Protocol

Author

Rosemary White

Overview

This protocol outlines how to use a sliding or sledge microtome to cut hard or soft plant tissues, creating sections between 5 μm to 50 μm thick. This technique is particularly useful for sectioning very hard tissues, such as wood. It is also useful if you wish to obtain complete cross sections from large branches, roots, stems, etc. as you have good control over the material and can get quite thin sections if you use a freshly sharpened large microtome blade.

Background

This is one of a series of protocols on sectioning unembedded plant tissues, prepared by Rosemary White.

Linked Protocols

Making hand sections without support material

Using other plant tissues as support tissue to make hand sections

Using support tissue in a hand microtome to make hand sections

Vibrating microtome sectioning of fresh or fixed tissues

Cryostat sectioning of frozen tissues

Materials/Equipment

• support material, for small tissue pieces – the best we’ve found is high density foam, which is very good for soft tissues (hard tissues, like woody roots or stems, may not need supporting material)
• sharpened microtome blades or disposable microtome blades
• water – conical flask with plastic or glass pipette, not a wash bottle
• watch glasses, spot plate, or small glass petri dishes with water to put sections in
• reasonably fine forceps
• fine paintbrush and/or sharpened orange sticks to transfer sections
• slides
• cover slips
• dissecting microscope – doesn’t have to be a fancy one
• some detergent – 1% Tween-20 or Triton-X-100 – use this if your leaf or other tissue is very hydrophobic, it will help to cover the tissue with water so you don’t get air bubbles between tissue and coverslip or slide

Units, terms, definitions

Microtome

Procedure

1. Place the tissue to be sectioned in a beaker of water, cut out a small piece for sectioning and place in a drop or small pool of water on the pink dental wax.
2. Cut out a small block of support material, then cut it in half. If sectioning a large or soft piece of tissue that might become compressed between the support material, make an appropriate-sized groove in one of the halves (note that since this material is somewhat compressible, it doesn’t squash the tissue as much as the carrot). Make sure the tissue to be sectioned and the support material remain wet at all times.
3. Sandwich your tissue between the two halves of the support material.
   
4. Place the sandwich into the specimen holder on the microtome and tighten until the material is held firmly, but without compressing the plant tissue unduly.
   
5. Adjust specimen height until it is just below the blade.
6. Pull the blade to the right (in this case) and increase specimen height manually until the blade is cutting the tissue plus support material. Push the blade back to the left before the next cutting stroke.A more ergonomic way to section is to stand with your right side towards the bench, and pull the blade towards you and push away from you. Best to avoid back strain if you have much sectioning to do!
7. Adjust section thickness as desired, this microtome sections up to 50 μm thick, and down to 5 μm or so depending on density of tissue being sectioned.
8. Collect the sections with forceps, or with the tip of a paintbrush (with few bristles – pull or cut the rest out), or with a sharpened orange stick into water in a watchglass, or into water in one section of the watch glass. If you use a transparent watchglass, it’s easier to pick the thinnest sections later on. Accumulate sections there – cut LOTS of sections.
9. Look at the sections under the dissecting microscope to select the very thinnest, they will be almost transparent – this is easy to see if you place the transparent watchglass onto black paper or other black background. Transfer some of these very thin sections to a SMALL drop of water on a microscope slide.
10. Cover with a coverslip – put one edge of the coverslip into one edge of the drop of water and lower the coverslip slowly with the forceps. If you drop the coverslip onto the sections you are likely to get air bubbles. If the water doesn’t extend to the edges of the coverslip, add a SMALL amount of water – don’t flood the sections or they will float around.
11. Observe on dissecting or compound microscope. If you need to add more contrast to the sections – more than you can get by closing down the iris diaphragm on the microscope, you can stain your sections (still to be added).

Literature references

Teaching Plant Anatomy (2008) by RL Peterson, CA Peterson and LH Melville, NRC Press, Ottawa, Canada ISBN 978-0-660-19798-2

This book is all about hand sectioning a wide range of tissues and observing either unstained or stained. It also has a CD with it.

Health, safety & hazardous waste disposal considerations

• To avoid back strain, stand with your right side towards the bench and pull and push the blade towards and away from you