Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
plant material: some hints
Does anyone know how to make nice sections of plant material? I tried to make paraffin sections but it seems that the thick cellulose walls of the cells are preventing the penetration of paraffin into the tissue.
Do I have use longer impregnation times for paraffin? Is it easier to make cryostat sections of plant material? Do I have to make the cellulose softer? And how do I do that? Are there any references that pertain specifically to botanical microtechnique?
I had a large project of plants several years ago. We processed and cut everything from jalapeno peppers to magnolia leaves. At
that time I tried to find a book on plant histotechnique and the only one available was "Botanical Microtechnique and Cytochemistry" by GP Berlyn and JP Miksche. The Iowa State University Press, Ames, IA, 1976. ISBN #8138-0220-2. We adapted our animal histology techniques to the plants and found that the most important phase was the paraffin. We used Paraplast, but the important part was three changes of paraffin, 1 hour in each. Vacuum was not used on the first paraffin, but was used on the final two.
You must also know that processing will decolorize the chlorophyll in the tissues.
For staining, I would suggest trying a safranin O - light green - alum hematoxylin sequence. It works the best for plant cells.
Here are a few general references for plant microtechnique. The methods are similar in principle to those for animal tissues, but allowance must be made for the high water content and fragility of plant specimens.
My own experience is very limited, but it fully supports the advice of Berlyn & Miksche. Cut your pieces with a VERY sharp razor blade, using a sawing motion, and do not expect decent sections from anywhere near the cut surfaces of the specimen.
Dehydrate as gently as possible, to avoid sudden collapse of the tissue, which distorts all the cells. There are three ways to dehydrate a plant specimen gently:
1. By evaporation, after immersing in 10% glycerol. This
takes many days. The glycerol is then gradually
displaced by alcohol, then xylene, then paraffin.
Although slow, this procedure is not unduly labor-intensive.
2. By using a long series of graded water-alcohol mixtures, from about 15% up to 100% alcohol. This
keeps someone busy for the best part of a day, and it is
easy to forget the plant specimens if you are doing other
3. Acid-catalyzed chemical dehydration with 2,2-dimethoxypropane is a single step,
usually less than one hour. It is nevertheless "gentle" to
the tissue, though perhaps a bit more traumatic than the
glycerol evaporation method.
Nostalgic note. Anyone who studied Biology in Britain or the Commonwealth from the 1940s to the early '70s (maybe even more recently?) will remember the practical component of the A-level (or HSC) public examination. This always included sectioning an alcohol-fixed piece of plant by hand (with a cut-throat razor; no embedding and no microtome). The free-floating sections then had to be stained, mounted, examined, and drawn with a pencil. These thick sections showed the plant anatomy pretty well under a X10 objective. Thinner paraffin sections provide better detail with a X40 objective, but only if the general tissue architecture is intact. The structural preservation seems to depend heavily on the way the specimen is processed into wax.
Berlyn,GP; Miksche,JP (1976): Botanical Microtechnique and Cytochemistry. Iowa State University Press, Ames,
Iowa. 336 pages. Has chapters on fixation, processing,
wax & plastic embedding, staining (methods with h'tox, safranine, light green etc; detailed accounts of 8
Clark,G (Ed.) (1973): Staining Procedures used by the Biological Stain Commission. 3rd ed. Williams &
Wilkins, Baltimore. 418 pages.
Jensen, WA (1962): Botanical Histochemistry. Freeman, San Francisco.
Kiernan, JA (1999): Histological and Histochemical Methods.
Theory and Practice. 3rd ed.
Vaughn,KC (Ed.) (1987): CRC Handbook of Plant Cytochemistry. 2 vols. CRC Press, Boca Raton, Florida. 176 & 184
Multi-author, 2 vols. Oxidative &
hydrolytic enzymes in Vol 1. Carbohydrates, lectins,
immunohistochem, Na, Ca, K in Vol 2.