Paul, I learned this trick from several of my expert bonehead colleagues who worked with some huge bone blocks in MMA. Be prepared to spend time doing this however.
It is a called the "layering" technic, but it prevents an explosion of bubbles when the MMA suddenly decides to polymerize. You can use prepolymerized layers, and we used Rubbermaid square plastic containers with snap top lids or Nalgene very wide mouth bottles/jars that have external threads for the white lid. Making prepolymerized layers is a good way to reuse your last MMA infiltration mixture waste. You can make the layers after embedding. It is important at any time to put a lid on top of your containers when making prepolymerized layers or when performing the layering technic. You must maintain the proper mixture balance or the monomer evaporates, leaving behind a seemingly properly polymerized plastic which in fact is not the case. You will end up with an ugly depressed surface that is funky looking, not a good thing to have happen and lousy as flat surface on which to lay your sample. Layering Embedding: Add approximately a half inch a to 3/4 of an inch of embedding mixture to the prepolymerized layer, place bone on top of this, and seal with lid. Stack inside a hood at RT. This embedding mixture should be polymerized before you add another layer of embedding mixture, so it may take several days to layer, polymerize, layer, polymerize, etc until you are finished. Buy a cheap metal cooking spoon or use a wooden tongue depressor to scoop i.e. baste MMA over the top of bone. Replace the lid, and be patient. The bone sometimes turns white due to exposure to the air or whatever but this is prevented by the described "basting" the bone a several times, daily or more - to coat it with the MMA embedding mixture. MMA in large quantities is touchy and really dislikes extra heat. Polymerization will slow if put in refrigerator (not good due to no fume control). Don't worry about adding non-polymerized plastic to the prepolymerized layer, as there is a smooth interface caused by the monomer dissolving a few mm of the pre- polymermized MMA. This is ideal since it forms a very tight bond after polymerization. You will see the differences in the layers as they polymerize, but if you add heat, a horrible mass of polymerized bubbles occurs, which means you have to grind the bubbles away and reembed the infiltrated bone. Been there, done that, and had to close a door due to inappropriate word usage shocking to my fellow workers. Your embedding mixture does not have to be made up daily, and make up no more than 500 mls at a time, but store in a tightly sealed bottle, in the refrigerator, and use it within 7 days. Always warm the MMA to RT before adding to the embedded bone - you must avoid water condensation contamination. We have blocks where some of the bone sticks out of the top of block, and it is coated with MMA, and without sample turning white. The block is perfecty clear, like a museum display piece. The screw top lid was the best method to prevent spills. I stacked containers up in the hood (always left ON) for both bone embedding/polymerization and making prepolymerized layers. After you are finished with embedding, make sure the top layer is above the bone unless you don't need that part of sample, and put the block in 60C oven for an hour or so to cure. You will smell the toxic fumes, so be very careful - the refrig should be vented into a hood or in the hood. Avoid a slightly tacky, sticky top block going into 60C - it will bubble at that part of layer. We did a baby alligator head along with some other large bovine and goat bone, and it took close to a month or longer, but we were ultimately very patient and had perfectly clear embedded sample. There is no STAT work when dealing with large bone in MMA. My favorite MMA mixture came from Diane Sterchi, and I never had problems after I started using her mixture and the layering method. I would be happy to send that procedure to you if you like. Her mixture contained prepolymerized PMMA powder which helped the polymerization get started. I will be off Histonet after this message, so respond to my personal email address Gayle M. Callis HTL(ASCP)HT, MT _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
