Rocking microtome practice session

[mrsonchus]

Hi all, getting the hang of my new rocker - thought you may like to see my practice session, cutting sections into ribbons then moving them to and manipulating them in a water-bath..
I started processing some plant parts today, they'll have fixed by the morning, should be able to dehydrate & maybe even start clearing tomorrow. I'll post more on the process as I fumble through it, and who knows - I may even make some proper sections soon (fingers crossed) .


Resulted in some really nice even sections:

ws_700x525_sections_in_flotation_bath (2).jpg (63.64 KiB) Viewed 4921 times

ws_700x525_sections_in_flotation_bath (5).jpg (97.86 KiB) Viewed 4921 times

These are from a larger wax-block I cut to a different shape:

ws_700x933_sections_in_flotation_bath (1).jpg (65.48 KiB) Viewed 4921 times

Won't be long before I'm firing-up the incubator! Real sections on the way (hopefully)..

Hope you like the updates - tell me to stop if they get a bit boring.

[75RR]

Hope you like the updates - tell me to stop if they get a bit boring.

No complaints here.

[Crater Eddie]

I, for one, am enjoying your updates very much. Love the video.
CE

[Seb28]

Well done,keep going.It will be very useful material for someone who takes the first steps in histology.

[mrsonchus]

Well done,keep going.It will be very useful material for someone who takes the first steps in histology.

Many thanks - samples are at this moment going through dehydration......

The dehydration series

ws_dehydration_01.jpg (41.68 KiB) Viewed 4895 times

Next step will be clearing....

[Naphthalene]

mrsonchus, I'm also watching the story
What are the samples you are processing currently? And what are their (approximate) dimensions? Maybe I'm wrong but It appears to me that your samples are kinda too large. In my experience I used samples below 1x1 cm in length/width, but I leave open the possibility that larger samples can be cut too.

[75RR]

Hi Naphthalene,
my Microtome has not arrived yet -
so can only mention that the manual says 20mm by 20mm (actually it says "cuts specimens up to 20mm diameter" see page 3.

Cambridge Rocking Microtome Manual: http://goo.gl/fkwpCJ

[mrsonchus]

mrsonchus, I'm also watching the story
What are the samples you are processing currently? And what are their (approximate) dimensions? Maybe I'm wrong but It appears to me that your samples are kinda too large. In my experience I used samples below 1x1 cm in length/width, but I leave open the possibility that larger samples can be cut too.

I trimmed the pieces to my expected usage sizes at the end of dehydration - saves a lot of expense on chemicals too. You are quite right, as pictured straight from fixer they are unsuitably large. I've samples now clearing (via a Histoclear/IPA transition) that are (approx) as follows:
The complete (red) Schlumbergera-flower ('Christmas Cactus' - although this variety is not truly the 'Christmas' variety) has been trimmed to pieces about 1cm maximum in any dimension - it's the stamens and carpels I'm mainly interested in here.. (although I'll take some pollen for comparison to earlier IPA-fixed slides).
I have some lengths of post-flowering (blue) Crocus leaves that are about 4mm wide and 1cm long;
sections of Schlumbergera leaf of about 1cm wide and 1cm long.
I also have some stems and leaves from a tiny Germander-Speedwell growing in a derelict plant pot in my garden - these are tiny pieces (albeit whole!) the stems are about 1mm diameter in lengths of about 7mm, the leaves are about 3mm x 5mm and about 500µ thick!
I've chosen a challenging mixture of specimens and I'm expecting trouble!
The Speedwell is simply tiny, the Schlumbergera leaves are large and soft-centered with quite a stiff cuticle, the Crocus leaves are quite hard and brittle and the Schlumbergera flower parts are tiny, awkward to handle and almost invisible after clearing (I suppose I should have applied temporary staining for visibility in the wax-blocks but I wanted to keep things (chemically at least) simple) - it's not going to be an easy first-attempt!
Almost forgot - I've also included several sections of small Aloe leaf, each about 1.5 cm long and about 8mm wide, they're quite fleshy but don't really have a hard cuticle - they are full of goo though - I managed to get very good results with similar sized & shaped Aloe samples using my hand microtome so thought it would be informative to try a comparison with the rocker's far thinner sections...

If it all goes horribly wrong - I'll simply try again until I get it right! - 'Microscopists know no fear!'
Can't wait for tomorrow - it's infiltration next!! I'm having a great time!

[75RR]

Hi Naphthalene,

What are the samples you are processing currently? And what are their (approximate) dimensions? Maybe I'm wrong but It appears to me that your samples are kinda too large. In my experience I used samples below 1x1 cm in length/width, but I leave open the possibility that larger samples can be cut too.

Had another read through your post and it got me wondering why you recomend sample sizes below 10mm x 10mm when the manual states 20mm x 20mm as max.
Is it that to cut 20mm x 20mm samples requires an extremely sharp blade - an impractically sharp blade, as in very hard to achieve?

[mrsonchus]

Hi all, on the subject of optimum or possible section-cutting sizes..
I rather suspect that the maximum would indeed be somewhere in the region of 2cm x 2cm as this size would appear perfectly reasonable when looking at the block-holder size for example. I like the 1x1 cm size also because it's about right for slides - although of course series of far smaller slices may also be mounted for comparisons on the same slide. All I can definitely say myself is that the rocker cuts superb smaller and larger block-sizes - as long as the consistency of the embedded specimen is close to the wax itself (the main point of infiltration rather than simple embedding as per my hand microtome adventures) I'd think that either size would have good potential - achieving complete inter & intra cellular infiltration seems to me to be the key. I'm going to run quite a few different trials of procedure for comparison as soon as I master the basics! I've still never cut an actual specimen-containing section at this time!
Going to bed now - I've just added the first wax-chips to my room-temp Histoclear-drowned specimens as per Ruzin's excellent book and I must sleep if I'm to dream of the perfect 6µ permanently-mounted, fully stained slide!
Hope my tuppence-worth helps a bit.
Back tomorrow with tales of infiltration and incubator-testing in preparation for adventures with liquid-wax!

[mrsonchus]

Here are a couple of pictures of the fixed Aloe leaf pieces - they look a bit poorly!

ws_aloe_fixed_sample_1.jpg (52.44 KiB) Viewed 4855 times

ws_aloe_fixed_sample_2.jpg (80.73 KiB) Viewed 4855 times

These seem to me to be about right to take into the clearing stages. There are still many large pieces in fixer left over for another time, they'll stay fine for years I suspect - and their larger size preserves the ability to choose exactly which aspect or piece I would like to study further. It seems to me that the big reduction in size is best made after dehydration & before clearing, the tissues also 'firm-up' a little by then but are still not too transparent to actually handle easily for final specimen selection...

I'll soon find out!