You are on page 1of 39

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

First part – preparing the epidermis, live cell structure, fixing and
staining with iodine

JUSTIFICATION
This work started as an attempt to make a preparation of onion skin
without the annoying air bubbles that almost always bother the
observation. You can work with them under your objective, but this
is annoying, isn’t it? I was then guided well beyond my initial
purpose, by a concatenation of suggestions dictated by studying the
material in itself, my memory... and Internet. Although, at a certain
point, work seemed to show a very serious unbalance in the "cost in
time / benefit" quotient, but the intrinsic fun generated by this
"light” research, that even drove me on joyful journeys through the
Web for many days, tempted me to continue. I believe that at the
end I had a lot of fun, I learnt several things, and in between there
were registered several points which may be of interest to other
amateur microscopists, biology students, or even biology teachers.
WARNING! Those who start reading this long series of articles must be advised that they
will be subject to the viewing of very similar images, with no artistic or novelty pretentions.
These are just witnesses of the use, on the same plant tissue, of many different techniques.
So, what is important, is the comparison between otherwise very similar images, to verify
(as long as a digital testimony can allow) the result of the different treatments.
Most pictures have been taken with a Canon Powershot A75 camera, handheld over the
10x ocular of my microscope. The 4x objective of the microscope plus the camera system
gives, depending on the intensity of illumination, a central hot spot (which is not evident
visually) even with the most carefully adjusted Köhler style illumination. Due to vignetting*,
images have 2Mpx out of the native 3.2 Mpx of the sensor. Most of the time I crop a 1024 x
768 px image, which for sake of better reading in the published article I reduce to 900x 600
px. If necessary for publication, I adjust the size additionally. My only interest is to better
show which I consider a significant testimony of the action of the reagents.
*Vignetting can be eliminated using the optical zoom of the camera. But I work until now
with a power supply, and in this configuration the camera does not admit its use.

INTRODUCTION
As part of 'another project' I’m working on, I needed a few onionskin preparations. Who does not need them some times?
But of what onion-skin (epidermis) are we talking about? For me it
was clear that the onion bulb offers us, for

our observations at least, 3 or 4 different types of epidermis. To
define and select the one to work with, was the subject of the
article “How many onion skins are there?” (MICSCAPE, February
2011).
And ... seeing the results, I asked myself who, when, and why,
the epidermis, specifically the innermost of one of the scales in
the onion bulb, a modest food ingredient, was proposed as a
successful model of plant tissue, and unbeatable elementary
demonstration of the organization of a cell.
Of course these are not big existential problems. This violent world
in which we live will not be better because I found an answer; but I
surrendered to my curiosity, and this was the starting point of the
article “Who invented the 'onion skin' biology
lab?” (MICSCAPE, March 2011) which retells the long history of this
practice, now inevitable in any course on elementary biology.
I recommend reading these two preliminary articles before trying
this one.
So, after that “divertissement”, I elected to work with the, for more
than a century, favorite inner epidermis of the middle of the
package of scales that made an onion, and try to work out a
technique satisfactory for me, and possibly useful to others, for not
to have to endure the uncomfortable and unsightly air bubbles.

THE FIRST OF MANY POSSIBLE WET MOUNTS OF AN ONION
SKIN
For the first mounts I made, I followed, with some
adjustments, the standard method, a thousand times
described on the Internet and in any elementary biology
book, exactly as recommended in 1890, or any of several
modifications tirelessly repeated by all those who, like me,
want to take advantage of the facilities offered by this
material.

or screws up as a tube. The use of needles and tweezers. I describe it. recommended many times to extend it. and can be ineffective. folds. The following technique. make up nasty preparations. Manipulation. tedious. or cause a disaster in the thin membrane. and when you manipulate it. I don't recommend it.Everyone who uses this technique must face the first problem: in the majority of onions the epidermis is very thin. the type of mounting medium. and other details. it folds in capricious forms. is difficult. helped me to solve this problem. . although laborious. And of course it also has its own limitations.

2 – A classic preparation. Picking up the fresh onion epidermis .Fig. Some of them in the upper layer. 10x objective. due to the difference in focus. oblique illumination. Living cells. The boxes made up by the cell walls enclose the textured cytoplasm with small granulations and the discoid nucleus near the cell wall. electronic colouration. some on the bottom one.

I take an onion bulb. because if the scales dry. would be coloured. making useless much of the surface of the used “peeling". and which. if dyes were used. and if it is large. even slightly. into quarters. I work immediately. the epidermis breaks off with thin layers of mesophyll adhered to its surface. I cut it in halves. . which then prevents the preparation being flat enough.

I cut a piece of scale (cataphyll) (usually the third or fourth from the outside of the bulb.Fig 3 – This is a stained peeling showing the stained mesophyll under the epithelium.5 cm wide (to fit comfortably on the slide) . to have a thickness which facilitates manipulation) of 1 to 1.

c. 2 cm from one end. on the convex side of the cataphyll. Foto 4 a. and do a cross cut from edge to edge.b.all the length of the scale. selecting a middle cataphyll. cutting dorsal epidermis .d– cutting the wedge.

or similar cutting tool. which has the width of the piece. I reject the intermediate piece of scale (now without the inner epidermis) and cut the other end of the cataphyll piece to have a length of other 1. and with a slight effort I break loose a sheet of epidermis.I break the piece. It’s necessary to work with care but it is not difficult. with care. Folding over the piece. and cutting off the excess.5 or 2 cm. which I give a length of about 2 or 3 cm at least. . Only needed is a more or less sharp scalpel.

At first I cut the cataphyll “handles” of only 0. .Foto 5a. so I decided to use the big pieces which guarantee an easy manipulation in all cases.b. peeling the epidermis. which will be extended by the weight of the other "bar".d breaking the slice. discarding the peeled portion.5 cm. With fine pointed tweezers I can take one of the handles and manipulate the piece. The piece of skin is now supported at both ends by two scale "bars".c. cutting the second bar. But I learned that their weight was not enough with other liquids. and this worked with some fluids.

Perhaps this will be not a technique to propose for use in classroom practices. Making the simplest onion peel mounting I make wettable a perfectly clean slide (pass it through the flame of a spirit lamp. can be moved and safely transferred between several reagents. When I finish working with a piece. 6b two prepared slides. fixed and stained. each with a typical piece of epidermis. but allows me to try comfortably different working techniques of my interest. and lay on it my sample. if necessary. a couple of times on both sides) put a drop of water (or stain) in the center. The epidermis. or a Bunsen burner. ready to be fixed or wet mounted. subtended between the two pieces of scale. I mount it over a slide and cut out the bars. if possible with the face that was attached to the mesophyll . Fig.Fig. 6a – finished piece. holding one of the bars with tweezers.

Carefully apply the coverslip with common precautions and if it seems necessary. you can do it. absorb a little liquid so that the weight of the coverslip flattens the epidermis. Of course I got. strewn with air bubbles (some more. It's a little tricky. . as so often in more than 60 years of attempts. This is the first of many types of wet mounts I can apply to my peel and it provides a good square or rectangular piece of epidermis. With high powers this assures a more clear focus of the epidermic cells. Add a small drop of water (or stain) so that it does not dry out. I use a Gillette or a sharp scalpel to cut with care next to the bar. You must seek and select fields which have the minimum amount of bubbles. epidermis peels mounted in water or diluted Glycerin. The result is a peeling of a very good size which is also extended almost without pleats (usually the cut edges bend a little). It is only easy using high magnifications (400x for example). but. without any chemical modifiers applied.5 cm wide x 2 cm long. and. Turn slide and repeat the maneuver. depending on luck. with care. some less. if I so wish. It can be almost impossible with the 4x. Holding one bar with my tweezers.downwards. usually more than 1. also with the 10x. but all with bubbles). freeing that end of the peel.

Fig. This and the following images had been subjected to a contrast enhancement. . as you know. fresh peeling mounted in water.4x Obj. 7 . to make more visible the structural details. No staining. As most of all living tissues the fresh onion skin is very transparent.

Phantom nuclei. No staining. 8 .10x obj. . Colours were a not programmed. from the illumination and the recording camera set up. unexpected bonus.Fig.

A nucleus attached to the lateral wall of the cell.Fig. .40x obj. 9 .

Fig. which are mostly mitochondria. 10 .40x obj. with many granulations included. you see in these fresh preparations. Strips of cytoplasm and included mitochondria are visible. surely embedded in the upper layer of the cytoplasm. With difficulty. You can even see a nucleolus in the upper one. that the cytoplasm is arranged in a delicate layer applied to the cell wall. This you can discern at least at 400x by slowly focusing through the . A frontal view of two nuclei.

Of course your work would be facilitated if you have a phase contrast system. And bands and strips of cytosol link the nucleus through the central vacuole to other parts of the cell. Included in one of the cytosol layers you find the nucleus. If you persevere in your efforts to optimise your illumination. you could discern all these details. focus with care. visible in frontal view as a disk. when applied to the lateral wall. and study many cells. through the sap of the central vacuole to the lower cytoplasm layer.depth of the cell. using a dark background stop (fig 11). . or in lateral view. and would be almost perfect if you have DIC. But the loose structure of the cytoplasm and its layers and strips can be visualized even with the 10x objective. as a shallow dome. An accumulation of cytosol and mitochondria is discerned around the nuclei. going from the upper cytoplasm layer.

11 – Darkfield (a technique easy to install in any modest microscope). 10x Obj.Fig. Fresh onion skin. Search the MICSCAPE library for technical suggestions on easy and cheap darkfield installations. .

com/watch?v=VXbQpRpUDmQ . See it. because is very difficult to have this experience oneself.youtube.Fig.12 – the spongy tri-dimensional structure of cytoplasm is better seen in this resized image of the cells. On YouTube there is an amazing video of the mitochondria streaming through the living cytosol. http://www.

but can be diluted 5 or more times its volume for use. absorb any excess solution.. and a cover. and stains them a light brown colour. for a long period! In a small capsule I mix 2 drops iodine solution.s. Allow 1 or 2 minutes for the colour to develop. . is a drop of a diluted pharmaceutical iodine-iodide solution (black iodine. transparent yellow colour. Metallic Iodine 2.p.0 g Potassium iodide 2. and obscure its texture. the more ancient. It shows what the live cell structure is. Make some trials.. as well as cell walls. and 10 drops water.The fresh preparation we have reviewed is our reference. the most simple. one more drop of the solution. and skin. and nucleoli even darker. and the cheapest. apply the epidermal strip.5 g Water c. Staining the “peeling” But it is normal to recommend adding some reagent to stain cell nuclei and make more visible the cytosol. The most recommended colouration. its inclusions. put 1 drop of this mix over the slide. As commercial solutions could be different. dilute your own solution until it is a beautiful. iodine tincture) Note: One of the black iodine solutions sold in Cancun denounce on its label this formula. Iodine precipitates the proteins of the protoplasm. Remember this. the nucleus in a more intense colour. 100 ml Beware! The iodine solution stains indelibly many fabrics . and see the result. Darker solutions stain nuclei dark. and cell membranes.

if yours is like mine. it is dissolved in a dense vehicle that causes plasmolysis in my preparations. But. The stains that it can produce are washable. . If possible. Active Iodine concentration drop with time.Take care. in my experience. or a relatively fresh bottle of reagent. It's the favorite of homemakers. use a new. don’t use Iodopovidone (iodinepolyvinylpyrrolidone). It is a pharmaceutical aqueous solution at 10-11%. equivalent to 1% of iodine.

Fig 13 . many (and this is a carefully selected area) air bubbles. Are they not a nuisance? .Typical iodine mount 4x.

air bubbles! . nucleus with two to four nucleoli. Well delineated cell walls.Fig. 14 .Iodine mount 10x.

. A stack of 3 Canon handheld pictures combined with CombineZP Roll mouse over image above for labels.Fig 15 .Cytoplasm streaming freezed by iodine.

See http://www. are seen in this image.sciencedaily. The folds and grooves. They are channels of endoplasmic reticulum which some times tunnel through the entire nucleus. Net image of nucleoli . 16 .Fig. that indent the nucleus.htm Cytosol nicely fixed.com/releases/2001/06/010614064109. 40x.Iodine mount. see the strips of mitochondria. Cell walls well evident.

Fig.A good iodine fixed and stained preparation . 17 . . 40x Obj.

18 .Cytoplasm streaming freezed by the iodine – a stack of 3 Canon handheld pictures combined with CombineZP. .

is concentrated around the nucleus. The image shows what is normal: the cytosol. combined in CombineZ. evidenced by his charge of mitochondria. Which seems strange to me is the nucleolus clearly divided in four parts. The image is a stack of four original ones. 100x.Fresh mount.Fig 19 . . A typical nucleus. with one nucleolus.

as leucoplasts and mitochondria. exposed to light. and which really are very unsightly. which not only detects the cytosol. the preparation. but many have folded surfaces. x 100) some are fairly circular. I think it was fortunate that the iodine solutions have been used since the 19th century.Three nuclei from the same epidermis (Obj. apart from the nasty bubbles that I hate. Even if I could not find clear statements about this. In the words of Bastin (1877) "Iodine solution also rapidly kills protoplasm without dissolving it. I must say. as you have seen. exploited to its full potential. but also their inclusions. made it ideal for staining the onion epidermis temporarily. and is therefore useful as a fixing agent". that. it finally fades. loud and clear.Fig 20 . can fulfill its task: . Certainly it was used in a much diluted form to detect starch (which iodine stains blue). it seems iodine is useless for permanent preparations because. but it was soon found that both protein precipitation and their strong colouration. The third one is a lateral view of one included in the cytosol layer adhered to the cell wall.

html [ Share: odine reacts with the starch present in onion cells. so it is difficult to properly visualize them without using a solution to increase contrast. of course.. and all their components even in very good detail! Giving them a polite apology for the bubbles. But to eliminate the bubbles was the original goal of this effort.uk/mag/artapr11/wd-onion3. http://www. of course. next month. to elementary biology students.microscopy-uk. Onion cells are naturally transparent. and it is a good practice.org. I promise that my next articles will be shorter than this one. producing a coloration that makes the cells easily visible under a microscope. of course! Most teachers use this technique. wasn’t it? Not yet fulfilled. CONTINUE READING KEEP LEARNING  What is an onion cell lab?  How does iodine react with starch in water?  What is iodine used in? . More efforts. with a very simple and very cheap technique. what a plant cell is..To show with very little effort.

then place a piece of onion skin on it. a wet mount microscope specimen must be prepared first. Take a small piece of onion and using forceps (tweezers). 3. First add a few drops of water or solution on the microscope slide to avoid dryness and wilting 2. The specimen is then protected with a cover slip and observed under the microscope. a drop of iodine solution is added. To ensure contrast. Method[edit] 1. peel off the membrane from the underside (the rough side). adding iodine creates a blue coloration. The person preparing the specimen must place a drop of water on a clean microscope slide. If the specimen contains a higher amount of starch.Credit: Ed Reschke Photolibrary Getty Images FULL ANSWER To obtain a clear image of onion cells. Lay the membrane flat on the surface of the slide .

 If the specimen does not come into view (does not focus). Then place the prepared slide onto the stage of the microscope.Could be dangerous if it is on you. 5. Add a drop of Iodine solution to the onion epidermis. The point of focus will be very near the cover glass. 1). 6. raise the tube a little with the coarse focus knob and attempt to focus again with the fine focus knob. slowly focus back (turn the fine focus knob to raise the optical tube) while looking through the eye piece. You should be able to make out a nucleus in each cell. you can make fine adjustments up or down with the fine focus knob without fear of damaging the slide or the microscope.  Initially.requires that the following techniques be followed:  Never use the coarse focus knob while looking through the eyepiece. 10. The cells should look something like lizard skin. Once the specimen comes into focus. Using a pin. Make sure the lowest power objective lens (the shortest lens if there are several present) is in line with the optical tube. switching objective lenses (to a higher power) should be possible without any further coarse adjustments. Once the object is in focus. Make sure there are no air bubbles (Fig.intended to prevent damage to the objective lenses . these dyes can stain your skin and clothes. and the microscope light is turned on. lower the optical tube until the objective lens is as close as you can get it to the cover glass without actually touching it. 8. Looking from the side. lower the thin glass cover slip or cover glass onto the slide. Do this carefully so as not to crack the cover glass (and possibly damage the objective lens). fine focusing knob to move the optical tube upwards until an image comes into focus. lower the tube using the coarse focus knob until the end of the objective lens is just above the cover glass. Starting with the low power objective lens is the fastest way to achieve proper focus. Now look through the eyepiece and turn ONLY the smaller. 9.Be very careful. Proper use of the microscope . . Swap the objective lens for a higher powered one so that you can see the cells at greater magnification. Looking from the side (NOT through the eyepiece).4. 7.

You should be able to make out a nucleus in each cell. Then place the prepared slide onto the stage of the microscope. Take a small piece of onion and using forceps (tweezers). peel off the membrane from the underside (the rough side). Swap the objective lens for a higher powered one so that you can see the cells at greater magnification. lower the tube using the coarse focus knob until the end of the objective lens is just above the cover glass. In this exercise you will make a wet mount on a microscope slide and look at the cells of the onion membrane magnified by the high power. 5.Be very careful. 8. Make sure there are no air bubbles (Fig. An onion is made of layers. Looking from the side (NOT through the eyepiece).Could be dangerous if it is on you. lower the thin glass cover slip or cover glass onto the slide. The cells should look something like lizard skin. compound microscope. The cells are easily visible under a microscope and the preparation of a thin section is straight forward. Add a drop of Iodine solution to the onion epidermis. . 10.Tissue from an onion is a good first exercise in using the microscope and viewing plant cells. Using a pin. each separated by a thin skin or membrane. Make sure the lowest power objective lens (the shortest lens if there are several present) is in line with the optical tube. First add a few drops of water or solution on the microscope slide to avoid dryness and wilting 2. Lay the membrane flat on the surface of the slide 4. 6. and the microscope light is turned on. 7. Method[edit] 1. 3. these dyes can stain your skin and clothes. 1). Now look through the eyepiece and turn ONLY the smaller. 9. Do this carefully so as not to crack the cover glass (and possibly damage the objective lens). fine focusing knob to move the optical tube upwards until an image comes into focus.

 If the specimen does not come into view (does not focus). The point of focus will be very near the cover glass.  Initially. switching objective lenses (to a higher power) should be possible without any further coarse adjustments.requires that the following techniques be followed:  Never use the coarse focus knob while looking through the eyepiece.wikibooks. you can make fine adjustments up or down with the fine focus knob without fear of damaging the slide or the microscope. slowly focus back (turn the fine focus knob to raise the optical tube) while looking through the eye piece. Once the object is in focus. https://en. lower the optical tube until the objective lens is as close as you can get it to the cover glass without actually touching it. raise the tube a little with the coarse focus knob and attempt to focus again with the fine focus knob.Proper use of the microscope . Once the specimen comes into focus.intended to prevent damage to the objective lenses . Starting with the low power objective lens is the fastest way to achieve proper focus. Looking from the side.org/wiki/School_Science/How_to_prepare_an_onion_cell_slide To Prepare Stained Temporary Mount of Onion Peel Materials Required .

 Peel off a leaf from half a piece of onion and using the forceps. transfer the peel into the watch glass containing the safranin solution.Real Lab Procedure  Pour some distilled water into a watch glass. .  Put the epidermis in the watch glass containing distilled water. pull out a piece of transparent onion peel (epidermis) from the leaf.  Take a few drops of safranin solution in a dropper and transfer this into another watch glass.  Using a brush.

 Using the brush.  Take the peel from the Safranin solution using the brush and place it in the watch glass containing the distilled water. place the peel onto the slide containing glycerine. and is surrounded by the cytoplasm.  Take a cover slip and place it gently on the peel with the aid of a needle.  Place this glass side on the stage of the compound microscope and view it.  Extra glycerine stain should be removed using blotting paper. Conclusion As cell walls and large vacuoles are clearly observed in all the cells. Let this remain in the Safranin solution for 30 seconds. Observations  There are a large number of regularly shaped cells lying side by side and each cell has a distinct cell wall. Precautions  Use a brush to transfer the peel from one apparatus to another. so that the peel is stained. To Prepare Stained Temporary Mount of Human Cheek Cells Materials Required .  A large vacuole is present at the centre of each cell.  Staining of peel should neither be too dark.  Lightly stained cytoplasm is observed in each cell. nor too light.  Remove the extra glycerine using a piece of blotting paper. the cells placed for observation are plant cells.  A distinct nucleus is present on the periphery of each cell.  Take a few drops of glycerine in a dropper and pour 2-3 drops at the center of a dry glass slide.

Real Lab Procedure  Gently scrape the inner side of the cheek using a toothpick.  Place the cells on a glass slide that has water on it. .  Mix the water and the cheek cells using a needle and spread them. which will collect some cheek cells.

 After 2-3 minutes remove any excess water and stain from the slide using a blotting paper.  To select the sample. Take a few drops of Methylene blue solution using a dropper and add this to the mixture on the slide.  No prominent vacuoles are observed in the cells.  Place this glass side on the stage of the compound microscope and view it. you can use the ‘Select sample’ drop down list.  To change the power of the lens.  Remove any extra liquid around the cover slip using a blotting paper.  Using a brush and needle.  Take a clean cover slip and lower it carefully on the mixture with the aid of a needle. you can choose a lens from the ‘Select objective lens’ drop down list. . However. nor a prominent vacuole. each cell has a thin cell membrane.  A deeply stained nucleus is observed at the centre of each cell. press the cover slip gently to spread the epithelial cells.  Take a few drops of glycerine using a dropper and add this to the test mixture.  The cells do not have a cell wall. the cells of the specimen on the slide are animal cells. Conclusion As the cells observed do not have a cell wall. Simulator Procedure (as performed through the Online Labs)  The ‘Select view’ drop down list allows you to select either the Microscope or Binocular View (It is the 'Binocular view' through which you can see the cell structure as viewed through the microscope). Observations  A large number of flat and irregular-shaped cells are observed.

you can either click on the up and down arrow of ‘Coarse adjustment’ knob.  For fine adjustments.in/?sub=79&brch=15&sim=125&cnt=2 .  You can redo the experiment by clicking on the ‘Reset’ button. you can click on the left and right arrows of ‘Fine adjustment’ seen on the left controls panel. For coarse adjustments.  Extra glycerine stain should be removed using blotting p http://amrita. or click on the left and right arrows of 'Course Adjustment' seen on the left controls panel. so it does not cause infection to the cheek.olabs. Precautions  Ensure toothpick used to scrape the cheek is clean.edu.