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Preliminary preparation
- Cut samples to a radial thickness of 1cm.
- Split samples with a blunt knife along the radial and tangential planes
so as to make blocks approximately 8 x 8 mm.
Precise anatomical orientation facilitates microtome sectioning.
Softening and embedding
- Dry wood of average hardness can be softened by immersion in boiling
water for 1-2 hours (marked with a soft pencil) or in a mixture of 96%
alcohol, water, and glycerin in volume proportions 1:1:3.
- Wood from living trees can be sectioned without softening.
- Tender and heterogeneous tissues (e.g. bark-cambiumwood) are embedded
in carbowax before cutting. Water saturated wood is immersed in a series
of Polyethelene 5000 solutions (progressively 30, 60, 100%) and left in
each solution for 24 hours. Since PEG is soluble in water, glycerine
is used as the lubricant for microtoming.
Sectioning
- Hand microtome: The fine sectionsthus obtained are sufficient for
microscopic observation but not for photography.
- Sliding microtome: With this type of microtome one can produce good
sectioned cuts of all the indigenous species.
(All the sections presented in this book were cut with a Reichert sliding
microtome.)
Only wedge-shaped blades which are very sharp produce good sections.
A blade is sufficiently sharp when it can cut a hair held in the hand.
A perfect blade sharpness is indispensible for the production of thin
sections of coni- ferous wood in which the secondary cell wall tends
to detach from the primary wall. In our laboratory, the best results
have been obtained with American Optical knives sharpened on an American
Optical Knife Sharp- ener. Much practice is necessary to make good sections;
however, some general guidelines can be indicated. The knife blade and
the cutting surface should form an angle of approximately 15° for
hardwood and approximately 8 ° for softwood. For wood of heterogeneous
density (e.g. larch) approximately only half of the cutting edge of
the knife is used to obtain a cut of 0.5 mm in width.
The optimal thickness of thin sections:
Transversal sections: 15µ in general; 10µ for species with
small pores.
Radial sections: 15µ for the structure of ray walls; 25µ
for the perforation and spiral thickenings.
Tangential sections: 15µ.
Staining Processes
For the study of normal cell structure, the cellular content is destroyed
before the section is embedded.
Safranin is an easily used stain.
Preliminary preparation
- Immerse in javel water for 15-30 minutes
- Rinse with water 2-3 times until the odor of the javel water disappears
- Stain with a solution of 1% safranin for 3-5 minutes
- Rinse once with water
- Rinse once with 50% alcohol
- Rinse with 96% alcohol 2-3 times until the excess stain disappears
- Rinse once with 100% alcohol
- Immerse in xylol. If the liquid is murky, rinse again with 100% alcohol
- Mount in Caedax (synthetic resin)
- Apply approximately 50 gr. of pressure on the coverslip and harden
the resin by drying in a 50-60°C oven for 24 hours.
If heartwood is present, the wood is often not stained. In that case,
the preparation process begins with the first rinsing with 50% alcohol.
Possible difficulties: Frequently the sections roll and it is difficult
to flatten them. This can be avoided by pressing on the sample being cut
with a paint brush above the microtome blade. The section is placed in
glycerine on an object slide, covered with a coverslip, rapidly passed
through a flame. To flatten rolled sections, place a point of a pair of
conical pincettes in the opening of the stained, rolled sections. Carefully
advance the section on the flat back of the pincettes and pour first 100%
alcohol over the section and then xylol. The section is then mounted in
Caedax on a slide.
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Wood attacked by fungi
The preliminary preparation and sectioning of the samples is the same
as for recent, healthy wood.
Staining
The thin sections are stained with safranin and picric acid-anilin blue.
The sections are dipped for a short time in an aqueous 1% solution of
safranin. Excess stain is washed off with water. The section is placed
in a drop of picric acid on a slide and heated over a flame to boiling.
Next, the section is immersed in a series of progressively increasing
concentrations of alcohol and finally in xylol. The section then is mounted
in Caedax. (Picric acid-anilin blue: 25 ml of aqueous, saturated anilin
blue and 100 ml of aqueous, saturated picric acid.)
Subfossil, uncarbonized wood
Hand cut sections are made with razor blades which have very hard butting
edges. Wood which is slightly compressed may be inflated and cleaned with
javel water so that the normal cellular structure can be observed. The
identification is done without staining. A phase-contrast microscope is
valuable for the observation of fine structures. In order to detect the
presence of bacteria, actinomyces and hyphae of fungi, the sections are
colored with anilin blue and picric acid. In general, only sections prepared
with a microtome can be photographed. The sections are embedded in paraffin
and colored with safranin.
Charcoal
The identification of charcoal requires reflected light. All the characteristics
are observed on fractured surfaces. Before examining a sample with a stereoscopic
dissection microscope, a fracture is made so as to obtain a transversal
surface. If it is necessary to examine longitudinal characteristics, the
sample is split with a scalpel under a stereoscopic microscope along radial
and tangential planes. The small fragments are to be placed horizontally
on a piece of wax on a slide. They are examined under a reflected light
microscope with a magnification range between 100-400 X. Reflected light,
however, is not convenient for photography and usually the sample must
be embedded in plexiglas and cut with a microtome.
Procedure
- Air dried samples are broken along the major orientation planes so
as to obtain cubes having 6-8 mm edges. The cubes are soaked twice for
3-4 days in absolute alcohol. (if a sample does not sink in the alcohol,
the air can be extracted in a vacuum tube.)
- The cubes are placed for a duration of two weeks in methy 1-metacry
late (pure metacrylic acid-methylic ester with 1% hydroquinone) with
activating agent 50% 2.4 dichlor-obenzol-peroxide in a softening agent
(product of Flucka, Buchs SG, Switzerland). The solution is replaced
after one week.
- The samples are then placed in gelatine capsules filled with methyl-methacrylate
and closed. The capsules are polymerized at 35°C in a vacuum for
2-3 days.
The presence of air bubbles in the sample blocks is a consequence of
incomplete dehydration, insufficient extraction of air, or excessive heating
during polymerization. A block is fixed on the microtome and a preliminary
cut is made to prepare the surface for sectioning. A piece of self-adhesive
tape is attached to the sectioning surface and when the knife is activated
15 microns below the tape, the sections adhere to the tape. The sections
are unglued from the tape with a couple of drops of glycerine and placed
on a slide.
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