The only way to guarantee a bone is not overexposed to acid is to perform an endpoint test. RDO has approximately 15% hydrochloric acid plus additives, so it is very fast, plus the color is very dark making it difficult to see the bone in the decalcifier. However, it can be diluted with water, even in half and still remain a rapid decalcifier with excellent staining results, but the endpoint test is needed.
Subjective testing is not considered accurate and these tests include mechanical testing where probing can create a probe track artifact in the bone, bending can disrupt tumor from bone or interface of bone and tissues/cells, plus create false trabecular fracture artifact. Slicing the bone to feel gritty-ness, this is accurate for detecting very tiny calcifications, or you can just plain miss them, same with a probe.
Bubble testing is inaccurate - as the calcium ionizes, CO2 is given off too, and resides as bubbles on the surface of the bone. As the calcium deposits become smaller the bubbles are smaller, and hard to see. One lab where I worked used this for tiny bone marrow biopsies which would float upwards at end of decalcification, however, there was often residual calcium deposits in bone, requiring surface decalcification of block face. Seeing bubbles in RDO is going to be difficult due to the color.
The most sensitive method is radiography with a FAXITRON, but most labs do not have this enclosed x-ray machine. We, unfortunately, gave our FAXITRON away, a terrible mistake and had to rely on another test which works well, although not the most accurate way to test.
Either do the chemical test which is easy to do, just read the test fluid against a black background, an old med tech trick, to detect cloudy ppt and use a clear water blank for comparison. Do not stir the fluid when doing this test and only one bone per container for accuracy, The aliquot should come from container bottom where Calcium settles during decalcification. If the bone is not decalcified, then you should rinse with tap water briefly, as calcium ionized by acid can reside on bone surfaces. You don't want that to happen when you put bone back into fresh decalcifier.
We now use the weight loss/weight gain method (developed for testing nitric acid, and was coupled with chemical testing at the very end.) We do not bother with further chemical test after the weight it gained. It works well, since one can stir (this also causes bubbles to disperse) and do more than one bone, suspended in decalcifying fluid, in a container. Often we have 50 murine bones in one container. The balance must weigh in milligrams for accuracy, and you need to blot bone with paper towel before weighing. The joy of this method is speed and efficiency and works perfectly well for EDTA decalcification which we do frequently for research bone protocols.
If you want the latter two methods, I will be happy to send them privately.
Whatever you do, don't "overdecalcify" the bones.
Gayle M. Callis
Bozeman MT 59715
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