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PostPosted: Sun Dec 02, 2018 3:23 pm 
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I recently purchased an AO820 microtome and I am now experimenting with it. I have been able to make a few sections with it but it turns out that the cell walls are shattered in all of them. Some of the sections are so bad that none of the cells are intact. I have no idea what I did wrong. Can someone help me?

I dehydrated the sections with the following series:
70% IPA for 2hrs
90% IPA for 2 hrs
100% IPA 2hrs,
100% IPA 2hrs,
100% IPA, overnight
toluene, 2hrs,
toluene, 2hrs,
molten paraffin wax 2hrs,
molten paraffin was 2hrs,
molten paraffin was 4 hrs

After sectioning I rehydrated using the following series:
2 changes of toluene for 30seconds
2 changes of 100% IPA for 30s
70%ipa for 30s
water for a few minutes

I also used a different way of rehydrating with xylene and ethanol with even worse results.


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PostPosted: Sun Dec 02, 2018 5:14 pm 
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Hi DeeJay,

I think you didn't mention what it was that you actually cut - the tips of your fingers, a shark tooth or a flower stem? :D

Bob


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PostPosted: Mon Dec 03, 2018 1:48 am 
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Hi, need more details to help. Have you any images, what are you sectioning, what type of blade are you using (e.g. disposable or steel re-sharpening), what thickness are your sections, how are you floating the sections before placing on slides, wax-type (melting/using temp for infiltration and embedding stages)....

As much detail as you have will help greatly.

John B.

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PostPosted: Mon Dec 03, 2018 9:28 pm 
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I am using a steel re-sharpening blade to cut plant material. I tried sectioning the stems of a hydrangea leaf. I also sectioned sellery. I tried both 20µm and 7µm cuts. Both had shattered cells. I included a picture of what it looks like. The first attempt I made with the 20µm cut was a bit better. It had much more stucture left. Unfortunately, I didn't take a picture of those.

I am floating the sections using a bowl of hot tap water since I don't have a tissue floating bath. I am using wax with a 56C melting point.
Attachment:
shattered cells_small.jpg
shattered cells_small.jpg [ 213.2 KiB | Viewed 375 times ]


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PostPosted: Mon Dec 03, 2018 10:13 pm 
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Is this tissue on the slide and de-waxed?
Ideally an image of a section still in the wax section but dried onto a slide before de-waxing.

From this image I can't really tell you anything I'm afraid. Also are you able to take an image of your knife's edge under a stereo 'scope so that I can see it's condition, although a complete wax section would suffice for this also.

This is a complex question with a similarly complex answer and much more information is needed, including your stage of processing right from cutting the plant into pieces before you begin....

I can help you with this, but have nothing to go-on with this image I'm afraid.

Here's a good section, with good tissue integrity, as imaged still in the wax as mentioned above - no tears or nicks in either the wax or the tissue it'self,
Attachment:
ws_sonchus_asper_in_wax (1)_stitch.jpg
ws_sonchus_asper_in_wax (1)_stitch.jpg [ 106.69 KiB | Viewed 370 times ]


This is a tragically poor section with scores in wax from a poor knife-edge, torn tissue from poor processing, etc,
Attachment:
ws_F1_in_wax_before_drying.jpg
ws_F1_in_wax_before_drying.jpg [ 331.97 KiB | Viewed 370 times ]


Images such as these will be needed to give you useful advice, of the sections on slides and dried but not yet de-waxed.....

John B.

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PostPosted: Tue Dec 04, 2018 9:16 am 
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I'm absolutely no expert on this matter but I would suggest to make thicker cuts like 40µ or 60µ as a starting point. You knife edge has to be really sharp and honed for good cuts. You rotary microtome can't do a slicing cut so the knife really has to be perfect. The knife edge needs a free angle to the material so the steel does not rub on the block after the cut is done. The necessary free angle depends on the shape of the knife directly behind the cutting edge. How do you keep your knives sharp and honed? Some knowledgable people hone the knife directly before cutting to acheive the best results.

Bob


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PostPosted: Tue Dec 04, 2018 9:19 am 
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Here is a link to a good beginners document in german language about knife sharpening: http://www.mikroskopie-bonn.de/_downloads/Abenteuer_Klingen_Schaerfen.pdf


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PostPosted: Tue Dec 04, 2018 7:10 pm 
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I tried inspecting the section under the microscope directly after cutting but it appeared to be opaque. I could not get a clear image so I proceeded to rehydrate it. I didn't heat it after cutting. Maybe that would make it clear again.

I own a stereomicroscope so I can get an image of the knife. It is not a trinocular one so I'll have to improvise. I don't think I have time for that before the weekend. To be honest I didn't sharpen or hone the knife. I just bought them second-hand online. The add said that the knife was sharp and ready for use but now I am not so sure.

So far, I have been unable to make ribbons when cutting. The sections always curl up or crumple. I remedy that by using a thin brush to push the start of the section down. The sections also tend to stick to the block rather than to each oher. I will make some thicker cuts and take a picture before hydrating.

Thank you for your tips. I wouldn't know where to start myself :).


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PostPosted: Tue Dec 04, 2018 7:47 pm 
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DeeJay wrote:
I wouldn't know where to start myself :).

This is a very good place to start:
http://user.xmission.com/~psneeley/Personal/AO%20820%20Microtome.pdf

MichaelG.

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PostPosted: Tue Dec 04, 2018 9:25 pm 
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Oh, when you refer to rehydration, have you floated the sectionson water at about 42 deg C to allow them to expand and relax, before picking them up with a slide and drying then at an angle so that they stick to the slide, then when they are dry they must be immersed in a wax solvent to remove the wax. The, the rehydration may begin, if you intend to stain them with aqueous stain/s.
The entire process is very long and must be done correctly, an un-sharpened knife is of no use whatsoever I'm afraid.

You need to be certain you understand the complete process, of which sectioning is only part, before having a chance to produce sections and permanent slides.....

Have a look over some of my older posts where I have progressed through the entire process from a total beginner to making permanent slides - there's an awful lot of learning and work to be done old chap. I started to learn sectioning in the first part of 2015 I think, that's where my posts will begin.

John B.

Can you give a complete description of your entire process, before and after sectioning, right from the selection of a plant to use - a lot I know, but there's no shortcut with sectioning I'm afraid. The more you are able to tell us the more we can help.

p.s.with an unprepared knife you will not be able to section successfully - believe me, I know this to be true....

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PostPosted: Fri Dec 07, 2018 8:04 pm 
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Yes, I have floated them in water to allow them to relax but the water was closer to 50c. The complete process I used is as follows:
I cut 5mm pieces of the stems of the leaves of the hydrangea leaves. I put them in FAA for a couple of days. Then I dehydrated the sections as described in my original post. I don't believe I left anything out.

I took some pictures of the microtome blade with my stereo microscope. I added them to this post. Two of them are zoomed out and show most of the area that i was using. The others are at 40x magnification. Now that i have been able to view them on the screen, they look horrible.

So what is the best way to sharpen them? I have a whetstone that I use for my kitchen knifes. Can I use it for microtome blades as well?

I will try to take some pictures of the sectioned slide still covered in wax tomorrow. I have an adapter for my camera now so I should be able to take good pictures. In the mean time I will take a look at John B's posts.


Attachments:
File comment: zoomed out
right side.JPG
right side.JPG [ 276.66 KiB | Viewed 224 times ]
File comment: zoomed out
left side.JPG
left side.JPG [ 264.4 KiB | Viewed 224 times ]
some more damage.JPG
some more damage.JPG [ 176.45 KiB | Viewed 224 times ]
rough edge.JPG
rough edge.JPG [ 179.6 KiB | Viewed 224 times ]
more or less undamaged.JPG
more or less undamaged.JPG [ 210.08 KiB | Viewed 224 times ]
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PostPosted: Fri Dec 07, 2018 8:23 pm 
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ARRRGGGHHHHH!! :o :shock: :!: :(
This brings back hideous memories..... :D

Hi, yes, the knife-edge is just the way mine went when I started with a 'rocking microtome' and such a steel resharpened blade.
I soon discovered that resharpening the blade is virtually impossible, to get both the finish and the angle needed for microtomy - this I learned very quickly!

My advice to you is to find then buy a disposable microtome-blade holder - of the 'low profile' type. Then of course you will be free of this tyranny and be able to use disposable blades, as do I with my Shandon rotary, very similar to your machine.

You'll see these details within my posts here as I started here from scratch and posted my long and hard progress. The acquisition of a reusable/disposable blade-holder and low-profile blades is absolutely essential in my experience.

John B.

Here's a holder that should fit your 'tome,
Here are the blades that go into it.

You can do it, but this is a vital requirement I'm afraid.....

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PostPosted: Fri Dec 07, 2018 8:37 pm 
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MicroBob wrote:
Here is a link to a good beginners document in german language about knife sharpening: http://www.mikroskopie-bonn.de/_downloads/Abenteuer_Klingen_Schaerfen.pdf


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PostPosted: Fri Dec 07, 2018 10:38 pm 
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MicroBob wrote:
Here is a link to a good beginners document in german language about knife sharpening: http://www.mikroskopie-bonn.de/_downloads/Abenteuer_Klingen_Schaerfen.pdf

DeepL will be working overtime ...

MichaelG.

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PostPosted: Fri Dec 07, 2018 11:30 pm 
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The Microscope and its Use, by Munoz and Charipper, has a large chapter on several methods for sharpening microtome knives. There are also instructions for examining the edge under the microscope and what to look for.

Unfortunately, I haven't been able to find a website where it can be downloaded for free. Archive.org has a method of "borrowing" an electronic copy, but I've never tried to use that.

https://archive.org/details/microscopeitsuse00mu

Abebooks has a copy for $6.00 plus $3.50 for shipping. I bought a copy from them several years ago, and the microtome knife use and sharpening information is very thorough. I haven't attempted to sharpen any of my microtome knives yet, that's on my to-do list for after New Year.

https://www.abebooks.com/book-search/ti ... noz-frank/

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A/O 10 Series Microstar
A/O 4 Series Microstar
A/O 4 Series Phasestar
A/O 4 Series Apostar
A/O Cycloptic Stereo
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PostPosted: Sat Dec 08, 2018 12:31 am 
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Thanks, Rick

I have found the Muñoz & Charipper book at Hathi Trust:
https://babel.hathitrust.org/cgi/pt?id=mdp.39015006851300;skin=default;view=image

Members of 'partner institutions' can download the whole book, but even we mere mortals can view and save individual pages.
... Slightly laborious, but very worthwhile.

MichaelG.

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PostPosted: Sat Dec 08, 2018 6:40 am 
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MichaelG. wrote:
DeepL will be working overtime ...MichaelG.


Hi Michael,
I just mention these documents in german language when I think the fumbling with the foreign language might be worthwhile for the reader or when it is just the only text I know of on this topic. The linked document on knife sharpening has the advantage that it tackles the topic from the amatuer standpoint of today, no lab technician or company representative at hand, a knife in unknown condition... Some microscopy recipes are difficult to follow for the amateur today because they are based on the idea that you have unlimited supply of chemicals, buy matching equipment new, you use it all the time and cost is not important. Also there are sometimes old rules that need to be challenged.

Bob


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PostPosted: Sat Dec 08, 2018 11:04 am 
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MicroBob wrote:
MichaelG. wrote:
DeepL will be working overtime ...MichaelG.


Hi Michael,
I just mention these documents in german language when I think the fumbling with the foreign language might be worthwhile for the reader or when it is just the only text I know of on this topic. [ ... ]

Fully understood, Bob ... and greatly appreciated.

With tools like Google Translate and DeepL freely available, we can all make use of texts in many languages:
My comment was entirely positive ... I probably would never have found that document myself: You presented it, and I thank you !!

MichaelG.

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PostPosted: Sat Dec 08, 2018 11:44 am 
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Hi Michael,
I didn't see you comment negative, I just wanted to make my intentions clear for everybody. I myself found electronic translations quite effective for languages I have a little understanding of or that are written at least in our script. With Japanese or Russian it starts to become confusing, which is a pity as there probably is a lot to be found in these languages. I used to have a Pentax K-30 DSLR, a type that tends to give problems with the aperture solenoid. The russians had gotten quite far in identifying and explaining the problem. In an english speaking Pentax-forum it was possible to find a russian speaker who was able to do the necessary clarifications of the electronic translation and set a couple of repair guys going. :roll:

Bob


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PostPosted: Sun Dec 09, 2018 11:43 am 
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Quote:
Here's a holder that should fit your 'tome,
Here are the blades that go into it.

You can do it, but this is a vital requirement I'm afraid.....


Ouch! That's more than I paid for the microtome :(.

So everybody here thinks that the microtome blade is the culprit here. That leaves me with two options: either I find a way to sharpen the blade or I switch to disosables. I did some reading about sharpening and it seems to involve a lot of skills and patience... and I have neither of them. I think I will look into getting a holder for disposable blades.

Another thing I can try is to send the blade to a company that sharpens blades as a service. The only thing I am afraid of is that they are used to sharpen blades for lawnmowers and chisels. I think sharpening microtome blades is not comparable. What do you guys think?


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PostPosted: Sun Dec 09, 2018 12:44 pm 
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Hi again,

I've been through this exact-same scenario as I was learning how to section from the paraffin-method,as are you now. I also considered sending blades to be sharpened, and soon discovered that that was a no-no due to cost and the fact that the steel knives really last only about 2 days with a perfect edge, even less (perhaps only 2 sections...) when learning.....
I then bought new ones at about £25 each and found the exact-same thing.....

By this stage I had to have a new approach, and thankfully I found one, as detailed in one of my posts from 2015.

Basically to identify the blade as the very significantly major factor I, perhaps somewhat speculatively but even so, sticky-taped a single-sided razorblade to the ruined steel knife and tried this to section using my microtome of the time which was a rocking-microtome, before as now I progressed to the 'Mighty Shandon' rotary that I now use...

The result was indeed a definitive answer - the blade was the culprit - no question.

Here's a link to this thread with pictures that will show you how I progressed

The sooner you get a disposable blade-holder (make certain it'll fit into the 'jaws' of your 'tome of course, but I think the std low-profile holder should be fine) and a pack of blades (I just bought another pack of 50 feather S35 low profile blades for £25 on e-bay as always) the sooner you'll be able to crack-on.

Check-out the link and in the meantime I'll look for some more of my results for you....

John B.

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PostPosted: Sun Dec 09, 2018 12:49 pm 
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Hi again, here's a post of mine where I stuck a scraper-blade to the rockers steel blade and got perfect sections, the next stage that beckoned was the acquisition of a 'proper' microtome that took disposable blades - the rest, including the 'Mighty Shandon's arrival, is history!

Have a look at this thread and my short video therein...

I'll look for some more,

Aha - here's a good one, this thread includes a complete protocol......

Here's a video of the disposable blades in action, https://youtu.be/170HrJbGlo4?list=PL1NN ... qlxZgCyeTo
I haven't embedded it here as this isn't my thread, but just follow this address to my video of the Mighty Shandon and a disposable blade in action....
John B.

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PostPosted: Sun Dec 09, 2018 10:02 pm 
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The images in that thread look amazing, especially considering that they were made with dirt cheap razorblades. You have convinced me: I am going the disposable route.

For a metalworker it shouldn't be hard to create a disposable blade holder. It is basically just two strips of metal with a blade sandwiched between them. I would prefer using the standard razor blades because they are cheap and readily available. Also, you cannot use most of the blade anyway so I don't seen any benefit in using the official disposable microtome blades. Unless they are sharper than the razor blades.

I'd like to try your sticky tape approach first. Except I am going to replace the tape with magnets. They are pretty strong and should be able to hold the blade in place.


Attachments:
improvised disposable blade holder2.jpg
improvised disposable blade holder2.jpg [ 197.9 KiB | Viewed 96 times ]
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PostPosted: Mon Dec 10, 2018 1:54 pm 
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Looks like good progress old chap!

The entire length of the disposables is used, in stages. The blades are able to be slid along the carrier 'from one end to the other' as it were.
I also use extra-long blades (because I bought a large number of them really cheap a while ago on e-bay.... :D ) of which I use essentially one end to 'rough and prepare' the wax block's face, and the other end to actually make the high-quality final cuts.... A sort-of two in one arrangement if you like.

You need to consider the requirement of a smooth and unobstructed 'run off' for the sections and ideally ribbon to travel across after cutting - your current arrangement seems a little difficult with regard to this aspect - I too found this to be a problem when I made my own clamp with screws, metal bar and wingnuts - then realised there was nowhere for the sections to go after the blade's edge! :oops:

Cheaper blade holders may be bought from India, and our own (U.K.) Brunel Microscopes sell such a holder.

Here's a screen-snip of the Brunel holder,
Attachment:
brunel blade holder.JPG
brunel blade holder.JPG [ 25.23 KiB | Viewed 60 times ]


As seen, there's a lever on the LH end that clamps/releases the blade - simple, without screws too....

Hope this helps a bit,

John B. :D

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