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

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.

 How many of these images taken with a regular light microscope, shows details which provide three-dimensional information that corresponds to the structure which the fluorescent image we included in the previous article shows?

 The closest snapshot I know of is a unique commercial picture you can reach at this link*, which shows me for the first time what a DIC microscope may show on a live onion cell.

http://www.sciencephoto.com/image/10537/530wm/B0600062-Cytoplasmic_streaming_in_onion_cells-SPL.jpg

That’s all. Only one snapshot of the dynamic features of an onion cell.

 No other published fixed image deals with this. And in stating that, in any case, I have used the expression an onion cell, I didn't say the cells, because there are many pictures of streaming but from tobacco cultured cells.

 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.

 Therefore, are there usually invisible with the light microscope, (especially at the amateur level) the "roads" of actin through which mitochondria flows? Are permanently present the trans-vacuolar stripes, or are they temporary? Is streaming in the onion cell a permanent phenomenon, or does it depend on any special physiological state of the cell?

 Supporting  pertinent information is only shown at 4 or 5 YouTube videos, displaying the flow of cytosol granules in a live cell, captured with great phase contrast equipment.

http://www.youtube.com/watch?v=f1xlZQJz3kw&NR=1 (desde 1.05 min en adelante)**

http://www.youtube.com/watch?v=wkbijKyM4eQ (Riveal contrast)**

http://www.youtube.com/watch?v=VXbQpRpUDmQ&NR=1*

http://www.youtube.com/watch?v=cLgHnJEmoI0

 Those videos show that live streaming, and active trans-vacuolar cytosol bands, can be shot with a video camera on a microscope that allows an appropriate contrast between the cytoplasm and other elements of the cell (of course the professional tools would be a phase contrast or a differential interference microscope)

 The best record I know of on the Internet of the activity of a living cell of onion, without phase microscopy, is contained in two small videos published two years ago, by someone so fortunate as I am (soon you will see why I say it), you can enjoy at this site:

 http://beyondthehumaneye.blogspot.com/2009/04/inner-workings-of-onion.html

 Why are they not normally seen in image captures of fixed and stained preparations that correspond to the images those videos display?

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 think that I'm talking about the unattainable dream pursued by the histologists for over 150 years!

 If that fixative had been already implemented we would have many images showing snapshots of the streaming in cells of onion. It’s a beautiful scenario. So...

 The experienced cytologists, who could use the best reagents, (those more expensive and difficult to use) placed their confidence in use of osmic acid to reveal the "real" structure of the cell. And this is still the most used fixative in ultra-structure research with the TEM (Transmission Electron Microscope). Its properties can be investigated easily on the Net; and its price noted! in the catalogues of suppliers. Outside research institutions nobody uses it now.

 Another fixative of confidence, especially for botanists, was chromic acid, in various combinations with other reagents also aggressive, and expensive.

 But even so, there is no (or perhaps I do not know) old images showing the trans-vacuolar strips in the onion cells fixed with the usual histological fixatives.

 All drawings I have been able to collect, used live streaming in the well known for many decades "tradescantia" stamen filaments *, or hairs of some plants, or cells of some algae, or the live cyclosis of chloroplasts in the cells of aquatic flowering plants (Elodea and similar, or Vallisneria).

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 seen as bands of cytoplasm inside of which are granules moving in a unidirectional flow.

 The bands are full of cytosol and are bounded by a membrane, which is part of the membrane which lines the vacuole, and is called tonoplast..

 With all the crude fixatives which I have used (of course I have not used the best professional fixatives), with the possible exception of Iodine and the Clarke, it’s clear thattthe strand’s tonoplast is gone. Although, as could be seen in many images throughout these articles, the location, and in some ways the morphology, of the cytosol which composed them, can be recognized by the ordered disposition of stained granulation patches. An example of a more or less clear is image 6c of the preceding article.

 In most botanical drawings the granulations are drawn, and not the limiting membrane of the strands, which implies that it was not taken into account conceptually.

 This good drawing (from 1890) of a cell in a staminal hair of “tradescantia” shows what I've stated (fig 1). 

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.)

taken from http://advancedlab.org/mediawiki/index.php/Brownian_Motion_in_Cells

 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.

 Even the Clarke’s does not seem to adequately comply with this task. I need a more competent reagent.

 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.

 This aldehyde, with subsequent integration of the glutaraldehyde, currently dominates the professional histological techniques.

 But both are closed for most of the amateurs, because of carcinogenic action of formalin, and because glutaraldehyde is very expensive, difficult to obtain, and especially difficult to use, if you do so correctly. To obtain a good cytological fixation it should be used together with complex stabilizing buffers. So these are used mainly to get the best images in electron microscopy, even if osmic acid continues to be the superstar.

 A poor relative, very cheap, Iodine, apparently demonstrated some qualities long times ago. The earliest histologists used it, as well as the osmic acid, to fix microorganisms using its vapours. The technique was (with osmic) to invert a coverslip with a hanging water drop containing the microorganism for some seconds over the mouth of a flask with the reagent. Iodine was used as crystals that are highly volatile and also produce fumes. But, as these are very heavy, the flask was tilted over the drop, and the fumes left to flow downward to reach the water. Not very easy or very safe.

 Then some researchers proposed Iodine as a fixer for protozoa and other microscopic creatures, using solutions of Iodine, and the appearance of one liquid formula due to Lugol provided a fixative of medium-quality for microalgae and plankton.

 Duly acetified it’s still used as a fixative of choice for plankton, but with some reluctance, because by their acidity it attacks many microorganisms which have calcified structures. It has then been proposed to fix and precipitate the organisms with Acetified Lugol, and then post fix (within a short time, at most a few days) with formaldehyde or another neutral fixative.

 Of course you know that the other preserved utility of iodine in today’s microscopy is colouring (prepared as "tincture of iodine") the epidermis of onion in elementary biology classes, and also, used in much more diluted solutions, to colour blue the starch, making it recognizable in plant cells.

 Formaldehyde, glutaraldehyde, and mercury dichloride, picric acid, and potassium dichromate based-fixatives, dominate the scene since the beginning of the 20th century until a couple of decades ago, and relegated the iodine into oblivion.

 The information contained in pictures 15 and 18 of the first article of this series made me think that perhaps the iodine could be used to fix the trabeculae..

 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.

 If successful, Acetic Lugol could save many manipulations which the use of “tincture of iodine” requires, (see the first article, you need one simple wet mount to have ready your slide), but, above all, I hoped that possibly it can also reveal a hint of the strand structure of the cytoplasm. An accepted formula is:

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.

 **http://www.liv.ac.uk/ciliate/methods.htm

 To fix and stain onion cells is a novel task for Acetified Lugol.

 A first test, made with a couple of drops of the formula, showed the effectiveness of the fixative, but the staining was extremely strong, preventing any hint of the structure of the nucleus, which only appeared as a dark spot. A less than half concentration gives equally a very dark tinting.

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)

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

Water                         100

Iodine                         0.5

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.

See  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2107641/pdf/633.pdf, (fig. 13)

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 ACDSee

“tradescantia” staminal hair

http://www.photomacrography.net/forum/viewtopic.php?p=46295&highlight=tradescantia#46295

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

http://www.youtube.com/watch?v=0pBQU08kVcg

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”