Molecular Techniques and Methods

Glutaraldehyde-Osmium Fixation of
Plant Tissue for Electron Microscopy

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Glutaraldehyde-Osmium fixation is the most popular procedure for electron microscopy and also yields excellent preparations for the light microscope.




MATERIALS AND SOLUTIONS

Glutaraldehyde Fixation Solution (10 ml)
3% Glutaraldehyde ------------------------------- 0.6 ml of 50% Glutaraldehyde
0.02 M Sodium Phosphate Buffer (pH 7.0-7.4) --- 0.2 ml of 1 M Sodium Phosphate Buffer
Distilled H2O ------------------------------------- 9.2 ml


Osmium Fixation Solution (10 ml)
1% OSO4 ---------------------------------------- 0.1 g
0.02 M Sodium Phosphate Buffer (pH 7.0-7.4) --- 0.2 ml of 1 M Sodium Phosphate Buffer
Distilled H2O ------------------------------------- 9.7 ml
  • Make in the hood.




    PROCEDURES

    1. Cut the tissue into very fine pieces.
  • For electron microscopy the pieces should be very fine indeed-about the size of a pinhead.
  • A convenient way to do this is to use sheets of dental wax.
  • Dissect the pieces on a pool of Glutaraldehyde Fixation Solution placed directly on the wax.
  • Use a clean sharp razor blade. Spray acetone on the blade and wipe off with Kimwipe to clean the blade.

    2. After mincing the tissue, use the blade as a scoop and place the tissue in a vial of Glutaraldehyde Fixation Solution at 4oC.

    3. Change the Glutaraldehyde Fixation Solution after 2-3 hours and allow to fix for another 12-16 hours in the refrigerator.

    4. Wash the tissue in cold 0.02 M Sodium Phosphate Buffer (pH 7.0-7.4) by making several changes at 30 min intervals.
  • If the glutaraldehyde is not removed it will reduce the osmium which is added at the next step, and the reduced osmium does not have good fixative action. In addition the tissues tend to become coated with reaction product, and this decreases their permeability.

    5. The tissue is postfixed in Osmium Fixation Solution for 1-2 hours at 4oC.
  • The osmium should be added in the hood; after use the waste osmium should be collected in a large screw-capped waste jar which should also be kept in the hood. Pasteur pipettes in conjunction with gum rubber bulbs are useful for transferring solutions.

    6. Wash out the osmium with three changes of dehydrating fluids (ethyl alcohol and acetone) at 10-30 minutes each at 4oC.

    7. Glutaraldehyde has rather slow penetration and may not suffice by itself for many subjects. The rate of fixation can be improved by adding formaldehyde and/or acrolein to the glutaraldehyde. A useful general procedure is as follows.

  • Place 1.0 g paraformaldehyde in 50 ml of H2O at 60-70oC.
  • Clear the solution with NaOH.
  • Cool the solution to room temperature.
  • Add 6 ml of 50% glutaraldehyde and dilute the whole solution to 100 ml with 0.02 M Sodium Phosphate Buffer (pH 7-7.4).

    8. Fix the tissue in the above solution for 2-6 hours at 4oC.

    9. Change to Glutaraldehyde Fixation Solution for 12-24 hours.

    10. Wash tissue and postfix in Osmium Fixation Solution at 4oC.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Berlyn GP, Miksche JP (1976) Botanical Microtechnique and Cytochemistry, Ames, Iowa, Iowa State University Press.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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