DEGENERATION 



95 



DEHYDRATION 



by the needle method. Over decal- 

 cification is not likely. 



For rapid decalcification he advises 

 using sat. aq. phloroglucin to which 

 6-30% Nitric Acid is added. A some- 

 what slower formula is : nitric acid, 5 

 cc; phloroglucin, 70 cc; 95% ale, 1 

 cc; and aq. dest., 30 cc. The phloro- 

 glucin allows use of stronger acids. 

 1-2% aq. hydrochloric acid decalcifies 

 quickly but it causes the tissue to swell. 

 Formic Acid 1-5% in 70% alcohol is, 

 according to Shipley, the best decal- 

 cifying agent for large masses of bone. 

 With 5%, the decalcification is com- 

 pleted in 4-5 days. Use 70% ale. not 

 water, to wash out the acid. 



Kramer and Shipley devised a Magne- 

 sium Citrate method of decalcification 

 in neutral solutions. To make the de- 

 calcifier dissolve 80 gms. citric acid in 

 100 cc. hot aq. dest., add 4 gms. magne- 

 sium oxide and stir until completely 

 dissolved. If the magnesium oxide 

 contains carbonate it should be freshly 

 ignited. Cool and add 100 cc. ammonium 

 hydroxide (density 0.90) and aq. dest. 

 to make 300 cc. Allow to stand 24 hrs. 

 and filter. Titrate filtrate with 

 hydrochloric acid to about pH 7.0- 

 7.6 and add equal volume aq. dest. In 

 decalcifjdng, this reagent should be 

 changed every 3 days. A dog's rib is 

 decalcified in approximately 15 days. 



After decalcification, by whatever 

 method, the bone, or the area of calcifica- 

 tion, must be thoroughly washed to 

 remove the decalcifer and imbedded in 

 paraffin or celloidin. Some investiga- 

 tors prefer the latter but celloidin 

 sections are not so easily handled. See 

 Bones, Teeth. 



The ion exchange resin technique for 

 bone is new and interesting (Dotti, L. 

 B., Paparo, G. P. and Clarke, B. E., 

 Am. J. Clin. Path., 1951, 21, 475-479). 

 Try keeping tissue in cork stoppered 

 bottle containing 10 gms. of the resin 

 (Win-3000 supplied by Winthrop- 

 Stearns, Inc. 1450 Broadway, New York 

 18) in 80 cc 10-20% formic acid for 1-4 

 days. The optimum time should be 

 determined for the particular kind of 

 bone and the size of piece. The authors 

 supply helpful figures illustrating the 

 dependence of intranuclear details on 

 time of decalcification. 

 Degeneration. Because the structural or- 

 ganization of various sorts of cells is, 

 like their function, so very different 

 the types of degeneration leading to 

 death are also different at least in many 

 of their aspects. See Nerve Fiber 

 Degeneration, Cloudy Swelling, Necro- 

 sis, Caseation, Parenchymatous Degen- 

 eration, Postmortem Changes. 



Dehydration is the removal of water from a 

 tissue preliminary to clearing and paraf- 

 fin or celloidin imbedding. This is 

 routinely done by treating the tissue 

 after Fixation and Washing by passing 

 it through a series of ethyl alcohols of 

 increasing concentration. Usually the 

 percentages are 30, 50, 70, 80, 95 and 

 absolute. The time depends upon the 

 size and kind of the tissue and the sort 

 of fixative. For slices of tissue less 

 than 3 mm. thick the dehydration can 

 be accomplished in 6-12 hours. The 

 alcohols for large slices fixed say in 

 Zenker's fluid are ordinarily changed 

 every morning and evening, but it is not 

 desirable to leave them in absolute 

 alcohol very long because it makes them 

 brittle. Three to 6 hours should be 

 sufficient. Tissues fixed in alcoholic 

 solutions take a shorter time to de- 

 hydrate. After fixation in alcohol- 

 formalin or in Carnoy's fluid the tissue 

 can be dehydrated and partly washed in 

 several changes of absolute alcohol 

 skipping the lower grades of alcohol 

 entirely. 



When, for some reason, it is desired 

 to eliminate treatment with absolute 

 alcohol the tissues can be passed directly 

 from 95% alcohol into Aniline Oil (say 

 30 min.) which is itself later removed, 

 at least partly, in 5-10 minutes by 

 washing in 2 changes of chloroform. 

 Clearing is continued in chloroform for 

 imbedding in paraffin, or the tissue may 

 be passed from 95% alcohol, even from 

 80%, into Terpineol and cleared in half 

 terpineol and xylol. Still another way 

 to avoid absolute alcohol is to transfer 

 from 95% alcohol to Bergamot Oil 

 which serves as a clearing agent. 



Several substitutes for ethyl alcohol 

 as a dehydrating agent are available. 

 Acetone is the best known. This is 

 strongljr recommended by R. D. Lillie, 

 p. 43. Four changes of acetone each 

 lasting 40 minutes are suggested for 

 routine work but this can be reduced 

 to 4 changes each 20 min. Thereafter 

 pass, the blocks of tissue to a paraffin 

 solvent such as benzene, toluene or 

 xylene before placing in melted para- 

 ffin. The schedules which he provides 

 (p. 46) for dehydration, clearing and 

 infiltrating with paraffin are useful. 

 See Cellosolve. 



Dioxan will not only take the place 

 of the alcohol but also that of the clear- 

 ing agent so that it is possible to greatly' 

 simplify the technique and make the 

 sequence: fixative to dioxan to paraffin. 

 See Dioxan and note as to possible 

 danger to those using it. Cellosolve 

 has also been proposed as a dehydrating 

 agent. Lee (p. 64) says that it is ex- 



