The inner epidermis of the onion bulb’s cataphylls

(the onion skin)

Easy and not so easy methods to work with

 Walter  Dioni     -      Cancún, México

3) fixing with citric acid. (with some pictures of Aspergillus cf. niger)

Continued from part 2 – fixing with hot water and staining with Blue 1

 As I said in the first article of this series, I am seeking a technique to make “onions skin” wet-mounts without (or with the least possible) air bubbles.

I said, in my previous article that, even if the hot water technique I tried allows me to do onion skin preparations totally, or almost totally free of bubbles, I think that it is a risky option to apply in a classroom. And even if this is not my main objective, it has become some of a personal responsibility, and I wish to make some tests on “cold methods” that equally fulfill the task. And I have some alternatives to explore.

A LITTLE MORE OF MEXICAN GASTRONOMY

Is it funny!, the “association of ideas” continues: In Mexico, of course, cooks also make "cocido" similar to the Spanish one (but pungent, with lots of "Chile" (Capsicum anuum) of which there are at least 14 varieties in the country!!), but above all, Mexicans are fond of "ceviche", a dish based on seafood, mostly fish and shrimp, that is “cooked cold” , as they say, submerging it raw, for a few hours in a lemon dressing. Without forgetting their dose of “chile”, of course!

Note: Take note that I say “chile”, not “Chili”. Chili is a tex-mex spicy dish (spiced with chile of course), made with meat, beans, and rice.

Well, you already realized that, in reality, although they don’t known the elaborate scientific term, Mexican housewives are “fixing” the seafood’s protein, precipitating it with the acid of lemon juice, and sometimes a little vinegar, which is also added to the sauce.

A recipe of fish ceviche in english:  http://www.motherearthnews.com/Real-Food/1980-05-01/Mexican-Ceviche.aspx

Fig 1 - A “ceviche” as is shown at  http://tucocinafacil.net/2009/04/03/ceviche-de-cuaresma/

Another recipe, of shrimp ceviche: http://recipes.epicurean.com/recipe/2957/ceviche-de-camarones.html

And one with vinegar: http://www.recipecottage.com/mexican/seviche10.html

Fig 2 - These are what in México are named “limones” (lĩmõ̞nẽ̞s, following the International Phonetic Alphabeth) (or li.’mon,as Wikipedia says) (lemons) o leemonais as I think can be pronounced. They have a diameter of at most 4 cm. In the USA these are called “limes”, but in Mexico this is not appropriate because “lime” translates for “lima” (lee mah) and “lima” in Mexico is another fruit something bigger than these, with the scent and some taste of the “limones” but nothing of its acidity. So I best show my material as it is.

Fixation with lemon juice

According to the recommendations of the histologists, who know about these things, I put in a capsule enough lemon juice (my proposed fixative) to make its volume at least 10 times the volume of the epidermis… i.e. I filter through a fine sieve enough juice to fill two or three tablespoons… and left the epidermis** in it, abandoned to their fate for 10 minutes. Remove the peel from the lemon juice, and wash it in clean water for 2 minutes, stirring from time to time. The sheet does plunge easily in the lemon juice, but tries to float in the washing water. The waxy cuticle, therefore, is not totally neutralized. So I give it 15 more minutes. This time it does not float.

**For this and all coming experiences, I use fresh cut onion cataphylls. Those that were left exposed to air, or preserved to work “next day” could fail, or have erratic responses to reagents.

Also, when sinking the epidermis in any reagent, I take the precaution to do that with the dorsal (external) side of the piece down. Think of the pieces I prepare, with two handles. Doing this the handles can surface, more or less, but the epidermis would be in the underside, well submerged in the liquid.

Fig 3 – This is the series I use to prepare the onion skin peels. Of course those who can buy the appropriate laboratory glass material must do so. But no one can say that there is an amateur so poor who cannot have the opportunity to conduct meaningful experiments.

Have lids available for all reagents, especially those of the dye and fixative to prevent dust from falling on the surface of the reagents.  And often change the first washing water, to minimize the carryover of fixative’s traces to the dye.

Then I pass it for 10 minutes to a capsule with water colored with Blue 1, which was a successful nuclear stain in my last trials.

Fig 4 – 4x obj. You can see two minimal bubbles (I search for them) This is a common image, but not all times is so favorable

Fig 5 - An image with the 10x objective (another preparation) no bubbles.  The shadows are coloured mesophyll in the underside of the skin

Fig. 6 – 40x obj. General fixation will be rated as fairly good, nuclei are neat and well differentiated from the cytoplasm. Cytosol is depicted as an irregular reticle, but at this magnification, nucleoli are shown faintly or not at all.

 Fig 7 - Nucleus fixed in lemon juice, 100x obj. The image is very similar to those given by the 60ºC hot water. But nucleoli in the juice are a lot less prominent

8 – Nucleus fixed in 60 ºC water (fig. 12 from last month's article)

Fig 9 – the cytosol near the cell wall. Lemon juice

It is evident that the lemon juice fixed the epidermis, probably not so well as the 60ºC water did, but good enough to show the general cell morphology in some detail. Fixation is cruder, and in some cells the cytoplasm is detached from the cell wall. Perhaps all is needed is a better timing, but I have verified what I suspected (plain lemon juice fixes cells), and also I see now what could have been a foreseeable problem

The “ceviche” of onion epidermis was quite effective!

But... a major drawback is that the exact composition of lemon juice, and even the exact concentration of acid in the juice, may vary (within tight limits of course) from variety to variety, from lemon to lemon, from production area to production area, even from country to country.

This is not trivial. My first trial was a success. By good fortune I had not ready the photographic cameras. So I needed to repeat the essay... with another lemon... and it caused great plasmolysis. 

Fig. 10 – 10x obj. See that. You don’t need the beautiful red onion outer epidermis to see plasmolysis.

DC3 Motic Camera, picture resized with ACDSee Lanczos

 I think that surely this lemon (a living thing) had a high concentration of solutes other than the acid, in its complex biochemical composition. And to succeed once more, I had to dilute the juice to twice its volume. The success of this trick is testified by the above pictures 4 to 9.

It also happens in some batches that the piece appears sprayed by fine refractive, mostly yellow drops, which after much thinking I guess that they may be of essential oils of the lemon peel that were incorporated into the juice when I squeeze it.

So, fixation and colorations are fairly viable, bubbles are fairly well reduced, but the overall process doesn’t merit to be retained. It is very insecure.

I think there is, however, a way to overcome these limitations of lemon juice, and build on what we have learned from ceviche until now.

Fixing with citric acid –

According to Wikipedia the lemon juice contains 5-6% citric acid , with a pH between 2.1 and 2.3, which is responsible for its tasty acidity. If this is also the main agent responsible for fixing, as I suspect, the pure product (which is cheap and easily achieved with suppliers of products for pastry) could perform the same function as the lemon juice. With a big advantage: the concentration can be fixed at will. I prepare 4 concentrations, 10%, 5%, 2% and 1%.

The 5% would be roughly equivalent to the lemon juice, but in a clean solution, and with exactly known and repeatable concentration, without any other salts or organics added.

The best results I have obtained with the 1% solution followed by the 2 %. I discarded for the moment the 5% and 10% concentrations.

Fig 11 - I made the following experiment (a silly experiment may be) I mixed fixative and color and mounted a strip of onion skin in this media. For most of the ten minutes the image was similar to the upper one. You can only see the surface of the cells. After this time and in a more or less rapid succession the nuclei start to stain and show through the cells wall. I think that the citric had killed the cells and allowed the dye to penetrate and bind to the proteins.

Citric acid 5%

In all the following protocols I used the same series of reagents used for the lemon juice trial, changing only the content of the first cup, using in each test the different fixative to be assayed.

The tested materials are epidermis peelings from the third or fourth cataphyle of an onion bulb

This is an example of a working protocol

Cítric 5%                                         25 min         

Washing (distilled water)             1 min

Blue 1                                               15 min

Washing (bottled commercial water)     

Rinse (bottled commercial water)

 

Fig. 12 – 40 x obj. - Good fixed cells. Nuclei well stained, but with a greenish colour. Nucleoli are hard to be distinguished.

To show both the cytoplasm and the nuclei response to citric acid I add the following six pictures taken through the 100x OI objective.

13

14

15

16

17

18

Nuclei (1000x). On the external face of the epidermis (pictures 13 and 15) you can see that there is a bacterial invasion. Bacteria, fixed by the citric acid, have been coloured by the Blue 1. The nuclei are well fixed, two nucleoli are clearly seen, although the contrast of the RNA of the nucleoli with chromatin in the nucleus is much lower than with other methods of preparation I already experienced. For some reason, which I leave to the biochemists to elucidate, citric acid decreases the affinity of the dye with the RNA.

 Yet, even if we do not go ahead, it is clear that following the ceviche’s track has provided an interesting material: both the lemon juice (with its obvious limitations to be scientifically used), and the irreproachable citric acid, produce a true and similar fixation of the cytoplasm, the nucleus and its nucleoli. And the Blue 1, also continues to demonstrate its nuclear coloring activity.

 Citric acid is not used as a cell fixative in normal histological technique. I did a long search on the Internet and old books of histology without finding any description of its use as a fixative, neither in animal histology nor in plant histology. It is only used as a component of the citric-citrate buffer.

If, used in the raw form that I have used it, it gives acceptable results as a fixative, I believe that the citric acid deserves, later, a more detailed investigation.

 That is why, at the risk of being boring, I present now the results from the fixation with the other 3 prepared concentrations. For a more compact presentation I limit the illustrations, in almost all cases, to those obtained through the 100x immersion oil objective, both in regards to the nucleus or the cytoplasm. 

 Working protocols:

Citric acid 2% - 25 minutes fixing, 5 minutes washing, and 15 minutes staining. Washing, rinse.

Fig. 19 - SIMILAR RESULTS TO THOSE OBTAINED WITH THE 5% CONCENTRATION

Citric acid  1% - 25 or 30 min. Fixative; 10 min. washing; 25 min. Blue 1

Fig 20 - 40x obj

Fig. 21 - six nuclei, 10x obj. 1% citric acid

The consequences of using this lower concentration appear to be several.

1)  The differentiation between the intensity of coloration of nucleus and cytoplasm was very high.

2)  The nuclei were intensely coloured, but in a transparent manner which gives a sense of three-dimensionality that previously were not achieved.

3)  The nucleoli are much better distinguished, even for the color, that is more reddish.

4)  The structure of chromatin is dense and does not appear hard grained as in all previous pictures.

    5)  The cytoplasm, doesn’t acquire any intense colour, nor did the cell wall. The structure of the cytosol is gently granulated.

 Citric acid  10% -

 I left for the end the concentration of 10%, because it is the only one which was  clearly inadequate in these working conditions. The cytoplasm was fixed in a cruder way, and the nuclei suffered a contraction of around 25% of its diameter, being coloured proportionally more dark. The nucleoli are not identifiable. I show a 1000x image.

22 - citric acid 10% - obj. 100x OI

I feel that the really good concentration is the 1%, which of course is the cheaper of this cheap reagent. Two per cent is almost as good. Pending a more intense investigation, I discard for the moment the 5% (only for economic reasons) and the 10% concentrations.

I think the above long sequence of pictures shows that quality (with the 2% and 1% citric acid in distilled water, specially the last) looks very similar and even better to that obtained with water at 60ºC. Nuclei in this piece were well fixed, are fully and clearly circular, (or oval in some cases, as is common in this epithelium), have well highlighted nucleoli and coloration has the appropriate depth, emphasizing them well and without obscuring surface and internal details.

Fig. 23 – In Citric 5% and 10% the circular gap of the cytoplasm around some nuclei are evident.

Fig 24– 40x, plasmolysis, a trial some months ago with the 10% citric acid and 10 minutes of exposition.

DC3 Motic camera resized with ACDSee Lanczos

My inevitable conclusion was that plain 1% citric acid is the best concentration. But... at least in the temperatures of Cancún, don’t think about having diluted aqueous citric acid solutions in stock! In some weeks, even days sometimes, mine become good sources of mould, specially Aspergillus sps., whose black sporangia, and felt-like mycelia, floating on the surface, denounces them immediately. So you must prepare your solution when required, or, at least, frequently ... or find some antiseptic that preserves the solutions (which I have not searched for, until now).

This is the Aspergillus sp. As I can see it.

Fig. 25 – Aspergillus, colony border

Fig 26 – A complete conidiophore, and a naked head showing the spherical form characteristic of the genus.

Fig. 27 – a complete head, and the same with text labels if move mouse over image.

Fig. 28 – conidiospores spread by the coverslip preassure. All pictures from Aspergillus mounted in Lactoglicerol – 100x obj.

Even so, using citric acid provide me with an easy to find reagent, not even previously assessed, as a long search on the Internet suggests, which in the simple and cheap 1% or 2% concentration gives very good fixation.

And I must declare that almost all preparations I made had no visible air bubbles, even with the sensitive 4x objective.

 So ... Do I have to say "Mission Accomplished” and close this boring series?

Perhaps...

But I am very curious about the action of the other acid included in many ceviche recipes: the acetic acid, and... citric acid is very promising, and I am developing and testing some fixative formulas which, if successful, would be shared in some subsequent articles.

So... you understand me...