The inner epidermis of the onion bulb cataphylls

(the onion skin)

Easy and not so easy methods to work with

Walter  Dioni   -  Cancún, México

5) fixing with alcohols

Continues from Part 4 – fixing with acetic acid

Alcohol as a fixative

The adventure with the “ceviche” (see previous articles) made me think of other ways to preserve food that may have been an incentive for the experimentation of the first histologists.

I discarded sodium chloride, the “salt” (dry salt, and brine), because their products are undoubtedly preserved, as salt dehydrates (even the bacteria, making it inoperative, thus its utility), but also hardens, and usually deforms. (Do you remember the dry salty pieces of cod?). However, I picked up some documents which show that salt was used earlier as a stabilizer, mixed with other products, to preserve animals and even humans for anatomical research.

But I remembered (Oh! Yes!) the beautiful cherries my aunt packed every year in sugar and alcohol. And I immediately visualized the rows of bottles in the collections of museums, many of which are full of alcohol to preserve their content, which thus lasts for dozens of years.

I think that the following short historical resume about the relationship of alcohol with human beings is clear and sufficient (more data in Wikipedia):

Ethanol has been used by humans since prehistory as the intoxicating ingredient in alcoholic beverages. Dried residues on 9000-year-old pottery found in northern China imply the use of alcoholic beverages even among Neolithic peoples. Its isolation as a relatively pure compound was first achieved by Islamic alchemists who developed the art of distillation during the Abbasid caliphate, the most notable of whom was Al-Razi. The writings attributed to Jabir Ibn Hayyan (Geber) (721-815) mention the flammable vapors of boiled wine. Al-Kindi (801-873) unambiguously described the distillation of wine. Distillation of ethanol from water yields a product that is at most 96% ethanol, because ethanol forms an azeotrope with water. Absolute ethanol was first obtained in 1796 by Johann Tobias Lowitz, by filtering distilled ethanol through charcoal.

http://e85.whipnet.net/ethanol.history/

I have records of its use in histology from the very beginnings. Absolute (100%), 96%, 90%, 70%, and “1/3 alcohol” were used for different purposes. Apparently a consensus was rapidly reached that to fix, the best concentration was Absolute Alcohol, even if this is very difficult to maintain “absolute”. Absolute alcohol was selected as the best plant material fixative in the 1884 edition of the famous Strasburger's "Botanische Prakticum".

Their more useful role was, and is until now, as a component of most of the most credited fixative formulas, in a variety of concentrations. In short, most writers say that its merit is to surprise the cytoplasm, taking out its water suddenly without giving opportunity for severe changes in their structure.

To be coherent with the design of this series I did a rapid test of the alcohols within my reach, as fixatives for the onion skin, using this to highlight the specific modalities for its use.

96% ALCOHOL

I am working with a plant tissue whose cells are bound by a quite rigid wall of cellulose. If alcohol pumps out water from the cells cytoplasm its volume must reduce.

So I did a first quick test to verify if, as one might think, strong alcohol produces plasmolysis. I fixed in pure 96% alcohol for a period of 15 minutes, and examined a sample in the same alcohol. I could detect no effect greater than that produced by the reagents I used before!, so I made a formal test slide, using alcohol 96% and the Blue 1, as I have until now.

I follow this protocol:

Fixation: alcohol 96, 15 minutes; alcohol 70, 5 min.; alcohol 35, 5 min.; distilled water, 5 minutes; Blue 1 (according to the result of a previous experience) 1.5 minutes. Washing until no more clouds of dye were released, rinse in another water bath and mounting.

Fig 1 – the reagent series

Hydration: To avoid too much tension to the nucleus and the cytoplasm if I go directly from the alcohol to the aqueous dye, I set 3 hydration steps. Descending the concentration from 96%, through 70%, to 35% and finally to water.

Normally, in professional histology, and especially in the treatment of sections made with a microtome from animal material, a much more delicate hydration series is performed when a high alcoholic fixative is used, varying the concentration by only 10 or at most a 20 per cent in each step. Not doing so produces artifacts by creating for example false cracks between naturally contiguous structures.

Fig. 2 – Duodenum, section of a Paneth gland, Harris Haematoxylin – Eosin. The arrow points to a crack artifact due to inadequate dehydration of the histological piece. 100xOI objective. DC3 camera. From a commercial slide.

However, still wishing to be careful in my preparations, with only 3 steps, I did not find severe deformations attributable to fixing.

A disappointing detail is that preparations, while they show clean spacious fields, are also riddled with bubbles.

Fig 3 - 4x objective, 96% alcohol

Fig 4 - With the 10x objective, however, you can find good fields to work with

Fig 5 - 100xOI obj. – 6 pictures (Logitech 9000) reduced to ¼ its size. Comments in the text

In almost all preparations made with the methods I used before (with the notable exception of the Iodine tincture – see first article) cytoplasm appears as a transparent area, perhaps with a weak more refractive layer of cytosol attached to the cell wall and some scattered granules or irregular granular spots and bands.

In this case it's remarkable the good appearance of the cytoplasm, in which a layer of fine granules along the cell walls are easily identified (pictures 2 and 3) with accumulations in the corners (see picture 5 of the upper series), and also coarsely reticulate granular areas close to the walls (top or bottom) of the cell, as well as the suggestion of bands of granules (without visible bounds), around the nucleus, and which extends radially from the same (fig 4).

Indeed, 96% alcohol has made the cytoplasm far more visible, than with previously used fixatives. Cytologists say that the small granules are mitochondria, and vesicles, and other granular materials included in the cytosol.

I know that live cytoplasm is structured. The two dark background pictures I posted in the first part show this. My hope is to show, with techniques that anyone can repeat, the delicate aspect of this living structure. Alcohol seems to be a step towards this end.

Nucleoli are very visible here, as small dark areas (much smaller than with the other fixatives).

 The most impressive detail is the appearance of the “grooves”, that with the other fixatives were seen as more or less deep folds, visible, but not ostentatious, which here are seen as neat, short, broad and deep incisions spanning the nuclear disk. I interpret this difference as due to the strong dehydration of the nuclei.

70% ALCOHOL

70% Alcohol is widely used in macroscopy, to preserve the sampled materials for further study. 10% Glycerine is generally added for to protect the pieces against alcohol evaporation.

Strangely it is not so recommended for histology, where histologists use the much weaker alcohol at 30% approx. As you can see below I believe alcohol 70, applied to the skin of onion is a simple but helpful Fixer.

Fig 6 – The reagent series

Fixing: 70% Alcohol: 15 minutos, 35 % alcohol, 5 min., water, 5 min, new dye solution, 2 min., washing, rinse.

There is a big difference between both pieces (96 and 70%). 70% Alcohol provided a more clean piece, with the nuclei very well fixed (according to the experience gathered so far), very evident nucleoli of normal size and strong staining, and a cytoplasm with a well fixed lattice (see photos 5 and 6 of the following series), and good relations with the nucleus, although 96% alcohol shows a more natural distribution of the mitochondria.

Fig 7 – 6 pictures of nuclei and cytoplasm fixed with 70% alcohol

35% ALCOHOL

French histologist Louis-Antoine Ranvier (1835-1922), proposed and promoted the use of the "1/3 alcohol" known at his time just as "Ranvier alcohol", which he produced by adding 1/3 of 90% alcohol to 2/3 of distilled water.

Today it's easier (and especially for an amateur) to use a 50% dilution of the common 70% alcohol, which is why I'm trying alcohol at 35%.

Bolles Lee (and the ten editions of his Manual that are published at the Internet Archive), is unavoidable. On any subject it has and offered an opinion. Many times it is not very understandable at first reading, because the described concepts have widely varied.

For example, he classified reagents as fixatives or hardeners. Both groups are currently considered as "fixatives". But in his time, especially at the time of the first editions, the fixatives fixed, to avoid the deterioration and loss of structures, and the hardeners (usually salts of heavy metals), were those reagents that were able to give the tissues a proper hardness to allow it to be manually cut into thin sections for microscopic study.

Yes, not only the plant tissues were cut by hand! also the animal ones!

The appearance and the rapid spread of mechanical microtomes, and the custom of giving consistency to tissues through its inclusion in paraffin, erased, step by step, the difference between fixing and hardening.

1/3 Alcohol (30% more or less) was regarded as a fixative. Unlike the absolute alcohol it did not produce "hardness", what did it useful only for certain organs.

Of course, using the thin skin of onion, with only one cell thick, it is not a distinction that bothers me. But it does matter to know the results of using it as a Fixer. I never used as a fixative an alcohol of so low concentration.

Protocol:

Fig 8 - The simple and reduced series of reagents

Fixing, 5 hours; water, 5 min.; Blue 1, 2 min; washing, rinsing

Fig 9 - 35% Alcohol – 40x obj.
 

Fig 10 - 35% ALCOHOL – 100xOI – It is the second time, that I find a nucleus like the one in the 3rd. image, when fixing with alcohol!!!

I tried 35% alcohol, for what it is, a weak alcohol, which surely would need time to make a complete fixation. And which could be dangerous, because it is not as good an antibacterial agent as 70% alcohol. The pictures show a satisfactory nuclear fixation, but poor cytoplasmic fixing, not very different, but inferior to that obtained with 70% alcohol. And the intriguing punched nucleus!

A SHORT SUMMARY

If I had to graduate the fixatives used until now to fix the onion skin, I would only rule out the boiling water and the lemon juice. I think citric acid would be a good option, but I would give 1% acetic acid or at most 2%, and to 96% alcohol, the best score as nuclear fixatives. 70% alcohol produced nucleii as good as acetic acid, even if some cells show the same deep grooves 96% alcohol show.

And I would better classify 96% alcohol, and the old Iodine as cytoplasm fixatives. Review the pictures. Possibly all that is needed is to stain the cytoplasm to make details more visible.

In the future, we will see...!

It is not worth working with 35% alcohol, except for economic reasons, and only for educational purposes.