Eosin as a
nuclear stain in botany
and as an
economical substitute for
indicated to the contrary, the included images are personal pictures
obtained with a digital camera of 0.4 Mpx. integrated into my National
Optical DC3-163-P microscope equipped with planachromatic optics,
10x, Objectives: x4 (NA 0.10), x10 (NA 0.25), x40 (NA 0.65) and x100
(NA 1.25)). The original ones have been captured at 640 x 480 px. and
reduced or trimmed as was necessary to include them in this work. An
entire picture’s formatting, including mosaics of several pictures was
made in Photo Paint. In each picture's legend, the objective whereupon
taken is indicated, just as a suggestion of the power used because of
diferent sizes of each picture. A number of contrast devices (Rheinberg
discs, darkfield discs, COL discs, and the Mathias arrow), could had
used to impart color, or relief to some
Eosin is an acidic aniline dye,
which is considered to have a selective
affinity for the cell cytoplasm. In contrast, one considers
as a basic dye, which consequently has an affinity with the cellular
acidic elements. As the nuclei are essentially formed by ribonucleic
acids, hematoxylin is used to color them.
These two dyes are the basis of the universally used staining technique generally known as Hematoxylin-Eosin, which provides nuclei stained blue, (or almost black, according to the formula employed, see fig 1) and reddish cytoplasm.
The use of a commercial 2% eosin solution that was sent to me by Jean-Marie Cavanihac showed that in the foliar epithelium of Aptenia cordifolia the eosin strongly stained the nuclei (and nucleoli) as well as the chloroplasts, and much more slightly or not at all the cytoplasm.
I have never known the use of eosin as a nuclear stain, and a very extended research through the World Wide Web did not provide any reference in this direction, largely reinforcing again the use of the eosin as a simple acidic dye.
In consultation with Jean-Marie Cavanihac he queried the existence of some additive in the commercial solution which could be responsible for such a behavior that one could consider "abnormal", and suggested trying some solutions directly prepared in the laboratory.
Consequently by using eosin in powder form, bought in an old pharmacy at Durango, I prepared two solutions of eosin:
1) In an ethanol-methanol mixture (MEM, ethanol 96%....70ml; methanol 85%....30 ml) and
2) Directly in distilled water.
Moreover I also used the European commercial solution.
Preparing solution in the alcohols proved to be difficult. Alcohol dissolved little of the dye and the solution seems to be a saturated one. The color was clear, transparent and yellowish. My eosin was definitely an “aqueous” eosin.
Water easily dissolved a larger quantity of eosin. The diluted solution showed at first the same color as the alcoholic solution, but, as it become concentrated, it turned a deep red as the commercial solution was.
1 – Foliar epithelia
Three pieces of foliar epithelia from Aptenia cordifolia were fixed in AFA, (Alcohol, Formalin, Acetic Acid) for one hour. They were washed in two water changes, and one of them was submerged in one of each staining solution for 5-6 minutes.
Each epidermis was then carefully washed in two changes of water, until they did not lose more dye and they were then mounted between slide and coverslip in 50% glycerin.
slides were manually compressed under a layer of absorbing paper
to obtain quite a thin and readable preparation. They had been sealed
nail polish to improve their permanence.
1) European Commercial Eosin : as in the preliminary test, nuclei and nucleoli, as well as the chloroplasts, were well colored, not therefore the cytoplasm.
2) Aqueous Eosin : The result is very similar to that of the commercial solution.
3) Alcoholic Eosin : in spite of its much clearer and yellowish color and the weak concentration of dissolved eosin, the result was very similar to the two preceding ones. Perhaps the nuclei are a little sharper and clearer than in the other two solutions. If the material supports an alcohol treatment this technique can save a lot of eosin.
2 – Gingival Epithelium
Cells obtained by softly scraping the interior of the cheek were mounted in diluted physiological solution 50/50 with aqueous eosin.
Results : The examination showed that the cells were suitably colored and that in a few seconds the nuclei and its nucleoli were quickly differentiated.
A parallel staining was carried out with methylene blue (the solution for aquarists) it also colored the nuclei, and the bacteria in the outside of the cells additionally, but the aspect is not so clear and clean as it was with the eosin.
Staining with eosin, and then with blue like a contrast stain, did not prove to be adequate. Blue was only partially fixed in the cytoplasm. The eosin dominated the staining and was not removed from the nuclei by the blue.
3 – Rotifers
A population of Adineta sp. was fixed by very hot water. Once the liquid was cold, 2% eosin was added drop by drop until a red, weak but definite color, was obtained. A small drop of the deposit was mixed with a similar drop of 50% glycerin, and it was covered with a coverslip aiming to obtain a very thin preparation.
Results : the examination of this preparation (and
other additional ones) showed
that the rotifers are colored intensely enough, taking the dye in a
diffuse manner in its tissues (it does not color the nuclei
providing a sound differentiation of internal organization. The
preparations look very similar to those of the Myers collection,
with carmine, although the color is more orange.
4 – Nematodes
Physaloptera squamata nematodes parasitic on the Cancun small lizard of the walls ( Anolis sp.) fixed in 70% alcohol, were placed in a solution of 0.5% eosin for 5 minutes. The nematodes took a strong color. They were then passed to 15% glycerin, and after 20 minutes they were mounted in 30% glycerin.
Results : Contrary to what is recommended in the bibliography, where staining nematodes is considered very difficult, and where it is recommended to puncture the cuticle to allow a better absorption of the dye, the eosin almost immediately colored even very intensely the specimens of Physaloptera squamata used.
The contrast filters can be used precisely as with fresh material, although they give other effects of color and they provide interesting additional possibilities for effects of oblique illumination.
Important details, related to these experiments are: 1) to use an aqueous fixer, 70% alcohol deformed the animals a little; 2) to wash out with water and to probably pass to a very diluted eosin (0.1 or 0.2% would be enough); 3) to supervise staining, transferring often to clean water, and to stop it, washing in 5% acetic acid (vinegar); 4) to eliminate the acetic acid by washing in pure water; 5) to pass to 10% glycerin and to let concentrate until approx. 50%; 5) to work up out of Glycerin, mounting in Glycerin Jelly or in PVA-G.
If one uses a more concentrated staining solution and over colors the specimens, it is possible to regress staining by fading it with 0.1% hydrochloric acid. The reversal must be supervised at X40 since it delays only a few minutes. Stop the regression by washing with abundant water. Mount as before.
In the case of the vegetable epidermal staining, eosin offers an important advantage over the examination of fresh products, without coloring. Nuclei, nucleoli, chloroplasts and cytoplasm are better differentiated. The aspect is also more attractive and if the materials were fixed beforehand (with AFA or GALA) and one treats them by adequate methods, permanent preparations can be made.
The staining and glycerin mounting of rotifers definitely gave better results than those obtained by mounting unstained individuals.
For the nematodes mounted in glycerin after clarifying them beforehand, some tissues are distinguished by a greater absorption of the dye or a different shade of the color. Body organs are thus clearly differentiated.
discs, and the Rheinberg method especially, made it possible to
improve even more the topographic study while offering very interesting
possibilities of color and oblique illumination.
In Physaloptera the analysis of the structure of the caudal wings, the nervous system and the excretory system, was much better with staining, than in the individual without color. Also some glandular structures are better seen.
However in the case of small nematodes, generally a preparation mounted in glycerin without any stains (the standard method of nematologists) has also a good definition of organography and it also allows an intelligent use of the contrast discs.
Followed-up by image processing in a photo-processor can offer in some cases and for some details even better results than staining.
The preceding tests indicate that it can be profitable to try additional groups of invertebrates (Temnocephalida, Digenea, Monogenea, Cestodaria, Turbellaria, Micro-oligochaeta, etc.) since eosin is a cheap stain which is obtained rather easily. It could be an economic substitute of the traditional carmine …. at least for amateurs.
PERMANENCE : Four months after the tests, the stain is maintained with the same intensity and still better definition than when they were freshly made.
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