[Histonet] Maximum bone sample size for methyl methacrylate embedding

[Jack Ratliff]

Orla,
Based upon my personal and professional experience, the sample size of undecalcified bone which can be properly processed into methyl methacrylate for sectioning and staining for Goldner's trichrome is completely dependent upon your microtomy capabilities. For example, if you only have access to a motorized rotary microtome, you are limited to a specimen size that will generally fit onto a 25mm x 75mm microscope slide. In reality, you are looking at a specimen that will be sectioned thin @ approximately 4-6 microns and have a polymerized resin block size no greater than  15mm in width and 20-25mm in cutting length. This means that your specimen needs to fit within these dimensions and at least leave 0.5 - 1.0mm in plastic surrounding the tissue. Again, this is if you are limited by having only a motorized rotary microtome like a Leica RM2255 or RM2265.
If you have something like an EXAKT cutting and grinding system for use with cutting thick sections of metallic medical device implants and then polishing sections down to 25-35 microns, you will likely use plastic slides that have a size limitation of 50mm x 100mm. This then means that your resin block will need to be contained within these dimensions of the slide, but without worry to a 0.5mm - 1.0mm plastic resin layer surrounding the tissue because you are cutting thick and grinding/polishing down to a desired final thickness. On the other hand, if you have access to a motorized sledge microtome like a Rechert-Jung Polycut S, E or a Leica SM 2500 for cutting thin 4-6 micron sections, you are only limited by the size of glass microscope slide you can purchase and of course the cutting width of the knife which is 200mm. However, most of my routine work with this piece of equipment is with a resin block limitation of 70mm x 90mm.
Please keep in mind that what I am trying to say here is that you are mostly limited by the sectioning capabilities of the equipment that you have access to for microtomy. However, I will caution you that there is also a limitation in size based upon the mastery of the acrylic resin polymerization process. As you go up in size, you increase the difficulty in the ability to manage and control the exothermic polymerization of the specimen and block. Processing and polymerization of larger specimens is absolutely something that can be accomplished without issue, but only by experience that is learned over time through repetition and patience or by training from another that has mastered the technique, something that I have done for others in the past. I hope you understand that working with MMA is not simply something where one can provide you with a protocol and then you can easily expect the same results from one person to another. While I use the same general acrylic resin protocol for every type of bone, I frequently have to adjust the processing and infiltration times to compensate for the size and density of the specimen. I also have to then change how I manage or control the exothermic reaction. This is done by controlling the rate of polymerization, proportionally to the size of the tissue and specimen block, using temperature controls like adjusting room temperature, using a water bath, etc.
Now to answer your question, the size you have defined as 2 cm x 1 cm is completely adequate and fairly routine to accomplish. If you message me back privately, I will provide you with a protocol to try. Additionally, if you are doing this for the first time and would like some initial help just to get you past this project until you can practice on your own, I can also offer my services to complete this project for you on contract. Of course, as I stated earlier, I can also train you on how to accomplish this technique as it is also something that I have done in the past for several labs both domestic and internationally.
Best Regards,
Jack

Jack L RatliffRatliff Histology Consultants, LLC317-281-1975
> Date: Thu, 13 Dec 2012 17:44:04 +0000
> From: o.m.gallagher <@t> sheffield.ac.uk
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Maximum bone sample size for methyl methacrylate	embedding
> 
> Dear Histonetters,
> 
> Would anyone advise on the maximum size sample of undecalcified bone which
> could be properly processed into methyl methacrylate for sectioning and
> staining for Goldner's trichrome? Would anyone have a protocol for
> processing large bone samples (possibly 2 x1cm) into MMA as most of the
> protocols I've seen are based on Bordier trephine 5mm iliac crest biopsies.
> 
> Thank you,
> Orla
> 
> -- 
> **************************
> Ms. Orla Gallagher
> Bone Analysis Laboratory
> Mellanby Centre for Bone Research
> D Floor Medical School
> University of Sheffield
> Beech Hill Road
> Sheffield
> S10 2RX
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> Website: http://mellanbycentre.dept.shef.ac.uk
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