Botanical Microtechnique Part I

Fixing, dehydrating & embedding plant material

The techniques herewith have been chosen as they are both reliable and as far as possible avoiding the use of xylene, which is now considered to be extremely hazardous to health. As permanent mounting media generally use xylene as a solvent, its use should be kept to an absolute minimum as the last stage of the staining schedules prior to mounting.

This small series of articles is designed to supplement (and in some cases update) the microscopy literature, to which the reader is referred - especially Eric Marson's excellent little book 'Practical Microscopy' and Johansen's 'Plant Microtechnique'. I will include a full bibliography and reading list in Part II.

I. Where to find suitable material

Suitable botanical material is never far away. Gardens, hedgerows, even garden centres will often allow you to take a few small clippings if you explain what it's for. It is important that specimens are correctly labelled, and if your knowledge of botany is as poor as mine, there are plenty of cheap secondhand books on garden plants & wild flowers available.

II. Collection & Fixation

The first step in preparing material for wax embedding is to 'fix' the material. This both kills and preserves the cells so that no further changes take place.

It is important to fix material as soon as possible, and is preferable to take the fixative to the plant than vice versa. 28ml McCartney bottles, which have rubber, lined metal screw caps, half filled with fixative, are an ideal way of taking the fixative into the field. Each bottle should have a blank label attached ready to take the name of the material collected. Each bottle should be used for one type of material only.

Specimens for fixation should not be more than 5mm thick, and no greater than 10mm at the widest point. Hard materials like roots and woody stems should not exceed 5mm². If using FAA or FPA (see below), the specimens can be kept stored quite safely in the fixative for months.

If specimens float in the fixative, a water pump can be used to extract the air (see Vacuum Infiltration). The specimens should be left in the fixative for at least 24 hours after they have sunk to the bottom before dehydrating.

There are several types of botanical fixative, but the most common 'all purpose' fixatives ate FAA - Formal Acetic Alcohol, and FPA - Formal Propionic Alcohol. Johansen (1940) recommends FPA (and considers this more effective than FAA), so I have included the formula below:

Formalin-Propiono-Alcohol Botanical Fixative

Note: Isopropyl alcohol is not suitable for fixatives (Johansen 1940).

IMS (Industrial Methylated Spirit) 50% 450ml

Propionic acid conc. 25ml

Formalin (approx.37% formaldehyde solution) 25ml

III. Dehydration

Once it has been fixed, the next stage is to slowly dehydrate the material to slowly remove all the water. In order not to destroy the delicate tissues, this is done gradually in a series of alcohol/water solutions of increasing strengths.

The following TBA / Isopropyl alcohol schedule is based on Johansen (1940).

	Step 1	Step 2	Step 3	Step 4	Step 5	Step 6	Step 7	Step 8
Approx Total % Alcohol	50	70	85	95	100	Pure TBA	Pure TBA	Pure TBA
Times	2hrs	o/night	1hr	1hr	1hr	1 hr	1 hr	1 hr

These are 'average' times and may be extended for harder tissues.

IV. Embedding

Step 9 Place material on top of cooled paraffin wax (just sufficiently solidified - not cooled completely) and just cover with the TBA from step 8. 1hr after material has sunk to the bottom.

Steps 10 & 11 Two further changes of pure paraffin wax over the next six hours or so (the first can be overnight).

Step 12 Finally one last change (30 mins) and the specimens are ready for making blocks, and sectioning. As a number of articles and many books have been written on making blocks and sectioning (i.e. Marson, J.E. 'Practical Microscopy'), so I will not reiterate the processes here.

These are 'average' timings, and may have to be extended for harder tissues, or reduced for delicate tissues.

NOTES

Heath & Safety

It is difficult writing heath & safety notes without possibly frightening off newcomers to practical microscopy ! However, like many household and garden chemicals, with a little care and attention these are quite safe to use.

Firstly, as some of the individual chemicals used in fixatives are very dangerous in their pure state, and unless you have a laboratory or secure preparation room at your disposal, it is not advisable to prepare fixatives yourself. I strongly advise buying fixatives ready made or ask your microscopical club if any of its members can help.

Secondly, a fixative that kills the cells in botanical material will equally kill the cells in a human, so great care must be exercised in the handling & use of all these materials. Ideally, Vitrile or rubber gloves should be used when handing fixatives, but this is not always desirable in the field. I would suggest wearing disposable rubber gloves when handling bottles of fixative, and wash your hands as soon as possible afterwards.

All comments to the author Jim Battersby are welcomed.