Dandelion slide making adventure - the beginning!

[mrsonchus]

Hi all, well, here in the UK Summer's fast approaching and our lawns are being invaded by the common Dandelion - 'Taraxacum.officinale'. Sounds like an opportunity to make some slides to me! Last year I spent a lot of time scrambling up the steep & very long learning-curve that comes with the desire to make permanently-mounted stained microscope slides - and this year is no exception, as I'm really still learning and trying to perfect my microtechnique.

Anyway, the journey begins with the Dandelion happily growing and basically 'minding it's own business'.....
Here is the target of this adventure - 'Taraxacum.officinale'.




At a glance it's easy to see that the Dandelion hasn't really got any stems as such - the leaves all coming from a basal rosette. The only 'stem' is the flower-stem ('Pedicel'), not regarded as a true stem. So when I refer to the stem - it's to this pedicel that I refer.
I begin with the selection and cutting of tissue pieces with a view to making slides from their sections ultimately, so the choice even at this initial stage must be made carefully.....
The sections/slides I would like to make are leaf TS, pedicel/stem TS, root TS & LS, flower-head LS & TS.
(TS - 'cross/transverse section')
Here are the notes I took while pondering this stage, just 3 small sheets that help me to keep track!





These requirements lead to these tissue-pieces...


Yes, that root-piece looks very gnarled, twisted and just plain filthy! It is! This, although it may not look like it, is a 'tap-root' - a main and normally straight root such as that of a carrot. Here is one from a Dandelion growing in rocky, stony soil (unfortunately typical Here in the Lake-District of Cumbria where we live....) that has clearly had to 'make some adjustments' to it's ideally straight and regular growth down into a softer more homogenous soil.

Nothing a wash and scrub can't put right though - the outer corky layer is not really important when making root-sections such as these are to be, as the main purpose is to make some nice slides detailing the often quite dramatic and always fascinating morphology of the root's interior, which can be very fibrous and usually stains really well - we shall see!

After a wash and scrub,


Cut into appropriate pieces,


Safely into fixative, which kills and preserves the tissue, the roots are on the far right, the other tissue-parts are also now in fixative, although from here-on I'm going to concentrate on showing the progress of the stem-sections only, in order to keep the size of this thread reasonable....
The contraption connected to the root jar is my means of applying vacuum to the root-pieces, as they are particularly difficult to penetrate completely with fluids and the vacuum will 'encourage' trapped gases within the tissue to be displaced by the liquid (in this case fixative) in which they are immersed.... (for more details re the application of the vacuum just ask and I'll put an extra thread up)


So far things are going pretty smoothly.... Back soon with more....

[mrsonchus]

Hi all, here's the next phase - so far to this point we've seen the following,
** Select a plant (Taraxacum.officinale - Dandelion) from which to make slides,
** Consider which sections will be needed then cut pieces of tissue to suit this aim,
** Clean tissue if necessary, beyond a quick rinse under the tap,
** Begin histological process with the killing and preservation of the tissue - this stage is known as 'faxation' and the tissue is immersed into a 'fixative' solution to achieve this, for a minimum of about 24-48hrs,

Here to recap are our tissue pieces in their respective fixative jars, although from this point I'm only going to follow the stem section tissues for the sake of brevity (and to save me a little time! )


Here are the stem pieces after a time in fixative, the next stage will be to totally remove the fixative and at the same time dehydrate the tissue with the use of a series of alcohol jars that begin with 75% OH(alcohol) and finish with three or four stages in 95% OH - the highest concentration available to the home microscopist here in the UK.
They are moved through these stage inside plastic tissue-cassettes which certainly make the whole journey easier and prevent damage to the tissue as they only need to be handled when placed into and taken out of the tissue-cassettes,

Here I have eight Dandelion-stem pieces that will be used for sectioning when finished,


After dehydration the alcohol must then be removed by displacement with a solvent of wax - this is called a 'clearing agent' and smells very much like gasoline - the one I use is called 'Histoclear II' and is the bridge between the alcohol-saturated tissue and their final destination, which is for them to be totally infiltrated with paraffin-wax and then cast into wax blocks for sectioning with my 'Mighty Shandon' rotary microtome.

back soon...

[hkv]

This is a very interesting story to follow! I want to learn this.

[billbillt]

Thanks so much is sharing your "tour de force" project!.. I never fails to generate great interest in me!... I would also be interested in learning your vacuum degassing method..


The Best!,
BillT

[mrsonchus]

This is a very interesting story to follow! I want to learn this.

Buckle-up hk' - more to come!

[mrsonchus]

Thanks so much is sharing your "tour de force" project!.. I never fails to generate great interest in me!... I would also be interested in learning your vacuum degassing method..


The Best!,
BillT

Hi Bill, I'll put some details up ASAP my friend.

[mrsonchus]

Hi all, well, the tissue-processing has been completed,from fixative (FAA50)--> dehydrant (OH) --> wax-antemedium (clearing-agent AKA 'Histoclear II' in this example - I used to use 'Histoclear I', but II is considerably cheaper and far, far less odorous) --> wax infiltration --> wax-block embedding.....

Here's the wax-oven containing liquid (paraffin histological) wax at approx 61 deg C into which the tissue is placed overnight and through 2/3 changes to thoroughly replace (displace) the Histoclear II with wax - this must be thorough, right down to the intra-cellular level for sectioning to be successful.



We need (steel) tissue-block moulds next, into which go the tissue pieces orientated correctly for sectioning. The mould is topped up with molten wax and the plastic tissue cassette lid put on to act as the base of the solidified wax block, and it's means of being clamped into the jaws of the microtome during sectioning....



into mould, these pieces are oriented for TS to be cut, these pieces are totally covered by molten wax, it's just so clear you can't really see it in the picture..


Lid is on - mould is full of molten wax. Into chilled water to set......

[mrsonchus]

Now, here are the results so far, a nice-looking (fingers crossed... ) set of tissue-blocks containing pieces of Dandelion stem (pedicel) ready to clamp into the waiting maw of the Mighty-Shandon (ahem... that's the name I've given to my Shandon 0320 rotary microtome... )


There are additional blocks in this picture - I have also been preparing similar stem tissue from another common wildflower here in the UK - the Valerianella locusta or 'Lamb-lettuce' - and edible 'weed' - although this thread only covers the Dandelion stem I may post a few images of the rather unusual stem morphology of the V.locusta later if I remember.

A closer look shows the tissue as seen looking at the cutting-face of the block, that is to say the face from which sections will be sliced by the action of the microtome, probably at thicknesses between 5µ and 15µ depending on early results.

and,


We're now very close to taking some early 'roughing' sections to assess the blocks and decide how to section them in more detail, such as section thickness, block temperature (e.g. at RT or chilled at about 4 deg C), blade angle - this can usually be set to zero offset but may be set shallower or steeper in it's angle of attack to the wax block if needed as a remedy for excessive section compression....

This is where things begin to get exciting , and also where complete devastation can occur if the blocks are a failure - and there are many reasons why they can be! Believe me - I know this from experience!

Next-up will hopefully be some sections, still in wax but havinh enough detail visible to make some early adjustments etc as needed...

Nearly there!

[billbillt]

Anxious to see this!!..

BillT

[mrsonchus]

Aha! Bill old friend - I've had a chance this evening to make a few roughing-sections of one of the blocks of both the Dandelion stem and the Lamb-lettuce stem - initial results look very promising indeed,

A few very early images of roughing-sections still to dry, although they have been drained, and a few as on the flotation-bath water-surface during 'relaxation' of the tissue/wax section before pick-up onto a slide...

Here's a nice ribbon of 7µ sections from the Dandelion's pedicel,


Just out of interest here's a section from the double-winged stem of the earlier mentioned 'Lamb-lettuce' (Valerianella.locusta) that also has sectioned rather well, also sectioned at 7µ,


A quick look through the stereo 'scope - remember these are still wet! This is the Dandelion,


and the Velerianella similarly viewed, apologies for the poor images, I quickly took these holding a little Koda C140 up to an eyepiece - important not to let the section be heated by the light, even the SMD round LED I have in this stereo 'scope gives off quite a heat,


So far looking pretty good for the stem sections.

Blocks are kept face-down on an ice block before sectioning begins,


The block is clamped into the Mighty Shandon's jaws,


My 'roughing' waterbath (OK - it's a beaker standing on a coffee warmer and filled with DI water... I use a big round lab waterbath for the 'proper' sectioning runs) held at about 42-48 deg C,


A few nice rough sections drained and ready to go into my air-dryer at about 38 deg C for an hour to give the drying process a good start - here they're under a dust cover,


So, if I get time I'll cut some 'proper sections' tomorrow - a day or so to dry after that and they are ready to stain & mount! An exciting stage!
If these rough sections are dry tomorrow and not going to fall off of the slide when de-waxed I may just stain and mount afew to get a first impression...

Stay tuned - the best bits are coming soon!

[JimT]

John, do you know the leaves are quite delicious. Either raw in a salad or steamed. Once the flower goes to seeds however they are too bitter.

Lots of work and I am looking forward to more of this adventure.

JimT

[billbillt]

Thanks for the update, John B.!!.. This is a very interesting adventure we are on!... It is a real pleasure to follow your exploits!....

BillT

[RudiV]

A really interesting post with lots to learn!

Looking forward to the next update, thanks for sharing,
Rudi

[mrsonchus]

Thanks fellows, glad to have you along - I managed to stain and mount a few this morning, rough-cuts but still of value and quite nice.
I stained the Dandelion petiole with 'old faithful' that is the Safranin & Fast-green combination, always valuable as I'm so familiar with both the application and interpretation of this method. Makes examination of early sections easier in some ways.

Having said that, I also used another very quick technique which is the use of the very nice metachromatic stain, Toluidine-blue (TBO) applied directly to the sections as they are still in the (now dried) wax on the slide - before dewaxing! The staining time must be increased by a factor of about 10 when compared to the usual aqueous application of the TBO at about 0.5% concentration. This is no problem however, as this is a matter of increasing from about 1 minute to about 10 minutes, still a very short staining-time.

Anyway, some images,
Here is a comparison of Dandelion pedicel stained with the Safranin + Fast-green on one side, and the TBO on the other. The sections are of reasonable quality considering they are roughing-sections and still wet!
I quite like them personally, see what you think,


Now, you may like to have a peek at those 'Lamb-lettuce' sections - I stained and mounted a few of those also this morning, but only stained them with the TBO technique, here's the whole TS of one - a far smaller stem than the Dandelion's pedicel, hence the whole stitch!


Here's a closer look at a section of the Lamb-lettuce's stem TS - notice there appear to be crystals present within the generously-wide cortex - unstained but just noticeable,


Time to quickly put the polarizer onto it, here they are really easy to see - apologies for the poor image, rather rushed as I'm a little short of lab-time today....


I need to go and do 'weekend stuff' for a while - back soon chaps, hope you like the adventure - perhaps some more staining (I've two rough slides left to play with...) variation would be fun. Over the next few days I'll take some 'proper sections' and run them through the full process to stain & mount rather than the extra-quick roughing protocol I use.

Back soon also with some higher mag images and a more detailed look at the tissue - best to let the slides cure for a little longer before slotting the immersion nosepiece into the big O I think - interesting also to process the other tissue, leaf, root etc - over the coming week I should be able to finish the complete set from the Dandelion and maybe the Valerianella too with luck!

Here's a larger view of the polarized birefringence, the thickened (lignin) secondary cell-walls of the Xylem are glowing brightly, while what I think are crystals (probably Calcium oxalate) can be seen to be predominantly in the intercellular spaces of the parenchymatous cells of the cortex. I wish I had a full-blown polarizing rig for the Orthoplan - still at least I have this basic capability of slide-in analyzer and rotating lollipop.


This is an interesting peek at the surface of the cell-wall (primary unthickened -- cellulose mainly) of a cortical parenchymatous cell from the Valerianella slide. The plasmodesmata seem quite distinct - they're 'holes' in the primary wall and middle-lamella of adjacent cells that together with the plant's cell contents form a huge plant-wide plasma-continuum called the 'symplastic pathway' for the translocation of certain smaller molecules including some sugars - amazing to see them - even in this somewhat blurry image!


If I mention TEM or SEM to my Darling Wife I suspect they may never find my body!
I love exploring slides, even the roughing ones yield much information, most of it of Taxonomic relevance, all of it fascinating! My goodness who'd imagine what fun and fascination a couple of 'weeds' plucked from our lawn would yield!

Thanks for joining in chaps, back soon.

p.s. Funny to think these were living plants in our gardens less than a week ago - slide-making is extremely satisfying and enormously interesting - every adventure full of mystery and discovery!

[billbillt]

I love all of these!... This is you usual perfect job!... I enjoy following your exploits...

BillT

[Crater Eddie]

I really enjoy these sequences John. Please keep it up.
CE

[mrsonchus]

Thanks Bill & Eddie - it's very nice to know folks like the adventures - I love going through them!
I've been cutting some 'proper' sections this evening and have managed to eliminate the slight tissue edge/wax border damage seen in some of the sections.
I had sectioned the wax blocks from the refrigerator - the blocks therefore being cut with their surface (and maybe more importantly core) temperature, as measured by the infrared thermometer seen in the pictures, at a rather cool 3-7 deg C approx. This made the wax & tissue too hard to section cleanly, even at the 15µ & 12µ thicknesses tried! At 5µ there was chaos - something horribly wrong!

Having removed them from the 'fridge' I left the blocks at my lab's temperature for the day (that's 21-22 deg C) and thankfully they sectioned very smoothly this evening at 10µ, 8µ and 5µ without problems - no tissue damage and beautifully clean-cut sections.
But, a new problem emerged, albeit only slightly - the sections were refusing to flatten fully, that is to say that they were 'wrinkled' around their outer cortex and epidermis - a little like a doily....

Anyway, with a small scream and a tiny sight I set about trying to remove this problem with limited success, although they may well dry OK - can't be certain 'til tomorrow.... First attempt was to raise the water bath temperature as the problem can be caused by unequal expansion of wax and tissue during flotation - which ideally behave as a homogenous material during sectioning and flotation/drying. This condition being an indication that they were not acting this way during flotation/stretching on the water-bath....

With the water-bath raised to about 48-50 deg C things improved a little but not completely - progress but no cure. Almost there - no tissue damage - sections are very nicely cut but these darned 'wrinkles'....

The tissue may be a little too hard and therefore stretched by the passage of the blade during sectioning - both wax and tissue cut cleanly, but upon placing onto the water-bath the wrinkles in the tissue appear whilst the wax relaxes to a perfect flatness - this I suspect is my scenario.... Especially as the tough (relatively) epidermis seems to be the problem - I suspect that a shorter 'gentler' processing protocol may be the answer - time will tell as I intend to section some of the other blocks tomorrow to investigate further. I had a similar problem when sectioning some very tough mature Sunflower stems last year, which sectioned beautifully cleanly but had a troublesome and 'wrinkly' epidermis and cortex just as the far softer (in life) Dandelion pedicels have here....

(I've had a suspicion that the Histoclear II that I've changed to from my original Histoclear I may be the problem - after clearing with Histoclear II this batch were noticeably hard and 'glass-like'....)

The problem's actually quite minor - but I want to fix it - excellent practice for improvement of my slides and a great deal may be learned from this problem I think!

I've rambled-on quite a bit without any images to enjoy - back soon with some images to demonstrate this problem and my investigation of it....

Back soon,

[charlie g]

Beautiful microtechnique, Mr.Sonchas/John B, thanks for sharing you work with us. I collected more Trilliums early 4/17, my dear magenta and white and yellow Trillium species. They rest in vials of FAA...as do last years sample !

You always give off great energy with your microtechnique adventures of our plant neighbors, bravo.

charlie guevara

[mrsonchus]

Hi Charlie, and thanks for those kind words my friend, pleased you like the adventure. I stained a few more carefully sectioned slides this morning, taken at 5µ, 8µ and 10µ and have some rather better results.
However, I really did rush these through staining & mounting with the priority being to examine the tissue condition as I've a suspicion that they have been over-processed and made too hard, particularly I think by the clearing stage which displaces the OH dehydrant with the 'Histoclear II' wax ante-medium (AKA clearing-agent)....

Well, here are a few images, and the tissue isn't too bad, but I do really think that a less severe protocol may be beneficial. These Dandelion pedicel sections are quite a challenge as they are really quite tender when living, although they contain numerous vascular bundles to supply the ultimate flower-head with it's dozens of individual florets, each with it's very own ovary and ovule (and then seed/achene of course if fertilized). There is of course no secondary tissue and relatively small vascular bundles (not being part of the main vasculature that takes water to the whole plant from the roots and the photosynthetic sugars into the plant in the opposite direction).
A balance in both processing, sectioning and staining must be achieved between the very soft pithy parenchyma, the slightly harder cortex with a small measure of primary-wall thickening, the usual vascular-bundles with their secondary-wall lignification and the epidermis which is not only tough (relatively) but has to 'face the full force of processing chemistry' being the outer surface of the in-process tissue.

Well, these tissues have cut quite well with greatly reduced disruption compared to the previously roughed sections that it now seems certain to me were far too cold (having been kept in the 'fridge' at 3 deg C then sectioned at about 5 deg C). These blocks were kept as earlier post mentions, at my lab's 22 deg C and have as a result sectioned far better. The probably over-hard tissue (and indeed wax) has been helped by the softening effect of this higher sectioning temperature.

Please excuse the rather brutal and messy staining - I really did just dump the stains (Safranin + Fast-green) on to give me some quick idea of the tissue condition, the dreadful and low-contrast staining will be refined later....

Finally, a few images,
This is at 5µ - too thin for the purpose of morphology I think, but with the over-chilled blocks I was unable to actually get sections to cut below 10µ - the tissue has sectioned nicely, the staining is far too weak, but at least is good enough to allow me to see the section's quality or lack of.

There are a few broken cell-walls, but the cells (epidermis and cortex) are not shrivelled and lead me to believe that the dehydration stages may have been quite reasonably carried out - although moving further into the softer pith there is a little shrivelling evident.... See following images for pith...

This image gives a complete epidermis --> pith view where the pith is also visible, but mostly there are nice plump-looking cells. If they are a little too hard, then I think the clearing may be the culprit - maybe....


A large vascular bundle,


5µ is a little too thin I think for these harder cell-walls, the epidermal cell-wall thickening here is in line with a vascular bundle and extends it seems to two layers


Here between vascular bundles the thickening seems only to be one epidermal cell thick,


So many factors involved if the highest quality is to be achieved with a very diverse selection of tissue, these British wildflower sections are quite unusual and not the first choice for commercial slide sets, but I love wildflower taxonomy, so it's here in among the 'weeds' of the UK that I stalk my Botanical prey!

I think I will have time tomorrow to stain a few better sections with a far higher contrast, maybe even venture into the blue stains.... I'm pretty sure to be running a fresh set of tissues through from the start also, as at this time there is a huge amount of Dandelion pedicel tissue in our lawns! It would be interesting to see the effect of a really extreme protocol, maybe even a shortening of the FAA-50 'killing' stage perhaps down to 12hrs - interesting stuff!

Anyway, lots to investigate here, I'll keep you posted. Thanks for looking Mr-G and all! Hussar and onward-ho!

John B

[Nance]

FASCINATING ! will be on the look-out for more recent posts of the mighty shandon and the mounting saga...