The Dicotyledon Stem
for the beginner botanist from a beginner botanist
WALTER  DIONI                       Durango (Dgo) México

Title image: a usable portion of an otherwise badly cut Portulaca stem. Note the two cell thick epidermis and the continuous ring of xylem. Click the image to view a larger version

The pictures were taken at a resolution of 640 X 480 px. and reduced to be inserted in this article. Some pictures are clickable. The width of the field with each objective is as following: 4x = 3400, 10x = 1333, 40x = 340 and 100x = 133 microns. The equipment and the filters used were described in a previous article.


Objective x10: due to thickness of the section, in brightfield
the vascular bundles are opaque.

Obviously there are many resources devoted to this subject. Many of them are beautifully illustrated with excellent photographs of microscopic preparations made by observing all the “rules of the art”: sections made in a professional microtome after paraffin embedding, probably with less than 10 microns of thickness, stained with differential stains for the various tissues, and so on. 

If I, a dedicated zoologist, dare to add a new page, it is only to show how a simple tool (a "microtome of a double razor blade") described by Michel Neuberg in the French MICROSCOPIES Magazine, can help an amateur equipped with a microscope and some contrast filters to carry out some investigations and to learn first hand about dicotyledon anatomy… and to support (at almost no cost) the passing of some days without access to biological water samples.

 It is also true that there are on the Web at least 4 or 5 other designs of differing complexity to build home-made microtomes, or references in almost all the microscopy groups to the old but useful Ranvier botanist’s microtome. But with the exception of Dominic Voisin, a member of the French Microscopies group, there are almost no examples of what the amateur can do with these instruments. I start here by using what is essentially a simple razor blade, but with an ingenious twist.

2 - With the COL-D1 filter some tissues are differentiated and the vascular bundle show its structure - X10. 3 - A similar section labeled for ease of understanding the structure of the stem - X 10 - Filter Rh-Q. Click on the image to enlarge it.
ep = skin of the stem - col = colenchyma - par = cortical parenchyma (colenchyme + parenchyma form the cortex) -cb = cambium interfasciculaire - Paq.vasc. = package or vascular bundle formed by the phloem (ph) and the xylem (xy). Later it will be seen with more details - moelle, the central cylinder of parenchyma (the pith). Cambium plus pith form a central cylinder: the stele. The raphides are oxalate of calcium needles secreted by some cells. Here, the edges of the razor blades have cut an epithelial cell full of raphides throwing them on the cuted surface (see images 6 to 9.)

The material used is the stem of a small ornamental dicotyledon, soft and flexible. I made transverse sections, and took some additional peels of the stem’s epithelium.

The “microtome” was prepared by modifying a little the instructions of Neuberg by covering two double edged razor blades with very thin "Scotch tape" separators. The new blades must be degreased with alcohol before being used. As Neuberg advises I used only the central part of the instrument to obtain thin sections of the stem. The cut must be made by sliding down the blades diagonally. Before cutting, dip the closed instrument in water to wet it. Several perfectly cylindrical cuts, with a neatly cut surface, were possible, but the cutting edge of the blades disappeared quickly, making further attempts useless.

 One must cut over a soft surface only as wide as the "zone of cut", to preserve the edge for further use. The cut must be continued until the section between the two blades is completely released from the two now separated parts of the stem. If this is not done, when opening the microtome, the section will be partially attached to one of the larger stem parts, making it much more difficult to separate without damage.


Photographs of the margin of the stem, showing the epithelium in transverse section (forming the outside limit of the stem, with a thickness of 17 to 20 microns), and the subjacent colenchyma. The colenchyma is a support tissue with very thick cellulose walls and specially reinforced corners. Picture at left (fig 4) was taken with the oblique light filter, the one at right (fig 5) with a Rheinberg blue-yellow filter –both with x40 objective.

Any cut carried out with blunt blades will prove deformed and unusable, because the cells would not present a plane face, but one inclined by the action of the defective edge.

I have succeeded to sharpen the blunt blades by passing their edge tilted approx. 30º along the edge of a sharpening stone, three or four times on each face, and then I refined the cutting edge using like a  razor strop the palm of my hand. It's up to you to consider if the effort is cost-effective.

 If the cut is carried out with a new instrument or one which still preserves a fine edge, even a relatively thick cut can be adequate. With tissues not deformed, the use of all the microscope objectives over the upper face of the cut will give adequate images.


A piece of the stem skin showing the epithelial cells, X4 (fig. 6) and X40 (fig. 7). Dark spots are specialized cells (called crystal idioblasts) which secrete calcium oxalate needles (raphides). The epithelial cells have a length of 170 to 220 microns and a wide of 25 to 53 microns. Their very refractive nuclei are visible like small circles of 7 to 8 microns.

The sections obtained were collected in water, and the thinnest one was chosen and mounted, without any additional treatment, in a 50% glycerin solution, between coverslip and slide. Don’t leave the sections to dry at any time or your wet mount will be full of air bubbles. Once the photographic studies were finished, I crushed the cut with two intentions: one, to see the longitudinal aspect of the xylem (see image 20), and another, to be able to measure the thickness of the stem. The flattened stem epithelium showed that the cut had a thickness of around 250 microns!!


Similar cuts and even thinner can be made freehand, but the thicknesses will be more variable, even from one side of the cut to the other, and in my experience it is more dangerous (for the thumb of the potential botanist) than to use the small instrument. The differences in thickness do not matter for the visual study of the materials; this is why the freehand cuts are traditional in the teaching of botany. But for photography they result in a catastrophe. On the Web J.A. Kiernan states that, by trying hard, sections one cell thick can be cut. Using the parenchyma cells of my sample as a rule this implies sections of 150-200 microns. So my cut was not so bad. It was only 1.3 to 1.6 cells deep.


8 - In polarized light the raphides glow.  9 – On the right a portion of the needles shown in photograph 3, also in polarized light. Both images with the 40x

Theoretically the cuts must be the thickness of the adhesive tapes used as separators. It could be that my razor blades were too flexible, or that I have failed to use an adequate amount of pressure.

Because the blades become blunt, I cannot prove any of these assumptions. One can buy old double edge razor blades at Durango, but it is impossible to do that in very modern Cancún.

With the harder stems of Portulaca one can obtain thinner cuts (probably finer than 100 microns) but generally they are incomplete.

The visible anatomical details in my preparations, and the filters and objectives used, are detailed for each image as it is required.

10 - The surface of the epithelium at X40 and with COL-D3 filter 11 - The medullar parenchyma, x40 and with the Rh-Q filter. Small oxalate crystals (the botanist's crystal sand) shine in the cytoplasm.

The studied section had a diameter of 4.356 mm. The stem has an epithelium of only one layer of cells, 17 to 20 microns thick (but 200 microns long, see fig 7), and a cortical area (that between the skin and the interior cylinder of cambium) of 0.535 mm. Cortical cells are ca. 100 microns in diameter. The pith has a uniform parenchyma, has a diameter of 3.700 mm, and its cells have a mean diameter of 180 microns.

All measurements were made from screen images using the software supplied with the microscope.

12 - A cell of the pith parenchyma, with it nucleus and small oxalate crystals. 13 - Another pith cell, full of oxalate needles.
14 – A strip of cambium, separate cortex (lower right) from pith. Some pith cells show crystal clusters. The image was recorded with the x10 and RhQ filter. 15 - The same sector, seen in polarized light. As you can see (at least in this material) The RhQ acceptably mimics the polarized light behavior

16 – The Cambium is a support and generating tissue for the vessels which transport the water minerals and elaborated materials up and down through the stems. It forms a cylinder of 70 to 90 microns thickness between the cortex and pith.

In other species, the strip of cambium supports vessels all over its circumference (see the Portulaca section in the title picture).
In this species they are concentrated in discrete zones (vascular bundles or packages), which we will see next, and are formed by the phloem, external to the belt of cambium, that conduct solutions downward, and the internal xylem with large vessels that have flexible walls supported by spiral ridges and conduct solutions upward. In other species the phloem is capped by stiff fibers, and it can be a phloem internal to the xylem.


17 - A mosaic of 4 images, to show the structure of the vascular package. Original images were taken with the X40 objective, and COL-D2 filter. Click on the image for an even larger and labeled image.   The labels are : phl - phloem, cb - cambium, M-xyl - meta xylem, P-xyl - proto xylem.

18 - the walls of the vessels are birefringent 19 - It is reinforced by spiral ridges.
Four levels, amalgamate with Combine Z.

After this experience I think that especially if Kiernan's statement is true, the use of a single edged razor blade, or (to protect fingers) a better skilled use of the Neuberg double razor blade can open up to the amateur a fascinating research area.

Now, of course, I want to use, one of these days, and to leave testimony of the results, what is generally known as a "nut-and-bolt microtome". It is very simple and a not expensive approximation to the Ranvier botanical microtome. 

Who knows? Perhaps I can acquire a second hand old but good Minot rotary microtome, absolute alcohol, xylene, paraffin and embedding oven, to finish my life as a botanist!

Comments to the author, Walter Dioni , are welcomed.


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