The inner epidermis of the onion bulb cataphylls

(the onion skin)

Easy and not so easy methods to work with

 Walter  Dioni   -  Cancún, México

 4) Fixing with acetic acid

 Follows from Part 3 – fixing with citric acid, staining with Blue 1

This is an exercise on developing an understanding of the principles and basic operations in histology, using the onion skin as a substrate.  I repeat the usual warning: we treat here with seemingly similar pictures what must be carefully examined to appreciate their similitude or difference due to the different treatments to which they are subject.

 My two or three readers (well, yes ... I am always optimistic!) if they have followed this inquiry on the best methods to obtain a NO-AIR-BUBBLES onion skin preparation, know now that, as well as the 60ºC hot water, I have found that the epidermal fixing with citric acid, which I have related in the preceding article, is also a “few bubbles” creditable technique.

 Following the fertile track of “ceviche” (a Mexican food, see previous article), now I am intending to check acetic acid.

 Acetic acid surely has as long as human history, been associated with knowledge of fruit fermentation techniques.

Acetobacter aceti, the responsible bacteria, is found world wide.

 Here you can see a picture of acetobacter

http://2.bp.blogspot.com/_31EfOCktsTA/TRveJ9llhpI/AAAAAAAABhI/SMqljd163io/s1600/Essigsaeurebakterien.jpg

 Cooks learned early to use the acidified wines (vinegar) as a condiment, and it seems a fact that more than 4000 years ago, cucumbers were pickled in Mesopotamia.

 Pickles and ceviche evidence a main advantage of acetic acid. It fixes and preserves the morphology of biological entities.

 It is not a surprise therefore that in many of the better histological fixatives which I learned to use many, many years ago, acetic acid was an ingredient.

(You can review in your browser the most famous formulae as the Bouin, Bouin-Hollande, Duboscq-Brazil, Zenker, Schaudinn, SUSA.)

 But what becomes intriguing is that acetic could be also the most despised of the fixing agents. I found that, in the classic Bolles-Lee’s (The Microtomis’s Vademecum, 8th ed. -1921) talking about the ability of acetic to make apparent the components of a cell, it says:

Injurious liquids which should never be used in cytological fixation are acetic acid, chloroform and alcohol. Acetic acid is nearly the most destructive.... and its use, except where chromosomes are being studied, is rarely indicated; any worker who uses acetic acid in his fixing mixtures cannot hope to get a correct picture of any part of his cell, possibly excepting the chromosomes (not the resting nucleus).(pag. 24). Bold additions are mine.

 This is a warning which was not included in any of the seven previous editions.

 And, to make this more confusing, he himself, when he treated the fixatives, has almost two pages dedicated to acetic acid, with some of the fixative’s formulae being still used.

 So, acetic acid has two faces (cytological, histological) and both were used by histologists continuously for 160 years... and surely earlier.

 For me, this is the equivalent of the required incentive to ask ... is it good or bad? and why? and for what? And to put my onion skin to work...

 From my experience with the citric acid, I did some tests at concentrations of 10%, 5%, 2% and 1%.

The references I have, suggests that acetic is a fast fixative. So I would start with 3 minutes.

Of course I used the same treatment series I used for the citric, changing the fixative. But I add another washing step. Acetic is strong and for the moment I don’t want to change the pH of the dye.

And... I cut a new onion bulb every time I start a test. Old cataphylls could behave erratically. My wife is creating new dishes, to use so many onions!

Acetic acid 10%

I dilute 2 ml of pure acetic and complete to 20 ml. Use a hypodermic syringe to do that.

I apply the usual protocol:

Fixing 3 minutes; first washing, 4 min; second washing 4 min; staining, 1:30 min; (Ouch! It's hard to time this) washing with plenty of water, rinse, mounting in water.

I stain with my usual solution of Blue 1: 10ml distilled water, 0.6 ml blue 1

(By the way, the blue solution must also be fresh (not more than 3 days old) because, once diluted the included preservative, the solution is a good culture medium for bacteria and yeast)

Fig 1 - Acetic acid 10% – 4x objective

Fig. 2 - Acetic acid 10% – 10x objective

Fig 3 - Acetic acid 10% – 40x objective

Fig. 4 – Four nuclei - Acetic acid 10% - 100xOI objective – pictures reduced to ¼ of the 1000x image

The second was the hue of the blue, more brilliant and pure than in any of the essays I did before, even if it is not evident in the pictures.

The third was a hard fixed cytoplasm, with visible but incomplete trails, born on the nucleus and pointing away from it. At the parietal layer some holes and ruptures were noted. The parietal layer is very conspicuous.

The fourth and the more disappointing were the distorted nuclei, folded many times on themselves, and with nucleoli difficult to discern. There is nothing that shows the disc shape I am accustomed, even with their typical superficial grooves. I add the evidential images, as always, for you to see “almost first hand” the result of the experiment.

I made more slides to confirm this nasty behaviour. I add two more pictures to confirm it. They are the best I could pick.

Fig. 5 – 2 more nuclei, 10% acetic, nucleoli and superficial grooves indistinct

There is not a big difference, the colour is the same, and the type of fixation of the cytoplasm appears to be equal. The morphology of the nucleus is also similar, but in these latest images it seems more "classical".

The most important thing to notice is the difference in appearance of the nucleoli, fixed with this high concentration of acetic. In addition being difficult to differentiate, because they have almost the same colour and intensity of the chromatin, the form of them is that of an oval organelle (instead of circular), without the clear and characteristic central point (which is due to a core of central dense fibres, according to the Transmission Electronic Microscope), which we have found so far. People say that acetic dissolves RNA, and nucleoli are pretty dense masses of filaments of RNA.

In my experience 10% is not a good concentration for the acetic to be used alone on the onion epidermis.

Fixing with white vinegar

The more easily found fixative in the domestic dominion, after 60ºC temperature, is straight white vinegar.

It is present without restriction in every kitchen, probably in every country.

So I must give it a tray. My commercial white vinegar is an aqueous solution, with 5% pure acetic acid which has a pH very near to 2.4. That is just the proportion which histologists most commonly use, when using acetic acid in their fixatives, as you could see in the above quoted formulae!

If we believe in honesty of the manufacturer, the vinegar I use is a 5% acetic acid solution, with 0.02% sodium metabisulfite added (to prevent moulds, yeasts, and nematodes to develop, which, growing in my mother’s kitchen, in the “mother”** of pure wine vinegar, many, many years ago, were so interesting subjects for my first little microscope).

**For the so, so young, that has never seen the “mother of vinegar” I translate: It was a dense gelatinous mass built by the development of Acetobacter aceti, which concentrates at the base of the vinegar bottle. It was many times colonized by Turbatrix aceti (In those times also known as Anguillula aceti), the “vinegar eel”, that eats the Acetobacter. Parts of this mass were transferred by the Cook to new portions of fresh wine, as a seed to enhance the acetification.

At the following address you can find a beautiful picture of this little (2 mm) worm

http://4.bp.blogspot.com/-Ddg7WDrYv58/TZ-8Ou3D9HI/AAAAAAAAATQ/y5vYYxQMvYc/s1600/Turbatrix_aceti.jpg

Acetic 5% (vinegar)

I opened a new bottle of commercial white Vinegar, and started with the straight concentration, fixing a fresh cut cataphyll

fixing 3 minutes, first washing 4 min, second washing 4 min, staining 1:30 min, washing, rinse, mounting in water.

Fig. 6 - 4x obj. shows some bubbles

fig. 7 40x obj.

 Fig 8- 5% acetic – 100xOI obj. - Images reduced to quarter of the 1000x pictures. The colour intensity of the nuclei is not bad. The nucleoli are better fixed than with citric acid, and many have the typical aspect, with the clear central point. But the cytoplasm is set in a very dense, unnatural form, as "crumpled". I can’t see well formed individualized granulations (mitochondria, leucosomes, sphaerosomes).

Fixing: 3 min, first washing 4’, second washing 4’, staining 3 min, washing and rinse. Thoroughly washing of fixative, and of dye.

. 9 - 100x obj – A rupture in the cytoplasm over the nucleus. CbZP

Fig 10 – 2% Acetic - 40x – very dark

On the other hand the colouration protocol we used so far is known as progressive.

With histological tissue sections, which are fixed to the slide, is simple to pass the preparation to the decolourizer, take it out periodically, verify the intensity, return it to the destaining solution, and so on. When the effect is the one the histologist prefers, he stops the action washing the removing agent, and proceeds with the rest of the protocol.

The onion skin is more difficult to manipulate. After being fixed in acetic (and coloured, and cut out from the handles to be mounted) it is somewhat rigid. The coverslip of the wet mount can be removed, with the aid of a mounted needle, and the piece of epidermis can be slipped with care to one little shallow dish with the destaining fluid. To recover it, slip the coverslip under the epidermis with one hand, and slide the epidermis over it with a mounted needle.

Believe me; the manoeuvre is easier than its description.

The removing fluid I used is a weak alcohol solution at 30%. It could take 30 to 40 seconds to obtain a good colour density.

Fig. 11 – 2% - 40x – excess stain removed with 30% alcohol

Fig. 12 – some, but few, nuclei had plied shapes, with a diffuse structure

Fig 13 – 2% acetic acid - ¼ reduced images of 4 nuclei. 100x obj., after decolouration.

The cytosol is a bluish gray mass with folds, in which mitochondria (RNA) and other granules are absent, and which often show “holes” in a totally abnormal fashion. The clear streaks of cytosol distinguished some times with iodine, and even 60ºC water, are absent.

The comparison between 5% and 2% shows however advantages for the latter, at both the nuclei and cytoplasm levels.

So, before leaving the acetic acid used alone, I will try a 1 % solution as my last exercise.

Acetic 1%

Fixing: 5-6 min, first washing 4’, second washing 4’, staining 2 min, washing and rinse. Verify colour. Alcohol 30% (if needed), rinse. Wet-mount

With similar characteristics to the 2%. Coloured nuclei. Distinct nucleoli. Dense cytoplasm, with folds, grooves and breaks which are difficult to interpret. I don't show the images with 4x and 10x, as they are as shown before.

Fig 15 – 100x – reduced to ¼ - 1% acetic

Although the cytoplasm looks something more normal (if I know what a normal cytoplasm is? I must say instead: more like with the citric, or even with iodine) it still shows artifacts* that are disturbing. The worst are the cracks and holes, evidently of the parietal layer of the cytosol. The nuclei do not have a bad morphology, and the nucleoli continue to be well featured. 

* artifacts are morphological details that do not seem natural, and are caused by a improper action of the reagent, or even by particles foreign to the preparation, included in it accidentally.

Acetic 1% from pure acid

To test the confidence I can put on the vinegar I test also a 1% dilution  of the pure acetic acid.

Fixative, 3’; washing1, 4’; washing2, 4’; staining, 2’; washing; rinsing

16 – 40x –acetic acid, pure, 1%, aqueous

17 – Nuclei with 100x obj., 1% pure acetic, aqueous, pictures reduced to 1/4 of size.

2% and 1% are clearly superior to the higher concentrations. The best images of nuclei I have are from the 2% acetic, after regressive colouration. More than 100 years ago Flemming (an enthusiast of the still now highly regarded strong osmic and chromic acids as cytological fixatives) selected for fixing nuclei in animal tissues, acetic in concentrations as low as 0.2% to 1%.

And the cytoplasm? As for citric acid, acetic is a nuclear fixative. Until now, only the Iodine tincture gives a somewhat more detailed representation, poor as it is, of the cytoplasm, more crisp and clear than the mere row of granules the other described fixatives showed (see the April article, fig.15 and 18).

The "ceviche’s way" has exhausted the possibilities. Both citric acid and acetic have shown their ability to fix the onion epidermis cells showing its basic elements: cell wall, cytoplasm (usually visible as a more refractive parietal layer attached to the cell wall), nucleus with its disc-shaped structure, and their 2 to 4 nucleoli disk-shaped, or spherical, with a clear point in its central area (of course, when they are well fixed and focused). Neither of them (citric or acetic) showed the cytoplasm with the same detail that the tincture of iodine does.

About the pejorative statement of Bolles Lee, history has only partially confirmed it, because as I have told you, acetic acid, alcohol, and chloroform, are in the formulae of very well known “old fixatives” used until now. And even if it is not the best in the world, the images I have taken from the epidermis fixed even in diluted vinegar, are good representations of the onion cell nucleus.

Acetic (and the other reagents) should have been tamed in some way.

I have a consolation. The games I’m playing now for my own illustration, were surely tested by the first microscopists, but their work was really frontier scientific research. They were building, and sharing, the first bricks of modern histology. Testing, accepting, and discarding results, some times being right, some times being wrong, step by step, they constructed (more than a century ago) the solid foundations of the extraordinarily useful techniques and science of modern histology.

Science, says someone, “is an error that is every time a lesser error”

For now, I must say goodbye to the ceviche!

But... there is a very big problem, you know...

Most of the professional fixatives of two decades ago (those which I have listed before in the introduction to this article) are now banned.. They have very toxic (mercury dichloride), or carcinogenic (formaldehyde), or addictive (chloral hydrate) components, or ingredients which may be used... to make terrifying explosives! (picric acid and picrates). Those components are not being sold to amateurs, and in many cases not even to professionals.

But, trust me; the holy fathers of histology had devised, in the very old times of the last half of the 19 century, some premonitory answers, which can now be applied to this modern social behaviour, then totally unexpected. Let me start exploring them.

And it's very disgusting, but air bubbles have returned!