The inner epidermis of the onion bulb cataphylls
(the onion skin)
Easy and not so easy methods to work with
Walter Dioni - Cancún, México
7) – Acetified Lugol –
Iodine alcohol, Blue 1
7) – Acetified Lugol – Iodine alcohol, Blue 1
Still images of cytosol streaming in epidermis of onion cell
Continued from part 6 – Fixing with Clarke’s fixative - Staining with Blue 1, and Eosin
I’m well aware that all samples of onion skin that could be taken will show the same arrangement of polygonal cells, with a cellulosic wall, cytoplasm, vacuoles and nuclei. In this series I have given extensive testimony of this, and virtually all of the images (whether drawings or pictures) that occur on the Internet show the same.
That’s all. Only one snapshot of the dynamic features of an onion cell.
Even a very good image published in the Forum photomacrography.net (Photography Through the Microscope) http://www.photomacrography.net/forum/viewtopic.php?t=12545&highlight=onion) using a DIC microscope, shows a compelling image of a living cell, but with little more information than that which one of my coloured cells at 400 or 1000x shows.
http://www.youtube.com/watch?v=f1xlZQJz3kw&NR=1 (desde 1.05 min en adelante)**
http://www.youtube.com/watch?v=wkbijKyM4eQ (Riveal contrast)**
Certainly to get fixed epidermis that shows it would require an ideal fixative which sets immediately, and unaltered, the cell structure, and which preserves it unchanged in time, and without adding artifacts that did not exist in the live cell.
I do not know pictures of preparations fixed and coloured, showing stills from the streaming in onion cell.
It is very clear for me that streaming was and is
regarded as what it really is: a dynamic phenomenon. This, as such, only could
be shown, in light microscopy, recorded on video.
It is very clear for me that streaming was and is regarded as what it really is: a dynamic phenomenon. This, as such, only could be shown, in light microscopy, recorded on video.
And also this better one, of the same time, from a Chelidonium (fig 2)
A modern image of the onion cell structure, that synthesizes the knowledge gathered through many years, and with the use of many techniques, including TEM, the live observations, and biochemical analysis is the following:
Fig 3.- (Source: N. S. Allen and D. T. Brown, 1988. Dynamics of the Endoplasmic Reticulum in living onion epidermal cells in relation to microtubules, microfilaments, and intracellular particle movement. Cell Motility and the Cytoskeleton 10:153-163 – Wiley-Liss Inc.)
Did you recognize the polygonal Endoplasmic Reticulum shown by eosin in Clarke’s fixed cells in the previous article, and the trans-vacuolar strands also shown by eosin, and also by iodine in the first article of this series?
If tonoplast is delimited, and allows therefore identifying the limits of the vacuole and the trabeculae, clearly the problem is to fix the cytosol while maintaining the tonoplast.
In normal histology, osmic acid and chromic acid were abandoned, except for special cases, at the end of the 19th century, when the low priced formaldehyde showed its superb qualities as a fixative.
COMBINED FIXING AND STAINING USING “Acetified Lugol”
I decided to try the Lugol fixative, acidified with acetic** because its formula makes it a fixative and a dye at the same time. It incorporates acetic acid ... normally deemed a good nuclear fixative!, and iodine, which as we saw in the first article of this series fixes the cytoplasm, and colour of both nucleus and cytoplasm.
Potassium Iodide 10 g
Water 100 ml
Iodine 5 g
Acetic acid 10 ml
Dissolve the KI in the warm water. Add the Iodine and dissolve completely. Do not reverse this order. Incorporate the acetic acid.
This formula is accepted as a very good fixative for plankton samples**. Stains and impregnate the organisms, and being iodine, a heavy metal (with a big molecular weight) facilitates the sedimentation and concentration of the stained organisms in the sample.
Fig. 4 - Contrast and intensity were severely diminished to make this picture acceptable
My expectations were fulfilled.
Setting aside the strong yellow colour, the cytoplasm images produced by the Acetified Lugol are, still now, the nearest to the live image, and even better than the ones recorded using the iodine tincture in the first part of this series, which I rated high. Not so the nuclear images. They are displayed as plump and dark discs, with none of the typical surface grooves the nucleii have. The grooves are a real features of nuclei. Almost all of the fixatives reported in the previous articles show this trait. (And see http://www.plantcell.org/content/12/12/2425.full)What is disappointing is, that as before, that although there are good fields without bubbles, there are many that have them (see the 4x total field), and, for now, the dark colouration of nuclei.
Fig. 5 - 4x total, Acetic-Lugol/10
I had to reduce a lot the concentration of the reagent to obtain acceptable results.
I made a test with a tenfold dilution:
Potassium Iodide 1
Acetic acid 1
There is no reason for a pharmacist not to produce this formula. No dangerous ingredients, not very expensive also. It is even an aqueous solution.
I also think that this formula merits a name of its own. I propose Acetic-Lugol/10
Fixation and staining were immediate. No other technique used so far produces a faster fixing, and a better preservation of which we know by the above videos is the living structure of the cytoplasm. Many well bounded cytosol strands are extended without distortion, full of granules, which not being coloured blue with iodine, could be leucoplasts (plastids with no starch) or mitochondria (people says that mitochondria are destroyed by acetic acid, but we have here a low concentration...) or Golgi bodies (plant cells has many of this, but are also a difficult target), ... or simply sphaerosomes or “vesicles”, a collective name applied by many writers for not to presume the nature of the granules.
With careful study, the refractive and dark granules allows the easy identification of the parietal layer of cytosol, and their accumulation around the nucleus, and in the angles of the cells. Figs.. 5-10
Fig. 6 – Acetic-Lugol/10, 10x obj. See the numerous cytoplasmic strands even at this low magnification
Fig. 7 - CombineZP – Acetic-Lugol/10, 40x obj., 3 images stacked
Fig. 8 – Acetic-Lugol/10, 40x obj., 3 images stacked in CombineZP
Fig. 9 – This is a detail of picture 7. See the easily identified delicate cytosol strips, the granulations, and the evident cytosol parietal layer.
Nuclei are as usual, in various shapes, from oval to strictly circular, well coloured, granular, with very distinct nucleoli, something darker and refractive.
Nucleoli are the place of formation of ribosomal RNA, and, according to some graphic representations, it is rolled as a dense ball of filaments. A careful visual observation at 1000x shows that they seem wrapped in a membrane, some with one or more portions condensed inside, and others with what is clearly a central clear point (is it a vacuole?) (not to be confused with a refraction glare which depends on the focusing). It is interesting that these nucleolar details can be identified at only 1000x because its description was made from Transmission Electronic Microscope images.
Fig. 10 – 40x obj., Acetic-Lugol/10, 4 images stacked in CombineZP.
Canon A75. Handheld camera
Fig. 11 - 100xOI obj – 3 images, combined with CombineZP and cropped from a 3Mpx image. Please! Remember that camera (Canon Powershot A75 in this case) was handheld.
Figs. 12 and 13 – Acetic-Lugol/10, yellow background eliminated with ACDSeeA DIC microscope can resolve the cytosol strands with excellence, and while living. I will show you this magnificent demonstration, made by Franz Neidl, an outstanding photomicrographist, of streaming strands in a
“tradescantia” staminal hair
Fig. 14 - Many thanks Franz for the beautiful image and kind permission
Compare with the above images (fig..6 to 13) and the drawings of figs. 1 and 2
Therefore my preparations, fixed and coloured with the fast ACETIFIED LUGOL, really correspond to the freezing, as a still image, of an instant in the dynamics of the living onion cell.
Some days after, I had the rare (at least for me) opportunity, that still now I can not repeat at will, of recording this video which showed me the magnificence of the live streaming. It was an impressive experience.
Streaming in an onion cell
(Editor's note: The above is a YouTube version for maximum cross platform compatibility. The author's master video is a larger video box, of higher quality and can be downloaded here for offline viewing and best viewed full screen, 13 Mbyte avi file. Use the right mouse button to click blue video link and save file locally.)
Since that day my wife is reluctant to use onions in ours meals. They are alive, she says, refusing to throw them in the boiling oil! And I couldn’t teach her any technique to euthanize them!
Fig 15 (a,b,c,d) 100xOI obj. Oil inmersión, Circular Oblique Lighting. Logitech Quick Cam Pro 9000. HD-960x720 video configuration – BW. Selected images from the video
Acetic-Lugol/10 could be, then, a fixative of choice for those that want to show with excellent detail, at 100x, 400x, or 1000x, the three-dimensional structure of an onion epidermic cell. Pity! ...The 4x objective image will be inevitably reveal many bubbles.
I hope that any amateur with a compound microscope equipped with a condenser and a diaphragm, capable of 400x, or better 1000x magnification, could see the live streaming using only a simple oblique light stop, or a circular oblique light stop, in the filter holder of his microscope. Please, take your time! Make a fine adjustment of your lighting before to start your quest. And be conscious that not many epidermis are in the mood. See this video that show an uncollaborative cell
it shows only Brownian movement of the “vesicles”, no streaming.
Which could be the clues for the good behaviour of the Acetic Lugol’s, diluted to 1/10 of its original concentration?
The high concentration of iodine in the original formula is clearly responsible for the dark staining of nuclei. The diluted solution is feebler and gives a better legible image. But, the iodine, itself, as a pharmaceutical tincture, and even diluted, gives not a so faithful representation of our “live patron” as the diluted Acetic Lugol did (see the first article of the series).
A high concentration of acetic (in the undiluted Acetic Lugol it is 10%) has shown in the former trials that it causes a dense precipitation of the cytoplasm very different from the live image, and that it destroys mitochondria and most “vesicles”. And even reduced to one percent, like it is in this particular formula, the acetic acid alone (see part 5) showed a very different behaviour than the diluted Acetic Lugol’s.
But... the best fixatives formulae are not of course simple, solitary reagents (they are a formula because of this, aren't they?). They are a mixture of reagents, each of which brings its special mode of action. They are synergistic agents.
And possibly is an effect of this type which is responsible for the last good images of some onion skin cells, fixed and stained with the 0.1% Acetified Lugol’s solution. (Acetic-Lugol/10)
We have seen the same phenomenon using Ethylic and Acetic in the Clarke’s fixative. Each component completes the action of the other.
Well! Acetic-Lugol/10 is excellent. There are other safe fixatives which merit to be tried on the “onion skin”, but this one has proved to work.
At the end, it's probable that you think, correctly, that I can live with a few air bubbles spoiling my images taken with the 4x objective! I give up!
But don’t miss the next article. As Scheherazade used to say: “there is another tale”
Comments to the author,
, are welcomed.
Published in the October 2011 edition of Micscape.
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