A new method for fixation and coloration of the
trans-vacuolar bands in cells of the onion epidermis
Photographing the streaming with the traditional light microscope
Walter Dioni Cancún, México
To round off this series without having to redirect the reader too often to earlier parts for the original images, I reuse here some media already presented. Unless otherwise stated, the images were captured with a Logitech QuickCam Pro 9000 webcam, adapted for photomicrography according to my articles published in MICSCAPE from February to April, 2010
In the eight part series "The inner epidermis of the onion bulb cataphylls" (Micscape, March to October 2011) I reviewed various methods at the amateur level and using a classic light microscope to investigate the structure of onion cells.
In the last article I identified a simple, cheap fixative based on iodine plus acetic acid (iodine-acetic/10) which produced excellent results, showing in detail, in preparations fixed and stained by iodine, not only the well known classic structure (cell wall, cytosol, nucleus), but even the trans-vacuolar bands seen in live video footage of physiologically active cells.
I haven't found on the Web, published examples of images similar to those which I included in the October article. This new article presents a method that has given me good results by staining with a more traditional stain rather than with iodine but still fixing with “lugol-acetic/10”.
THE ONION LIVE CELL
I include here a link to the video, and some video stills, which show the structures that we try to fix and stain.
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.)
1 - 2. Two stills from the video. Parameters used for the video were: camera Logitech 9000, video set to HD-960x720px, 100x OI objective. 10x eyepiece. Simple immersion, Circular Oblique Lighting (COL). This stills were captured from the video using Avidemux 4.0.
3 - This “in vivo” still was taken with oblique illumination at 1000x, creating a small panorama using PhotoPaint with 3 assembled pictures (the original has 600x 2400 px)
As shown below, the fixation with “acetic acid acidified Lugol” lets you capture stills of onion epidermis showing very similar morphological details with the images of living cells taken with a DIC microscope (discounting the different quality of the optics and special contrast technique).
FIXATION WITH “lugol-acid/10”
The “Lugol-acid/10” is prepared according to the following formula:
KI 1.0 g
Water (distilled) 99.0 ml
I2 0.5 g
Acetic acid 1.0 ml
Dissolve the KI in 80 ml of warm distilled water, add the iodine and fully dissolve it. Don’t reverse the order. Add acetic and complete the volume to 100ml. But I have a ten fold solution that I dilute at will, when I need it.
I think that the images previously obtained are the result of the speed of action of the iodine, which avoids the disintegration of the tonoplast, i.e. the membrane that defines the cell vacuole and wraps the cytoplasmic bands where the “streaming” moves, while maintaining true cell structure and size of the granulations flowing in the cytosol, and therefore the three-dimensional structure. Or is there another explanation?
4 - 5 - These two images clearly shows the three-dimensional character retained by the fixation, with the nucleus supported at the intersection of 3 trans-vacuolar bands. Secondary bands "sink" into the "vacuolar medium. The cell walls are of course hazy. The second image has been edited to eliminate the yellow background of the vacuolar medium.
But, apart from the fact that the uniform yellow stain of the preparations is not very pleasant, although it is entirely acceptable for educational demonstrations, iodine does not provide lasting preparations; even for the medium term. On the one hand this is because iodine is bleached by light and moreover, if you try to make a permanent mount, the alcohols that are generally used for this purpose dissolve it and discolor the tissue.
I thought that decoloration could be useful, as alcohol can be in itself a fixative (a post fixative in this case), it would not damage the cytosol already set by the iodine, and once this was washed with alcohol, the cell would maintain its structure and could be coloured in a traditional form.
I therefore tried the following protocol, using as a colorant the brilliant blue # 1 (Blue 1, a dye normally used for food) whose use I justified in the article:
Fix with “Lugol-acid/10”: immerse for only a few seconds**. Wash with 30% alcohol for just a few seconds until the epidermis is discolored. Wash in water for a minute. Immerse in “brilliant blue # 1” for 20 seconds. Wash in distilled water, Mount in water on a slide, and Cover.
**the times I give here are only indicative. The onion bulb you use, the dye batch, etc. are important factors so each user needs to optimise their own protocol.
The best microscopy images will be seen with the 40x and 100x objective. An unexpected and interesting detail was that Blue 1 acted with great speed and intensity, producing a blue tone more profound than that produced without the prior action of iodine. Compare these times with those used with other fixatives throughout this series.
Note: Given the speed of handling, I also successfully tried to cut the onion skin (usually a square of 1.5 x 1.5 cm) from its supports (see the April article where I explain my way of manipulating the epidermis) placing it on the slide, and applying reagents and washes with drippers. Handling was easy with the only requirement to retain the epidermis with a mounted needle when tipping the slide to drain fluids.
Use of Mordants
In the dyeing of textiles (from which techniques were derived many of the earliest histological staining methods) the action of “mordants” is well used. These are products that chemically modify the substrate, giving it a greater affinity for the dyes that are used later.
Not all biological substances accept a mordant but this can be a useful feature. If you check on the Web, you will find that an iodine mordant is the basis for the well known technique of bacteriological coloration (Gram stain) due to Dr. H.G.J. Gram, which was developed in 1884 (Wikipedia). Nothing is new! Old microscopists knew 'everything'!
In bacteria, the characteristics of their cell membranes divide them into two groups. When a smear of bacteria are stained with Crystal-Violet, all are stained blue. If they are treated now with iodine, and later the stain is washed with alcohol, bacteria that accepts the mordant retain the dye; the so-called Gram+ (or Gram positive). Cells called gram-negative (Gram-) are those whose membranes are not sensitized with iodine and lose the dye. It will be colorless or tinted in pink by a contrast dye (Safranin was originally used) as in the following picture:
6 - Image taken from the manual of laboratory practice
The mordant action of other substances (e.g. the currently prohibited mercury dichloride HgCl2) was what turned many of the histological fixatives normally used, to those with histological colorations that textbooks qualified as "brilliant".
The difference in tone, the intensity, and speed of the Brilliant Blue # 1 staining,when using iodine, indicates that this is acting as a mordant, (compare with the times I needed to apply after other fixatives used in the previous articles.)
7 - 9 - Observe the delicate fixation of the cytosol and the granules and vesicles, that “Lugol-acetic/10” produces, which shows images similar to those seen when watching the streaming in the living cell; as well as the deep color obtained, and the accentuated three-dimensional appearance. (Each picture is a 3 images stack combined with CombineZ.)
10. Further evidence of the fine fixation, strong color, and three-dimensional differentiation which is obtained with the technique. A single image. The delicate filament was contained entirely within the depth of focus of the 100x objective, suspended in the fluid of the central vacuole.
This form of preparation produces a very complete cell fixation of the cytoplasm. The nucleus is too dark, but the cytoplasm shows a very good structure. Note first, the layer of cytosol along the cell wall, full of colored granules, the medium filled central vacuole of course, and the trans-vacuolar trabeculae (bands, strands) of cytoplasm, shown as other much more elaborate and costly techniques (e.g. DIC or epifluorescence. e.g.) would show them.
The physiological status of the different cells of the same skin produces multiple different images. In the same sample there are cells in repose, very active cells, and all the intermediate grades. And you could have a skin that has only sluggish cells. Only a few actually show some more or less complete and active cytoplasmic streaming images that merit a video study.
For those who try to watch live streaming, this short excerpt from instructions to a college practical lesson can help them to be patient:
"... and then by using the 40x objective examine individual cells until one showing cytoplasmic streaming is evident. (bold is mine)
Pg 17, and see fig 17 from the book 'Teaching plant anatomy through creative laboratory exercises' by R. Larry Peterson, Carol Anne Peterson, Lewis H. Melville (I cite this from a Google Books excerpt)"
So far my experience is that few live samples can provide a three-dimensional photographic record that is demonstrative. It is symptomatic that there are none on the Web, and I imagine that neither in publications nor papers, there are images similar to those displayed above.
The images below are not so effective in showing the features as the above examples, from a white onion, two months later.
11 - Image at 400x. The next images are 1000x images in oil inmersion.
12 – 13 - The granules are especially large and a strange angular shape. I used for these pictures a dye which I denominate VB1. (It is Blue 1 at a 1% solution with 1% of acetic acid. It has a better durability. The pure B1 solution is often infected by huge quantities of a large bacterium, or by the mould Aspergillus sp.)
I have not seen examples of living onion cells imaged with DIC that show these features. This is unlike Tradescantia sp., which as the image taken by Franz Neidl shows and presented in the previous article, (which I still consider to be the best of its genre), this technique can display the cellular dynamics which we discuss here. Even with the difficulties involved in selection and mounting of the stamen hairs, Tradescantia has provided for at least the last 150 years, the best documentary evidence of cytoplasmic streaming. There's also a beautiful series of images with DIC, which shows mitosis in living plant cells. This site is an essential visit:
The onion is not so lucky. There are only (on YouTube) five beautiful videos, in phase contrast, which I link to here showing “streaming” similar to that seen in my video above:
And another one filmed in “brightfield”, which is very similar to mine, and is dated two years earlier.
Although much scientific attention has been paid to the epidermis of the onion cataphyll, published studies of the morphological and physiological characteristics of the cells and their components, using the light microscope with phase contrast, or DIC, or even through the transmission electron microscope, do not show capture features of cytoplasmic streaming. There are also few examples from the amateur microscopy community.
15 - Note in this picture the large size of the vesicles and their different colours
Comments to the author,
, are welcomed.
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