TEETH, DECALCIFICATION 



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TEETH, DECALCIFICATION 



phuric, or five per cent chromic, acetic 

 or citric, is run under a propped cover 

 slip over the section. Action may be 

 stopped at any point by substituting 

 water for acid and the remaining mate- 

 rial stained and mounted as de- 

 sired without disturbance. Boedeker's 

 method of "celloidin-decalcifying" is 

 also said to give good results (Funda- 

 mentals of Dental Histology and Embry- 

 ology, New York, The MacMillan Co., 

 1926, p. 223) and allows sectioning of the 

 organic remainder in any plane. See 

 Enamel. 



For the examination of sections of 

 whole teeth without enamel or for teeth 

 in relation to the bone of the jaws five 

 per cent nitric acid in water has been 

 found by most investigators to give con- 

 sistent results. Hydrochloric acid may 

 be used but causes too much swelling. 

 For delicate objects one to five per cent 

 nitric acid in 70 per cent alcohol may 

 prove superior. 



Recently, excellent results have been 

 obtained with the use of formic acid 

 according to the technique of Morse, 

 J. Dent. Res., 1945, 24, 143-153. Two 

 solutions are made as follows: Solution 

 A: 1 part 90% formic acid C.P. and 1 

 part aq. dest.. Solution B: 20 grams 

 sodium citrate C.P. and 100 c.c. aq. 

 dest. At the time of use combine 

 equal parts of A and B. Change solu- 

 tion daily until decalcification is com- 

 plete as shown by chemical test. (See 

 below.) 



Celloidin imbedding before decal- 

 cification helps preserve tissue relation- 

 ships (See Teeth, celloidin technique). 

 Arnim has perfected a technique of 

 double imbedding for rat jaws and teeth 

 which, though tedious, yields beautiful 

 results. Enamel matrix is frequently 

 preserved. (Anat. Rec, 62, pp. 321- 

 330, 1935.) This method has been 

 modified by Burket for larger teeth 

 (McClung, p. 366). 



Tooth buds may be decalcified after 

 paraffin imbedding by the following 

 method given by Dr. L. R. Boling in a 

 personal communication. Carefully re- 

 move from the tooth bud all surround- 

 ing bone. Fix, dehydrate, clear and 

 imbed in paraffin in the usual way. 

 Shave away paraffin and soft tissue from 

 one surface of the specimen so that 

 enamel is exposed. Immerse block in 5 

 per cent aq. nitric acid until decalci- 

 fication is complete. Place in 5 per cent 

 aq. sodium sulphate for a few hours. 

 Wash over night in running water and 

 reimbed, handling the tissue as gently 

 as possible in order not to disturb rela- 

 tionship of hard and soft tissues. This 

 method permits demonstration of Golgi 



apparatus and mitochondria in amelo- 

 blasts and odontoblasts in situ. It 

 works best with teeth of small animals 

 easily penetrated by fixative. The 

 paraffin protects the soft tissues but does 

 not interfere with action of acid on 

 enamel and dentin. (See also Teeth, 

 Developing.) 



Successful preparation of decalcified 

 tooth sections depends as much or more 

 on the care of the tissues before and 

 after decalcification than on the actual 

 process. Good fixation of the pulp 

 tissue is difficult but essential to pre- 

 vent shrinkage. Ten per cent formalin 

 in physiological salt solution may be 

 used for several days or weeks without 

 injury to the soft tissue and allow 

 thorough penetration. Better results 

 are obtained in a short time if the fixa- 

 tive can be perfused through the blood 

 vessels. In the preparation of human 

 or other large teeth fixation artifacts 

 are minimized if the tooth is ground 

 longitudinally on a flat stone until the 

 pulp is just exposed. Two opposite 

 surfaces may be ground. Grinding 

 should be done on a sharp stone under 

 running water to prevent heating. 

 Cutting of holes through the dentin to 

 the pulp or the amputation of the tips 

 of teeth is often resorted to in order to 

 get better penetration but these meth- 

 ods are apt to disturb the position of 

 the pulp and should be avoided if pos- 

 sible. After decalcification the teeth 

 should be carefully handled and the de- 

 hydration process should be slow to 

 prevent separation of tissues of different 

 densities. The substitution of n-butyl 

 alcohol for ethyl alcohol and xylol in 

 dehydration and clearing processes has 

 proven advantageous (Morse, loc. cit.). 

 By this method dehydration may be 

 prolonged with less hardening. 



Over decalcification should be care- 

 fully avoided because it will partially 

 destroy the dentin matrix, cause sepa- 

 ration of tissues of differing consistency 

 and disturb staining reactions. Testing 

 for completion of decalcification by prob- 

 ing with needles or bending and squeez- 

 ing in the fingers should be avoided at 

 all costs if tissue relationships are de- 

 sired. The progress of decalcification 

 can be followed radiographically but the 

 end point can not be accurately deter- 

 mined. The best method of testing is 

 that described by Arnim (loc. cit.). 

 Five cc. of the acid used in decalcifica- 

 tion is placed in a clean test tube and 

 neutralized with ammonium hydroxide, 

 and .1 cc. of a saturated solution of 

 ammonium oxalate added. If no pre- 

 cipitate forms additional .1 cc. portions 

 of oxalate are added at 15 minute inter- 



