acetic acid over the sediment in tube ‘B’, centrifuge it and decant off the acid.
13. Add water once or twice, centrifuge it every time and decant off the water. Disperse the grains in water.
14. Transfer one half of this dispersion to another centrifuge tube marked ‘C’, and leave the centrifuge tube B’ along with centrifuge tube ‘A’ in the rack.
(c) Chlorination or Oxidation:
15. Take the contents of tube ‘C’, centrifuge them and decant off the water.
16. Add about 5 ml glacial acetic acid followed by 2-4 drops of saturated solution of sodium chlorate or potassium chlorate in water and then 1 or 2 drops of concentrated hydrochloric acid. Wait for about 5 minutes, centrifuge and decant.
17. Wash with water once or twice and decant off the water.
18. Add a few drops of methyl green on the sediment and wait for about 5 minutes.
19. Wash the sediment with water twice or thrice till the water becomes colourless and centrifuge every time.
21. Transfer the pollen grains from centrifuge tube ‘A’ to ‘B’ and then from tube ‘B’ to tube ‘C’. Centrifuge the mixture of tubes ‘A’, ‘B’ and ‘C’.
22. Place the pollen grains in glycerine gelly, mount with a thin (zero number) cover slip and seal with wax.
The acetolysed pollen grains are brown in colour, unacetolysed grains take red stain of safranin and the chlorinated or oxidized grains are green in colour.
For different purposes of study, the preparations can be made of only safranin-stained grains (A) or in combination with acetolysed grains (A + B) or also along with oxidized or chlorinated grains (A + B + C) as needed.