Protocol

Author

Rosemary White

Overview

This protocol outlines in detail how to obtain hand sections of plant tissues by using other plant material, such as pith, carrot or potato, as support tissue for your desired tissue. This method is very useful if you wish to section small tissues, such as roots, or soft, thin tissues, such as finer leaves. In some cases, you can hold the tissues down gently on dental wax to section using the dissecting microscope, as in the first module, Hand sectioning fresh tissues.

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 support tissue in a hand microtome 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

Procedure

Before starting, take some of the carrot or potato pieces out of the 70% ethanol and soak in water, as in Making hand sections without support material

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. Get your fingers and the half-razor blade wet, everything should be wet.
   Take one of the pieces of carrot from the beaker of water.
   
   
2. 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, as shown below, left and centre. Make sure the tissue to be sectioned and the carrot remain wet at all times.
   
   
3. Place a piece of tissue in the groove, and sandwich it in with the other half of the support tissue, as above, at right. (In th image here, the support tissue could be trimmed down a little more.)
4. Make sure your fingers, the carrot and the razor blade are wet. With the tissue in one hand, and the blade in the other, rest your forearms on the edge of the lab bench, and rest the outer edge of your hands on the bench. As when cutting on the dental wax, you want to slice through the plant tissue plus support tissue with one smooth stroke of the blade – NO SAWING! To get a smooth, even stroke, it’s best to have your hands supported as much as possible – you will also be less likely to wobble and cut yourself.
   You can either pull the blade towards you through the tissue, as shown below, or you can push it away from you. As shown, use your non-cutting forefinger as a guide for the blade. Make sure your non-cutting thumb is below the level of the carrot!
   
   
5. Collect the sections plus carrot in a watchglass, or in water in one section of the watch glass. Accumulate sections there – cut LOTS of sections, see below. It’s much easier to see the tissue and sections against a dark background – here, it’s a piece of black paper, but black plastic is good too, and doesn’t fall apart when it gets wet. If necessary, you can place the pink dental wax on a black background to detect small tissues, e.g. fine roots, more easily.
   
   
6. Look at the sections under the dissecting microscope to select the very thinnest, they will be almost transparent. It may be easier to see the thinnest sections if you examine them on a black background. Transfer some of these very thin sections to a SMALL drop of water on a microscope slide.
7. 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.
8. 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).

Notes and troubleshooting tips

You can use a wide range of support material to section soft or thin plant tissues (e.g. small roots, leaves, shoot meristems, etc.). Polystyrene “peanuts” are quite good for supporting softer tissues, and harder polystyrene or other packing material is useful as well. You can purchase pith, which is often from elder (Sambucus nigra) or possibly from cassava (Manihot utilissima) or ricepaper tree (Tetrapanax papyriferum), these tissues are used because they contain few or no lignified vascular tissue, so they are fairly soft and can be sectioned very thinly without collapsing.

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

• Take particular care when using razor blade held against bare hands