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Journal of Dental Research http://jdr.sagepub.

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Decalcification of Bone and Teeth Under Vacuum- A Rapid Method for Producing Hard Tissue Preparations
Jens Waerhaug J DENT RES 1949 28: 525 DOI: 10.1177/00220345490280051601 The online version of this article can be found at: http://jdr.sagepub.com/content/28/5/525.citation

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DECALCIFICATION OF BONE AND TEETH UNDER VACUUM-A RAPID METHOD FOR PRODUCING HARD TISSUE PREPARATIONS
JENS WAERHAUG, L.D.S. From the Histo-Pathological Department, State Dental Institute of Stockholm, Stockholm, Sweden

THE time required for decalcification of histologic specimens must be considered a technical problem of great importance. For those doing experimental work, it is rather nervebreaking that several weeks often will elapse before slides are available. This delay is especially inconvenient because planning of new experiments depends to a great extent on observations gained from earlier ones. The modification of the paraffin method for embedding has been a forward step, but even by using this method, it is practically impossible to have slides made in less than 45 days because decalcification takes from about 30 to 40 days if nitric acid is used. Trichloracetic acid requires about double that time. In order to shorten the time for decalcification, the author tried to carry out the same process under vacuum. The result was very promising. A rather large piece of a jaw with teeth which would have required 35 days was ready in less than 3 days. The equipment needed is shown in Fig. 1. If the water pressure is sufficiently high, a vacuum pump fixed to the water tap can be used. It must be constructed with a valve in order to avoid water flowing back into the bottles in case the pressure should temporarily decrease. As seen from Fig. 1, the bottles may be coupled in series making it possible to use the same pump for an indefinite number of bottles. These should be provided with rubber corks because ordinary corks are not sufficiently tight. For larger specimens containing bone, it is convenient to have bottles of larger size than those shown in the illustration to permit decalcification without changing acid. Histological slides from bone and teeth decalcified under vacuum are in all cases of as high quality as those produced in the usual way and in many cases they seem to be better. This may be because the tissues are in contact with the acid for a shorter period of time. There is no indication of damage caused to tissues or cells because of the faster chemical process. The reason that dissolution of inorganic bone salts is accelerated under vacuum may be that the carbon dioxide evaporates more easily and new acid comes in contact with undissolved bone at an increased rate. During the decalcification, bubbles containing carbon dioxide are seen rising continuously from the object. As soon as the decalcification is finished, this current of bubbles will stop. As a margin of safety it is recommended to continue the process from 12 to 24 hours after the bubbling has stopped.
Received for publication, December 20, 1948.

525
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It is a commoit experience that the time requr ed for decalcification may be shortened if nitric acid is used. This is because nitric acid usually is applied in a much stronger concentration. than trichioracetie acid. Both have about the same degree of dissociation, and both are among the most dissociated acids known. As a rule they are used in paper cent solution. bntl the nitric aci(d 5 usually is diluted 'by volume per cenet, Iwhile the triebloracetic acid is diluted by weight per cent because of its cry stalline foram. If their molecular weight had been the same, the nitrixc acid (spec. weight 1.4) a prior would have been 1.4 times stronger. The molecular weight of trichloracetic acid is 1.63 and that of nitric acid is 63. This neans that 163 grams of triebloracetic acid are required to gixc th~e same number ot acid radicals as 63 grams of nitric acid.

The -satme w eight of both aci&h d1i.soilved in equal qiuantititbs of Watter nmakczs the nitric acid 2.6 (163 63= 2.6) ttmes utore conceeitratcd than the triebloracetic acul. Nitric acid is used in. a concentrattion, 3.64 (2.6 x1.4) times stronger than triehloracetic acid. This may lend soite exphlatatiott as to why better results are obtained with trichioracetie aeid.
SUMMtARY

By erforning the decaletficatmot under xacuum it has beet possible to reduce the tinte required for this process to otme-tenmth. The rate of time process is }prop-ortioital to the xaeuuna. For that reasotm it is recommended to use a rather hight 'degree of v acuut. (about 74 cm,. Hg.) The quality of the slides obtajued imt this wxay ms usually better, no disadiautages have beet observed, after using the method for 6 months. It is pout ted out that nitric acid usually is applied hm a cotteentration. more tltam tlmree times stronger that triebloracetie acid. This may explaiti the faster deealeifleation with nitric acid ammd time better result obtianed with trichlroracetie acid.
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