Microscopy plant fibers

Protocol Macerations

  1. Put the plant fibers (about one third the size of a match) in a test tube and add 2-3 ml (or enough to cover the sample) of Schulz's solution:
     
    • Potassium chlorate (KClO3): 24g
    • Distilled water: 200 ml
    • Fuming nitric acid (100%): 200ml
  2. For wood: Incubate (test tube heater) at 40°C to 60°C for 5 min. For most plant fibres is this not necessary and it increase the risk of solving the fibre.
  3. When the maceration is finished, the plant fibres are translucent, and the fibres begin to detach from the sample. If the fibre is not as described above, continue the maceration, and observe every 5 min (or less) until you get the right result.
  4. Wash 3 times with distilled water. To do this, pipette the liquid without removing the plant fibre (glass pipette) and then fill the test tube with distilled water (it may be useful to leave the samples overnight in the rinse water)
  5. Place the sample in the staining solution (Safranin O & Alcian Blue) for 3 min (use small sieves).
  6. Dehydrate the sample (5 min) in a succession of 50%, 75%, 96% and 100% alcohol solutions.
  7. Place a small amount of plant fibre fragment on a slide and gently pull the pieces apart with a tweezer. It may be helpful to add a little 100% alcohol to the slide.
  8.  Add a few drops of Euparal, carefully place the slide, and allow the preparation to dry.

    (note: as fuming nitric acid is more expensive and more dangerous, it may be better to use 60% or 50% nitric acid and therefore adapt the dilution of the solution!)

Protocol Cross-sections

  1. Solve Polyethylene glycol (PEG) 5000 in the oven (50-70°C)
  2. Put the plant fibre in a silicon tray for ice cubes. They must be laid vertical or horizontal but not diagonal on the bottom.
  3. Pour the PEG into the trays, the fibre must be covered completely.
  4. Put the tray in the over overnight (50-70°c) so that the PEG can integrate into the fibre.
  5. Put the tray out of the oven and leave it for 5 minutes at room temperature. The fibres may not move anymore. If they do, place the fibre back horizontal of vertical on the bottom.
  6. Place the tray for 20-30 minutes in the refrigerator to coagulate the PEG.
  7. Remove the blocks from the tray and cut the edges even with a blade. Then place the block on the semi-automatic microtome.
  8. For plant fibres slices of 20nm are made with tape. The fibre will be stuck to the tape and the PEG will dissolve in water.
  9. Put the sample on the slide and cover it with a sieve, that will be hold with two rubber bands.
  10. Place the sample in the staining solution (Safranin O & Alcian Blue) for 5 min.
  11. Dehydrate the sample (3 min) in a succession of 50%, 75%, 96% and 100% alcohol solutions.
  12. Add a few drops of Euparal, carefully place the covering slide, and allow the preparation to dry.