You may want to try to decrease specimen thickness and use PMMA:

http://www.ddk.com/PDFs/guidetoplastic.pdf

And it looks like you have fungus infestation of your processing tools. You need to threat tools and vessels with bleach and wash them with alcohol.

Vladimir Dusevich Thank you very much for your answer. Well i'm working in a small lab so i can't afford for all this chemicals that MMA needs. Also i have read that for MMA i should have a chromatography column, that is not mentioned in the document you sent to me (witch is very helpful).

Also by saying about fungus, i mean about the fungus activity during the diagenesis of the bone and decomposition of the body.

I clean my tools and vessels with acetone, i think that's enough, right?

I'm sorry, I'm not anatomopathologist, I am Oral and Maxillofaccial Surgeon

Dear Panagiota Bantavanou , I even do not really understand what is your task (it's beyond the scope of my experience). As for fighting fungus, I prefer bleach.

And of course no need of chromatography column for embedding.

Vladimir Dusevich You are right i wasn't clear enough. I'm taking my master dissertation right now and i'm working on undecalcified bone morphometrics. Specifically, i'm trying age estimation from the histology of the bone. So i have to embed a small part of the femur (1cmx1cm), in resin so that the bone be elastic enough and not fragile. Because after that i'm taking thin sections and grinding them until the 15-10μm. So my problem is that even i reach the 15μm. i can't see the microstructure well enough (or at all). I believe that the problem is not the resin because i changed the resin (but still epoxy), but something ales. Maybe not good dehydration? I'm not sure.

Also i'm work on archaeological material and they are full of bacterial destruction (tunnels). Witch makes the analysis even more difficult.

Well i have attached one article about the Tecnovit, that uses the chromatographic column. I don't know any better protocol for MMA. If you know please suggest me, if you like.

Panagiota Bantavanou In paper you had attached they used column for resin preparation, not for embedding. Normally people do not produce their own resin, they just buy it. If you have Technovit (used in the paper) then proper embedding protocol is here: https://www.emsdiasum.com/microscopy/technical/datasheet/14655.aspx

No air drying. Best results (whether with epoxy or PMMA) are usually achieved without air drying.

Simple protocol of bone preparation (no embedding at all):

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artmay12/ug-Bone.html

I agree with Vladimir Dusevich the Technovit 9100 protocol seems good. When you still have problems I would recommend sharing some of the images you obtain now, to see what could be the problem. I'm a bit worried about the 10-15µm for a ground section that sounds really thin to me.

I do undecalcefied thin ground section myself and maybe of help.

Good luck!

Uwe

Uwe Yacine Schwarze Thank you very much for your answer. As i referred previously now i use epoxy resin. For now i can't afford an MMA or Technovit resin. But at the end i don't believe that only resin is my problem but the preparation of the sample. The procedure before the embedding, maybe prevents the resin penetration or leave artifacts or not enough dehydration (i don't really know).

As i've mentioned at a previous comment i'm working on histomorphometrics, so i need to have very thin sections to see the osteons and etc., i have no problem on grinding but still at the 10μm. the mictostructure is not visible enough for analysis.

So generally i was hoping to have any suggestion about my sample preparation. Right now i don't have any image to show you, i'm not at the lab and i don't have access. But i will send you as soon as i get them.

Dear Panagiota Bantavanou thank you for the additional information. I'm an anthropologist by education so I know you need to count how often osteons are remodeled to get the age estimation. Depending on your experience a general staining like toluidin blue or a kind of fuchsin staining is enough a good but a bit more complicated staining is a silver staining but the lamellae and osteons will be nicely visible when done right.

In my experience when you go too low with the section you actually get rid of the sample itself and what you see in the microscope is only the imprint of the glue of the bone which is not stainable. This happens at about 50µm and less, depending on your glue thickness, precision of grinding tools, and skill. I would recommend that you grind a slice until 60 µm, polish with 4000 grid paper, put it in a light microscope, and close a bit the aperture and you should be able to look and move through about 5 to 10 cell layers. There you should be able to see Volkmann and Havers channels (without staining just haven high contrast with an almost closed aperture). Whean all this is the case, you should decide between two ways to go forward. 1. stain it and put an opaque (white translucend) slice underneath which diffuses the light and transfers it to the top stained cell layer so you do not see the others (this can be done also with 150µm thick slices) or 2. grind down to 1-3 cell layers, polish, stain and then you have hopefully the result you want but a higher risk that the bone get lost. Always check between if there is still bone on it. I would bet that with 10-15µm there is no real bone left on the slice usually that is the thickness of the glue.

With the resin it's a different problem. I rarely used epoxy resin to embed but its possible. You have two problems with epoxy: moisture and the curing process. When you have dry archeological samples I would clean them and dry them in a drying box completely (be careful cracks may occur) and put them directly in resin. When you have wet samples you need to dehydrate them with a substance that mixes with the resin you will use later, at least in the last step. This can be ethanol, isopropanol, xylol, acetone, and so on.

The next problem is that you can't control the curing as easily as with other resins. It really depends on what kind of resin you use. What you want is that the resin cures slowly and in the whole sample also in the inner part. When for example you have a small amount of hardener and mix it with a lot of resin and the curing is fast then there is a high probability that the resin with the hardener doesn't reach the inner part of the sample. You have to find a way to prepare the sample that everything gets hard. Maybe you could try to use less hardener in a step let the sample soak in for a while and then use the total amount; something like that. I'm sorry for the long post but there are so many things you should do different depending on the resin and sample and there are still a lot of details I omitted. Hope that helps. Best wishes Uwe

Uwe Yacine Schwarze Thank you very much for your help! All the information are very helpful.

You mentioned about staining. I'm very hesitant about that, i'm afraid that it will be a mess because the stain will fill the tunnels from the bacteria and the image will be very bad.

May i contact you again as soon as i have images to share with you, and some more details if you want?

Dear Panagiota Bantavanou, the stain should not go into the tunnels (usually from fungus) because they are filled with resin. Remember with a usual thin ground section you don't do deplastination.

Yeah sure you can also contact me under my e-mail address uwe.schwarze(a)medunigraz.at

Good luck and best wishes

Uwe