Protocol

Author

Rosemary White

Overview

This protocol outlines how to use a hand microtome or a small bench microtome to obtain tissue sections of a precise thickness for dissecting or compound microscopy. These small microtomes are useful for serial sectioning large, solid tissues, or for sectioning very soft tissues held in support material.

This is one of a series of protocols on sectioning fresh 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

Sliding or sledge microtome sectioning of fresh or fixed tissues

Vibrating microtome sectioning of fresh or fixed tissues

Cryostat sectioning of frozen tissues

Materials/Equipment

• piece of pink dental wax
• water – conical flask with plastic pipette, not a wash bottle – plastic pipettes come in a wide range of volumes and tip sizes and are safer than glass pipettes
• watch glasses, spot plates, or small glass petri dishes with water to put sections in
• double-edged razor blades – lots – break in half while in their wax paper wrapping – use lots, stop using a blade as soon as you feel any resistance to cutting the tissue
• 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 (e.g. rice), it will help to cover the tissue with water so you don’t get air bubbles between tissue and coverslip or slide, although it may damage membranes if you plan to stain the living tissues with membrane stains or live/dead stains or look at chloroplast or other autofluorescence
• support tissue for sectioning – pith, carrot or potato – the latter two can be stored in 70% ethanol for many months until required
• hand microtome
• cork borer with same inner diameter as hand microtome

Units, terms, definitions

microtome

Procedure

Before starting, take some of the carrot or potato pieces out of the 70% ethanol and soak in water.

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. a. The image below is of a hand-held microtome. The tissue is squashed into the top hollow, and the blade pulled across the top to cut off protruding tissue. Section thickness is determined by how much you raise the tissue by turning the knurled knob at the base of the microtome.
   Make appropriate-sized carrot pieces using a cork borer of the same diameter as the hand microtome, then cut the resulting carrot cylinder in half, lengthwise.
   If the tissue is large or liable to be squashed, make a groove in one of the carrot halves, place the tissue between, then push the carrot cylinder plus tissue into the hand microtome.
   
   
   
   
   
   
3. b. This hand microtome attaches to the bench, as shown above. Here, the tissue is clamped into the top, and you slide a blade across to cut off protruding tissue. Section thickness is determined by raising the tissue with the knurled knob at the bottom.
   Prepare the carrot support tissue exactly as for free-hand sectioning. Cut out a small block of carrot, then cut it in half. If sectioning a large or soft piece of tissue that might become compressed between the carrot, make an appropriate-sized groove in one of the halves. Make sure the tissue to be sectioned and the support tissue remain wet at all times.
   Place a piece of tissue in the groove, and sandwich it in with the other half of the support tissue. Clamp this into the hand microtome.
4. Make sure your hands, the support tissue and the blade are wet. A stiff blade works best for both of these hand microtomes, either an old-fashioned shaving razor or a sharpened old microtome blade is best, but a reasonably inflexible disposable blade works OK. The blade must be sharp.
5. Cut sections by either pulling the blade towards you through the tissue, or pushing it away from you. In either case, the tissue must be cut with a single, smooth, even stroke.
6. Collect the sections plus support tissue in a watchglass, or in 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.
7. 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.
8. 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.
9. 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. See instructions about staining (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.