BIOL2053: Hand
sectioning and staining of sections
This topic is
well covered by Dave
Webb at the University of Hawaii
Preparing
permanent microscopic sections is time-consuming involving fixation,
dehydration, embedding, sectioning and staining of the final sections. Hand
sectioning allows you to observe the plant substructure within minutes and is an
important skill to acquire. While invariably most of your section will be too
thick, you should have portions thin enough to allow you to see or measure what
you want. Practice makes perfect.
Sectioning
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1.
Hold the tissue, supported in a Styrofoam or carrot sandwich,
with thumb
and first two fingers of the left hand (if right-handed).
2.
Trim the Styrofoam/carrot to a pyramid shape so that you can
cut a very
small area.
3.
Thoroughly wet the razor-blade (degreased by xylene soaking),
tissue and
fingers with water. The water serves as a lubricant.)
4
Sit comfortably with your elbows resting on the lab bench.
5.
Cut slowly and smoothly (discard the first section) using a
smooth,
slicing action.
6.
Float the sections off in a watch glass filled with water.
7.
Once the edge of the razor starts to deteriorate move on to a
new part of
the blade.
8.
Use a fine artist=s
paint brush to choose your section.
|
 |
Staining with toluidine blue
O
The objective of staining is to distinguish parts of
the cell/tissue that are chemically different. Toluidine blue is a particularly
useful general purpose stain. It is called a metachromatic dye because certain
cellular components stain a colour which differs from that of the dye in
solution. (How is that achieved?)
-
Lignin/phenol -
green/ blue-green
-
Pectins
- pink, reddish purple
-
DNA
- green/purplish blue
1)
Place your section on a slide and add cover with a drop of 0.05%
toluidine blue O in 50 mM citrate (pH 5) buffer. Leave a 30 s to 2 minutes and
then dab off the dye using the corner of paper towel.
2)
Cover the section with a drop of water to rinse off excess dye, removing
the water with absorbent paper again.
3)
Add another drop of water to the section and cover with a cover slip.
You can alternatively mount the section in toluidine blue
and then by a technique termed "irrigation" replace the dye with
water as shown below.
 |
| Irrigation - Filter paper is used to replace the solution
under the coverslip with a new solution. |
Examples of
toluidine blue O staining
 |
 |
| Unstained makaloa stem © Dave Webb |
Makaloa stem, toluidine blue stained © Dave Webb |
Staining with phloroglucinol
Phloroglucinol is a cytochemical stain for lignin,
giving a red colour wherever there is lignification.
1)
Place a drop of saturated phloroglucinol in 20% HCl solution on a freshly
cut section (CAUTION ACID!).
2)
Mount section in water. Lignin stains red.
Return to BIOL2053 Plant Project
© Sean Carrington 2004
Revised 10/09/04
