DEAD CELLS 



78 



DEHYDRATION 



of the lower epidermis of the scale of 

 the onion bulb is peeled off and placed, 

 cutin side down, on a slide. A drop of 

 a .5 per cent, slightly alkaline, aqueous 

 solution of neutral red is deposited on 

 the piece of epidermis and left there for 

 2 minutes; then it is blotted off and re- 

 placed by a drop of a .4 per cent potas- 

 sium hydroxide solution, which is imme- 

 diately removed (also with a blotter) ; 

 then the preparation is washed with 

 tap water. The living cells take with 

 that treatment a bright cerise red color, 

 while the dead cells are of an intense 

 orange yellow. The contrasts are vio- 

 lent. There are intermediate tints 

 which correspond to the dying cells. 

 See Necrosis, Necrobiosis, Survival 

 of tissue. 

 Decalcification. The removal of calcium so 

 that bony tissues can be cut in sections. 

 There are many methods almost all of 

 which involve acid treatment. It is 

 generally better to apply the de- 

 calcifying agent after fi.xation, particu- 

 larily so when the agent is a poor fi.xative. 

 The volume of decalcifier should be 

 about 100 times that of the tissue. The 

 usual, crude, way of testing the progress 

 of decalcification is to stick a fine needle 

 into the bone being careful to avoid the 

 area that will be cut in sections; but 

 less objectionable methods can be used, 

 see Teeth, Decalcification. 



Saturated aq. sulphurous, 5% tri- 

 chlorlactic, 5% hydrochloric and equal 

 parts of 1% hydrochloric and 1% 

 chromic acids are all fairly good de- 

 calcifiers. Lactic, acetic, phosphoric 

 and picric acids are usually unsatis- 

 factory. Shipley (McClung, p. 347) 

 recommends slow decalcification by 

 long immersion in MuIIer's Fluid 

 through liberation of small amounts of 

 chromic acid from the bichromate. 

 The bones of an adult rat require 21-30 

 days. The process can be hurried 

 somewhat by using an incubator at 

 37°C. Adequate decalcification is de- 

 tected by sliglit bending of the bone or 

 by the needle method. Over decal- 

 cification is not likely. 



For rapid decalcification he advises 

 using sat. aq. phloroglucin to which 

 5-30% 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 o.xide and stir until completely 

 dissolved. If the magnesium oxide 

 contains carbonate it sliould 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 

 decalcifying, 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 dccalcifer and imbedded in 

 paraffin or celloidin. Some investiga- 

 tors prefer the latter but celloidin 

 sections are not so easily handled. See 

 Bones, Teeth. 

 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 fi.xative. 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 



