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Lab protocol for basic wood anatomy procedures: making and staining of
micro-sections of wood samples

Method · October 2010

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Nele Schmitz
Thünen Institute
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Sampling wood for microtomy

Abbreviations
DI water, distilled water. FAA, Formaldehyde Alcohol Acetic Acid. FG, fast green. PEG,
PolyEthyleneGlycol. Rd, radial. Tg, tangential. Tv, transverse.

General comment: Use small paper tags marked with pencil to label tissue samples. Place the
tag in the vial/ziplock bag with the tissue specimen so that the tag follows the sample through
all handling steps. Writings directly on the wood sample or with marker pen on glass vials
only are unreliable since they can fade away in the oven or when touched with ethanol!).

1. Sample fixation & storage

 The best chemical fixative when cell contents should be preserved, besides tissue
structure, is FAA (see safety issues below): 10 ml Formaldehyde (37-40 %), 50
ml Alcohol (ethanol 95 %), 5 ml glacial Acetic acid, and 35 ml distilled water.
Depending on the size of the samples, they can be transferred to a less toxic solution
for storage after a few days or weeks (see solutions below).
 Alternative chemicals, of equal quality, but lower toxicity can be used when intra-
cellular studies are not envisaged:
o 50-70 % ethanol
o Copenhagen Mix: 99% ethanol: DI water: glycerol (70:28:2)
Copenhagen Mix is recommended for hard wood samples since the glycerol
will avoid the wood to become even harder due to the dehydrating effect of the
alcohol.

2. Sample preparation
General
Depending on the hardness of the wood, cut little blocks of 25-100 mm² (shape depends on
the purpose) and of at least 1 cm but max. 2 cm high (otherwise it will not fit in the
microtome). You can do this with a handsaw or if the samples are already small you can also
use a single-side-razor blade. Use a marble or glass plate as cutting board.
For liquid preserved samples, take them out of the solution only one by one to be cut, to limit
the time they are out of liquid. We don’t want them to become hard.

The orientation of the wood block when cutting is of high importance to be able to make

Tv Tg Rd
sections that show exactly the anatomy as seen in transverse, tangential or radial plane.

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 For cubes, two sides should be cut parallel to the wood rays, two sides perpendicular
to the rays and the last two sides perpendicular to the other sides already created.
 For blocks of other shapes, think about which side you want to section and how the
block should then be positioned in the microtome (and thus which side has to be bit
longer to clamp).

PEG infiltration and embedding

1. tissue dehydration
Comment: Dehydration is not really necessary if instead you leave samples infiltrating with
PEG for a longer time (see further).

 If FAA was used as fixative: first wash samples in the fixative solvent, 50% ethanol,
for about 10 min. (in the meantime you can prepare the different ethanol
concentrations).
 Dehydrate in graded ethanol series.
o For all but the final ethanol concentrations commercial 95% may be used
(100% ethanol is expensive!).
o Samples are put in the oven at 60 °C in little glass vials, several together in a
large beaker with a large watch glass on top to prevent evaporation.

% Ethanol Time Notes

50 1h
70 1h Tissues can be stored for several days in 70% ethanol or higher.
96 1h
100 2-4 h
100 weekend

2. infiltration and embedding

Primary remark if samples = microcores
Before putting the cores in PEG you have to mark the side of the core you want to make
slides of (Tv, Tg or Rd). If the cores are wet of the alcohol you can see the direction of the
fibers and in this way you know where the Tv plane is. You can now put a dot with a
permanent marker pen on that side and/or cut a piece of the core with a scalpel such that the
transverse plane (or Rd or Tg) is already straight (not round anymore).

Procedure
 To melt pure PEG1500, put a beaker with enough PEG grains to cover the bottom in
another bigger beaker with warm water). It is faster to gradually add PEG grains to the
liquid PEG and stir with a glass rod, than to melt immediately a whole beaker full of
PEG grains.
 PEG is corrosive to all metals and is only liquid at high temperature, so
clean all beakers and utensils like tweezers immediately after use with
plenty of water.
 PEG should never boil!

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 Do not add PEG grains, to the beaker in the water bath, directly from
the PEG1500 bottle but use another beaker (the warm air above the
water bath will affect the grains inside the bottle).
 Just before the PEG liquid is ready, place the wood samples WITH marked paper tags
in an ice cube tray, pour the hot PEG over them and fill almost to the border of the
tray. If the samples are floating don’t worry, after one hour they will sink. Place the
tray in the oven at 60 °C for a period of at least two days (f.ex. over the weekend).
 Take the samples out of the oven and transfer the wood samples to another ice cube
tray, sprayed with some Para Gard1 (paraffin repellent). It will prevent sticking of the
PEG blocks to the tray and allow for easy release. Make new marked paper tags, with
a long unwritten part that you place against one wall of the tray so that the marked part
sticks out of the tray.
 Melt and pour new hot PEG over the wood samples and let the PEG cool down at
room temperature overnight.
o Place the samples in the tray so that the side you want to make slides of is
upside down, facing the bottom of the tray.
o For small samples: first drop a thin layer of liquid PEG in the tray, put the
samples in the desired position and let the PEG harden (If needed: while
holding the sample perpendicular to the bottom of the tray with tweezers.).
Now you can fill the tray with PEG without the samples moving while pouring
the PEG (Remember: good orientation of the samples in the PEG cubes is
essential to make perfect Tv, Rd or Tg sections!).
 After cooling of the PEG, the cubes which have hardened can be wriggled out by
moving and turning with the tray.

Softening of wood samples

This is only needed for air-dried wood samples and if sectioning of chemically fixed samples
does not give good results.
 Start with soaking samples in DI water for one night in the oven at 60°C or until the
samples sink to the bottom of the glass vials.
By using a vacuum pump this procedure can be quickened. To do this, switch the
vacuum pump on for a few minutes, with the samples inside in a big jar with DI water,
to put the bell jar under vacuum. Close the valve of the bell jar and turn off the pump.
Leave for a few hours. Repeat this procedure until all samples have sunken.
 If still too hard to section, put samples in oven again in a solution of 1:3:1 (95%
alcohol:glycerine:DI water) until you can make good sections.

3. Sectioning

1. Sharpening the knife

Be careful not to cut yourself!
 Make coarse and fine grinding solution by adding 3 g of 1µ or 0.3µ Aluminium
powder, respectively to 25 ml fine oil and shake until well mixed. It is not useful to
make a bigger volume at once since the powder will clog at the bottom of the vial after

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As an alternative you can use glycerine or some detergent.

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a while. So use a plastic bottle with lid which can be thrown away after oil and powder
can not be mixed anymore.
 Place the glass plate with the coarse side up in the sharpening machine. Pour some
coarse grinding compound (not too much! Just draw a line of 2 cm width and 10 cm
length) on the glass plate. Put both the push-button and the draw-button on ‘coarse’
and set the timer with a cork stop stuck for a period of 1 to several days depending on
the state of the knife.
 After a day, check under the microscope if there are still grooves. If not, clean the
glass plate, the knife and everything else with paper towels.
 Turn the glass plate to the fine side and pour, in the same way as before, fine
sharpening compound on it and sharpen for 0.5-1 day. Check the knife edge under the
microscope, the cutting edge should be uniformly smooth and show no notches.
TIP: If there are still some notches you can mark these spots with marker pen
on the knife so that when cutting you know which positions of the knife to
avoid.

2. Sectioning PEG embedded samples

 Trim the PEG block so that the sides which will be clamped in the microtome are
straight. Carefully remove some PEG around all four sides of the wood sample (Only
for big samples! If you remove PEG with small samples they may be pulled out while
sectioning). If not, the PEG can distort sensitive tissues (eg. cambium) because of the
pressure when the PEG is cut with the microtome.
 While sectioning use a brush for putting some glycerine on the sample and on the
knife. It will make the section slide easier on the knife (Don’t use water or ethanol
for it will dissolve the PEG!).
 When samples are from decayed wood or when it is important to section both wood
and bark (that easily detaches from the wood), a piece of transparent adhesive tape can
be applied to the surface of the cube before cutting. Lift the tip of the tape piece a little
bit with the tweezers (so that the knife goes under the tape), cut and pick the slide
attached to the tape with tweezers from the knife.
o Tesa crystal is the best since it does not dissolve in Parasolve (see further).
o Cut a piece of tape, just a bit bigger than the sample. Big tape pieces will
make the staining and dehydration afterwards more difficult.
o In this case don’t use glycerine, the PEG block should be dry as well as the
knife.
o Sometimes the tape detaches from the section, sometimes not. If it does,
remove the tape from the cell strainer so that it will not interfere with the
staining procedures

3. Sectioning air-dried or liquid preserved samples

 Use 1:3:1 (95% alcohol:glycerine:DI water) solution to wet the sample with your
brush all the time while cutting to prevent drying and thus hardening of the sample.
Also wet the knife each time before making a section to help the section sliding over
the knife.

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 Start cutting in the same direction as the rays or as other features of different density
(f.ex. conspicuous parenchyma or vessel bands or phloem bands)
 Start with a thickness of 25 µm for transverse samples and 16 µm for longitudinal
sections.
 Approach the sample with the knife (C-profile) until one tiny piece of wood at the
corner of the block is cut, wet your brush, sample and knife, put the brush on the
sample and against the knife and cut in one smooth pull while moving your hand with
brush in the opposite direction guiding the section onto the knife.
 Put the slide in distilled water in a sieve WITH marked paper tag. Make 3-4 sections
per sample as some may be spoiled while staining or preparing slides.

4. Problem solving
When cutting creates lines on the sample and/or cutting makes noise:
 Change the knife angle to a lower one. If you get successive thin and thick sections
you made the knife angle too shallow.
 Change the orientation of the sample in the sample-holder. Try once parallel with the
rays and once perpendicular with the rays. Depending on the presence of axial
parenchyma, changes in density, … the best orientation to cut varies.
 Make smaller blocks (if you didn’t do yet). Cut blocks with a surgical blade to 25
mm².
 Use a freshly sharpened knife.
 If you tried all of the above and still do not get good sections, the wood is still too hard
and should be softened more.

4. Staining and dehydration

1. Method 1
The double stain Safranin-FG is especially good for meristematic tissues.

Step %
nr. Ethanol Other chemicals time
1 50 / 5 min.
2 / 1g/l aqueous Safranin solution 10 min.
2x5
3 50 / min.
4 75 / 5 min.
5 / Fast Green, 0.1g in 100 ml 96 % ethanol 4 min.
6 96 / 5 min.
7 100 / 15 min.
8 / Parasolve 2-3 h

 Don’t use Parasolve if the sections are attached to adhesive tape since Parasolve dissolves
some brands (f.ex. Sellotape, Tesafilm Cristalclear is Ok)

The above times are just an indication. For each species different times should be tried out to
find the optimal staining conditions.

2. Method 2

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*
0.5 g Fast Green in 50ml clove oil + 50 ml 100% alcohol
†
50ml clove oil + 25 ml 100% alcohol + 25 ml Parasolve

Minutes Treatment Notes

DI water from sectioning onwards
10 Safranin (1g/l water)
2x5 50% alcohol
5 75% alcohol
5 96% alcohol
2x5 100% alcohol
1-2 Fast Green solution*  in glas or porcelain cups
2x10 Differentiation solution†  in glas or porcelain cups
30 Parasolve in little glass bottles

TIPs: Sections with tape can be treated with Parasolve depending on which brand of tape.
Sellotape is affected by Parasolve but Tesafilm is not.

Excess FG can be removed by putting it in differentiation solution

3. Method 3

If no double staining is needed Method 2 can be used up to the 100% alcohol step. Afterwards
the section can be mounted immediately on a slide by using Euparal (see below).

4. Method 4

Minutes Treatment Notes

5 DI water*
5 Staining solution  mix of safranin&alcian blue**
1 DI water  just rinse
3 50% alcohol
3 75% alcohol
3 96% alcohol
3 100% alcohol
*
Only for sections stored in alcohol, not needed if section was kept in water from sectioning
onwards
**
Dissolve 0.35g Safranin in 35ml 50% alcohol in one little bottle. Dissolve 0.65g Alcian
Blue in 65 ml DI H20 in another little bottle. Add both solutions and mix well.

TIP: Use a separate six-well plate for the staining solution so that it can be recuperated and re-
used afterwards. Use also for each alcohol step a different plate to avoid confusion.

 Don’t leave sections much longer (certainly not 30 min.) in 100% alcohol because the
Safranin stain will fade gradually.

5. Making slides

1. General

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Never let your slides dry to avoid air bubbles coming in. Especially when the last step was
100% ethanol, after putting the sections on a slide immediately cover them with Euparal.

Label slides with pencil and not with marker pen since this can dissolve in ethanol.

2. Permanent slides
 Take the section carefully with tweezers and put it on a slide WITH correct pencil
mark
 If sections are curled, carefully flatten them with two brushes and cut the curled sides
(which do not uncurl) with a razor blade (the little ones you use to put on a scalpel
knife holder). Alternatively, you can put razor blades on top of curly sections and
leave them for a minute or so (if you prepare other sections meanwhile, make sure you
add droplets of ethanol/parasolve to keep the section moist). Take the razor carefully
from the section and if you are lucky the section now stays flat.
 Put some Canadabalsem (if Parasolve was used as last step) or Euparal (if alcohol was
used in the last step2) on the section. For Euparal use of a Pasteur pipette is easiest.
Just a few drops is enough: flooding over the cover glass but also air under the cover
glass should be limited.
 To mount the coverslip, gently place one edge of the coverslip on the slide and with a
pointer or forceps slowly lower the coverslip. Leave the slides like this for some
minutes so that the mounting medium can spread.
 Put the slides on two wooden beams (if just on a plate they can stick to it when
Euparal is squeezed out when putting weights) in the oven and put 1-2 weights on top
of the coverglasses. Leave the slides at 60°C for one night to dry and to let potential
air bubbles disappear. Place a post-it on the oven when slides are in so that other users
know they have to be careful when opening the oven.
 After 1 night, carefully remove the weights. If some weights are stuck turn carefully
around, DON’T pull because you will suck air under the coverglass. With a cotton-
bud add some 100% alcohol (recuperated one) or Parasolve to remove Euparal or
Canadabalsem. BUT only do this if it covered the section, when only on the sides or
just not reaching the section leave it because cleaning will also create greasy stripes on
the cover glass.

3. Non-permanent slides
Put the section on the slide and dry the slide around the section with some pieces of filter
paper. Do not dry the section itself to avoid air in the section. Add some drops of glycerine
and cover with a coverslip.

2
Euparal is not soluble in Parasolve, but it is in alcohol.

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Health, safety & hazardous waste disposal considerations

Health and safety
 Always wear a lab coat
 Wear gloves when working with any chemical (most chemicals are at least irritating)
and safety glasses (when preparing FAA f.ex.)
 No food and no drinks in the lab!
 Always use formaldehyde in the fume hood and also leave the bottle there (very
volatile and carcinogenic!!)
 Also when working with irritating chemicals, use the fume hood (f.ex. FAA, acetic
acid, …)
 When making permanent slides with Parasolve or Canadabalsem, ventilate the lab by
opening a window
 Mark bottles with content name (f.ex. alcohol 75%, PEG 1500,…)
 Turn off heating plate during evenings and weekends (fire risk!)

Nice and tidy

 Clean all material after use and put it back in the cupboards after drying. There is a
washing machine for big dishes.
 Remove writings on glass beakers/bottles with recuperated alcohol (don’t use new
alcohol for this!!)
 Throw away things you don’t need anymore and don’t let them hang around in the lab
for years! (f.ex. bottles with parasolve because it dries and makes the bottles useless!)
 Clean knife-sharpener after use with tissue paper
 Clean microtome and don’t forget to remove the PEG remains and fallen sections in
between the grooves and holes of the microtome holder. The wheels to orientate the
sample get stuck if you don’t do it! (TIP: easy with tweezers)
 Remove Canadabalsem/Euparal on pipette, table, glass plate, brushes,… with recup
parasolve/alcohol
 Also keep heating plate tidy by cleaning it with parasolve/alcohol
 Open boxes with coverslips just to take one and put the lid back immediately, just
loosely is ok, to avoid dust

Environment friendly
 Gloves can often be used more than once (by blowing you can pop-up the fingers
again)
 Dispose of all alcohol in the specific waste bottles. Since the alcohol can be distilled to
remove staining and recuperate 96% ethanol, it is very important to separate alcohol
from ALL other products used! If you have alcohol mixed with something else
dispose it in another empty bottle and label it clearly with its content.

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 Recuperate parasolve and staining solutions in their specific bottles (by putting
staining solutions in separate six-well plates, not together with alcohol, you can easily
pour them back in a beaker and then in one of the recup-bottles)
 Use dishwasher only when completely filled
 Don’t throw away chemicals in the sink! (They finally end up in our drinking water!!)
There are waste-bottles in which we collect formaldehyde, parasolve, acetic acid,
FAA, … . At specific moments in time they are collected by a special institution.

Some references
Books & Articles
De Micco V, Aronne G, 2007. Combined histochemistry and autofluorescence for identifying
lignin distribution in cell walls. Biotechnic & Histochemistry 82(4-5), 209-216.
Jansen S, Kitin P, De Pauw H, Idris M, Beekman H, Smets E. 1998. Preparation of wood
specimens for transmitted light microscopy and scanning electron microscopy. Belg. Journ.
Bot. 131: 41-49.
Rapp AO, Behrmann K. 1998. Preparation of wood for microscopic analysis after decay
testing. Holz als Roh- und Werkstoff 56: 277-278.
Ruzin SE. 1999. Plant microtechnique and microscopy. New York, USA: Oxford University
Press.

Websites
http://www.woodanatomy.ch/preparation.html
http://www.microscopie.be/
http://em-outreach.ucsd.edu/web-course/toc.html
http://prometheuswiki.publish.csiro.au/tiki-index.php

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