The inner epidermis of the onion bulb cataphylls
(the onion skin)
Easy and not so easy methods to work with
Walter Dioni - Cancún, México
6) – fixing with Clarke’s fixative - Staining with Blue 1, and Eosin
Follows from part 5 – Fixing with Alcohols
We have reviewed (in the first 5 articles) some physical factors and some chemicals, used alone, as fixatives. Of course, many more products were tested, by hundreds of histologists, through decades of histological practice. It is clear that the temptation to mix, and try to combine the characteristics of the different reagents, to improve the fixation of the investigated tissues, must have occurred very early, if I judge by my own tendency to try just now.
The best reagents needed for success were selected over a long period. Even now new reagents are incorporated to meet the requirements of new targets. And many others had been discarded under the pressure of novelties, availability, prices, or, as in the last decades, social considerations.
I think it might be instructive to review some of the earlier attempts, to understand the efforts of our earlier predecessors.
And we can make these, with little effort, with the reagents at the reach of an amateur.
Allow me a small recapitulation: I have tested boiling water, and 60ºC water, as fixatives for the epidermis of the onion cataphyll (first article), later I followed the “Ceviche’s track”, assessing citric acid (2nd article), and, after this, the well known acetic acid.(3rd article) I accepted the 60ºC water as a good fixative, and the citric acid as a good approximation. But I rejected the boiling water, and only partially accepted the acetic acid (as a nuclear fixative), used alone in the rough conditions of these tests.
96% Alcohol (4th article) has shown a capacity to fix and make evident the cytoplasm, better than the acids do.
I also tested, and approved, at least for my use, the Blue 1 food dye, as a dye for the onion cells, because it gives fairly good images of the cytoplasm and very good pictures of the nuclei. (May 2011 Micscape )
At first sight (after the tests I have presented formerly) what should seem disappointing, because it stands for a long time as an histologists favourite ingredient, is the fact that alcohol, especially 96% alcohol, as shown in the previous article, has given a better representation of the cell as a whole, than acetic acid, even weakly shows.
After so many years of use by so many histologists, there is a long time that a consensus was attained, and is now totally accepted that the acetic acid, used alone, is not a good general fixative.
It seems that it fixes cytoplasm very badly, especially in animal samples; it swells cells, and dissolves totally or partially, some components, even RNA (which includes not only the nucleoli, but also many cytoplasmic granules and the mitochondria), and you can only use and recommend it as a mitotic nucleus fixative, a fixative for DNA. (This is why the derogatory affirmation of Bolles Lee, did you remember? If not... read the former articles.)
But, in itself, this specialization is good, and this is why (as they will be seen by my two readers, whether they followed me and reviewed the formulae as I recommended), acetic is part of most of the best histological fixatives of general use.
By the way, without to go any further: one of the histological fixatives preferred by modern botanists for general use (when they discard formaldehyde) is what many call Carnoy 1, and which others (also many), who respect the chronology and priorities, and especially the zoologists and human histologists, call Clarke’s Fixative (1851). The year in brackets indicates the date on which the author published the formula. This is the citation of the original publication:
CLARKE, J. L., 1851. 'Researches into the structure of the spinal cord.' Phil. Trans., 141:607
Carnoy, used and published in 1886 (35 years later!), the formula of the Clarke’s (alcohol plus acetic acid in a 3:1 ratio), reinventing it, or, most probably, copying it without reporting the origin), and in 1887 he published the formula that must be properly called Carnoy (which others call Carnoy 2), which includes alcohol:chloroform:acetic acid (in the proportions 6:3:1) and is a most used and recommended fixative for important tasks.
Farmer and Shove (1905) used for studies of mitosis, two formulas of Alcohol-acetic, which applying the nomenclature I applied before, should be called alcohol: acetic 16:1 and alcohol: acetic 2:1 according to the formulas provided by Gray, 1954. (I have had no access to the original article, and I depend on Gray (1954) not registering a 3:1 formulation)
Who knows why, and who was the first to be wrong, but some botanists started to name the 3:1 as Farmer (forgetting Shove, and the real formulae implied) while some others call it Carnoy 1... and to use, with those wrong names, the Clarke’s formula... and so they do enthusiastically to this day in books and scientific papers. A short review on your browsers, and reading a few pages, will confirm this. My browser denounces over 300 articles that cite it when I search for (don’t forget quotes) “Clarke’s fixative”, 470 citations of “Farmer’s fixative”, 4800 when I search for “3-1 fixative” but 198,000 when searching for “Carnoy’s 3-1”. (Tell me, who said “Crime does not pay”?)
And at last, what is the Clarke?
Clarke’s Fixative - (aka Farmer, aka Carnoy 1, aka 3:1) was introduced 160 years ago, in 1851, as a fixative for parts of the nervous system. Old as it is, simple as it is, it is good enough to be currently used professionally in the fixing of animal and plant histological pieces, especially for the study of cell division by the mechanisms of mitosis and meiosis: it fixes very well the chromatin.
It is also interesting that it was the first published formula for a histological Fixative. Its formula is
95 or 96% Alcohol (not less) 3 parts 75%
Acetic acid, pure 1 part 25%
The "not less than" indicates that really the original formula of Clarke used absolute alcohol. But, if it is now difficult to find 96% alcohol, 100% alcohol is currently unreachable, for its price, and is indeed relatively useless for an amateur. Just open the bottle and the ambient humidity starts to contaminate absolute alcohol, which is highly hygroscopic. I could indicate ways to keep dry the absolute alcohol, or even how to prepare it from 96% alcohol, but they are not very easy, and for normal amateur applications, the 4% of water that has 96% alcohol does not bother at all. For this same reason, many professionals, reasonably, use this formula prepared with alcohol 96. (you know... 96% alcohol is (almost) 96 ml of absolute alcohol + (almost) 4 ml water for every 100ml). This is a well known azeotrope mixture, and when alcohol is concentrated through distillation, alcohol cannot be concentrated purer than this.
Post-treatment of pieces fixed in an alcoholic fixative
As the fixative uses alcohol with a concentration of 96%, when I withdraw the epidermis off the fixative I wash it in 96% alcohol (a couple of minutes) and then in alcohol 70% (another two minutes). What I try is not to distort the fixed cells with strong and sudden changes of alcohol concentration... and, also, not to carry over acetic acid.
Fig 1 - This is my series: Clarke’s (with lid), 96% alcohol (with lid), 70% alcohol, collector flask (with tight lid)
It is an important advantage of working with 70% alcohol. Because of its water proportion it doesn’t make brittle the epidermis as 96% can do in the long term, and doesn’t allow bacterial development, which 96% can do.. It seems a paradox, but those differences of action are related to a different mode of interaction between the two alcohol’s degrees and the membrane of bacterial cells. 70% alcohol is a better bactericide, and so a better preservative.
It is also a norm that dyes should be in the same alcohol graduation than the piece to be stained.
I mount in water, as I do all over this series. See these example pictures.
Fig. 3 – Clarke – 4x obj
Fig 4 – Clarke – Blue 1, 10x obj.
Fig. 5 – Clarke – Blue 1 – 40x obj
Most notable at this magnification, is the inclusion in the mostly homogeneous cytosol of numerous dark granules, which are identified at a higher magnification as small dark spheres, different and distinct from the finer grains you see in the cytoplasm bands. Several preparations, from different onions, show the same.
By its disposition and size it's a big temptation to think they are "Golgi bodies". But without other techniques at hand to certify this I only can dream about it. They are some fluorescent images that induce my dream, but...
Chromatin is finely granular, nucleoli look good. In a couple of images, the colour of the nucleoli, verified by direct observation, tends to a dark reddish tint. In the cytoplasm, cytoplasm bands are confirmed. Nuclei, however, appear as discs in which they are noticed with difficulty the grooves which we know well from other fixatives, and which are a normal characteristic in the onion, as confirmed by this work http://www.plantcell.org/content/12/12/2425.full
It seems that eosin is more or less easily at the reach of amateurs. I buy it in a drugstore at Durango, and it seems to be sold in Europe as a Pharmaceutical 2% solution as a disinfectant.
Fig. 8 - Fixation with Clarke and staining with eosin shows, in some onion epidermis, a detailed cytoplasmic structure. In picture above (taken through the 40x objective), we are lucky that there are two cells focused on different levels of depth. The lower, to the left of picture, shows the cytosol layer near the cell wall, forming a fine mesh of delicate trabeculae. The other, located immediately above, displays a classic median optical cut, with bands or trabeculae that cross the vacuole and bind the nucleus to the parietal cytosol. An interesting detail that we saw earlier in the test fixation with 96% alcohol, is the accumulation of cytosol and granules in cell angles.
However who will undertake the work of reviewing them, will often see that many times before, I also presented evidence showing a reticulated or trabecular structure. Fixation with iodine (see 1st. part) and fixing with alcohol 96 (see. 4th part), as well as fixing with citric acid (see fig 6 and 9 of the 3rd. part) showed glimpses of what the eosin appears to confirm now in some skins, fixed with Clarke.(fig 8)
But most of the images with fixatives tested before show a rather homogeneous cytoplasm, and fine or medium granules, mostly isolated, or gathered in spots of different sizes.
Many scientists, over many years had complained about the difficulty to know if what they see in their fixed and stained preparations had something in common with the live organism they started with. Bolles Lee (“the Bible”) put it in this way:
"Copyright American Society of Plant Biologists.”
The images portray the many paths actively built by actin (a linear, filamentous protein) bound to the cellular walls, coiled around the nucleus, and crossing through the central vacuole of an "arabidopsis" cell. Physiological and chemical studies had revealed that cytoplasm granules (mitochondria, Golgi bodies, “vesicles”, and other granular materials), travel along these pathways driven by “motors” of myosin.Oops! this is a deep insight, and a modern model of the live dynamics of vegetal cells!
This is a good explanation of the phenomenon known as “Cytoplasmic Streaming” checked by kids, in school labs, from many decades ago, for example, in elementary biology courses, watching the movement of the chloroplasts in the cells of the leaves of "Elodea" or "Vallisneria".
Perhaps... Perhaps... This sounds disappointing for me.
Comments to the author,
, are welcomed.
Published in the September 2011 edition of Micscape.
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