STAINS, ETC. 



with o.l per cent, hydrochloric acid, in which it is in- 

 soluble ; the lecithins and albuminoids are dissolved 

 out. Methyl-green is a test for chromatin, as it stains 

 nothing else in the nucleus. 2. Glycogen in Leukocytes. 

 Treat with I per cent, solution of iodin, containing 

 2 gm. of potassium iodid, a preparation of frogs' blood. 

 The white corpuscles are killed, and many stained yel- 

 low. In some of them mahogany-colored granules of 

 stained glycogen are seen. 3. Glycogen in Liver- 

 cells. Harden the liver of a well-fed frog in osmic 

 acid, make a thin section and wash it with iodin, and the 

 granules of glycogen in the hepatic protoplasm will 

 be colored brown. 4. Tizzoni' s Reaction for Ir n. 

 Harden the spleen, kidney, or liver of a young animal 

 in alcohol. Place the sections in a freshly prepared so- 

 lution of 3 c.c. of I : 12 potassium ferricyanid, 90 c.c. 

 of water, and 1. 5 c.c. of 25 per cent, hydrochloric acid. 

 Particles of free iron are colored blue. Spermato- 

 logic Methods. Tease fresh material on a slide, 

 fix with a strong solution of potassium permanganate, 

 and stain with gentian-violet or dahlia. For aqueous 

 mounts, use Ripart and Petit's solution or one of 

 Pacini's mercurial fluids. Sections of the hemaphrodite 

 gland of Lamellibranchiata may be stained for from 2 

 to 3 hours in a mixture of equal parts of a concentrated 

 alcoholic solution of methyl-green and safranin diluted 

 with 8 volumes of water ; wash out in alcohol 5 to IO 

 minutes, and mount in balsam. Nuclei of ova are 

 red; heads of spermatozoa, bluish-green. Watase's 

 Method for Differentiating Sexual Cells. The 

 sexual cells are fixed in Auerbach's fluid or simply in a 

 saturated aqueous solution of sublimate, and the pieces 

 embedded in paraffin. The sections are fixed to the 

 slide with dilute alcohol or distilled water. Saturated 

 solution of anilin-oil in water is added to alcohol until 

 the latter is 20 per cent, in strength ; with this 

 menstruum one per cent, solution of cyanin ,3 3 is 

 made for a blue stain ; and the same strength solution 

 of chromotrop RR for a red stain ; or for a more 

 powerful red stain, use erythrosin in one per cent, 

 solution. For contrast-staining it is immaterial which 

 solution, cyanin or chromotrop, is used first ; erythrosin 

 must be used before the blue stain. Stain quickly, 

 and wash in 50 per cent, alcohol. The nucleus of 

 the ovum becomes red, while that of the spermatozoon 

 becomes blue, showing the former to be erythrophilous, 

 the latter cyanophilous. 



DECALCIFYING FLUIDS. 

 These fluids usually consist of an acid, combined with 

 a hardening agent, to prevent swelling of the tissue. 

 The specimen should be previously fixed by any 

 of the usual methods, tested from time to time with 

 a needle or razor, and when the process of decalci- 

 fication is complete should be washed in water and 

 placed in 67 per cent, alcohol, gradually increasing the 

 strength to 82 per cent. Bayerls' Fluid. Chromic 

 acid, 1 part ; hydrochloric acid, I part ; water. 100 

 parts. Von Ebner's Fluid prevents swelling of the 

 tissue. It has the following composition : Hydrochloric 

 acid, 2.5 c.c, alcohol, 500 c.c, distilled Water, 100 

 c.c, sodium chlorid, 2.5 grams. Use a large volume 

 of the fluid and renew it frequently. If it be desired to 

 examine the fibrillar structure of bone, mount the sec- 

 tions in 10 per cent, sodium chlorid solution ; other- 

 wise the ordinary mounting-media may be employed. 

 Fol's Fluid. Nitric acid, 3 c.c, one per cent, 

 chromic acid, 70 c.c, water, 100 c.c Let the tissues 

 remain in this fluid for two weeks, renewing it every 

 other day. Wash thoroughly and preserve in 90 per 

 cent, alcohol. Gage's Fluid. This agent answers 

 well for the preparation of small salamanders, etc, 



1371 DECALCIFYING FLUIDS 



for sectioning. It consists of 67 per cent, alcohol, 

 100 c.c, and pure nitric acid, 3 c.c. More rapid in 

 action, and recommended for the decalcification of 

 teeth, is a fluid composed of nitric acid, 5 c.c, satu- 

 rated aqueous solution of alum, 100 c.c, and water, 

 loo c.c. It should be renewed every two days. 

 Haug's Phloroglucin Fluid. One of the most 

 rapid decalcifying agents, and without injurious action 

 on the tissue-elements, with the exception of blood. 

 It is prepared as follows : Warm slowly and care- 

 fully one gram of phloroglucin in 10 c.c of pure 

 nitric acid, and to the resulting ruby-colored solu- 

 tion add 50 c.c. of distilled water. If a larger quan- 

 tity is desired, add nitric acid and water to the fore- 

 going proportion until the volume measures 300 c.c, 

 the limit of the protective influence of the phloro- 

 glucin. Previously to being brought into this fluid, 

 the tissues should be well fixed. Fetal bones and those 

 of lower vertebrates are decalcified in half an hour. 

 Older and harder bones require several hours. When 

 decalcification is completed, wash in running water for 

 two days. The sections stain well. Another formula, 

 useful for teeth when rapid action is not necessary, 

 consists of phloroglucin, I gram, nitric acid, 5 c.c, 

 95 per cent, alcohol, 70 c.c, distilled water, 30 c.c. 

 The function of the phloroglucin is to protect the 

 organic tissue-elements against the action of the acid. 

 Hydrochloric Acid. A rapid decalcifying agent, 

 usually employed in combination with chromic acid or 

 alcohol. A 10 per cent, solution of sodium chlorid in 

 3 per cent, hydrochloric acid is recommended. Hydro- 

 chloric acid and glycerin, 5 c.c. of the acid to 95 

 c.c. of the glycerin, constitute a good mixture for 

 softening teeth. Mayer's Desilicification Method. 

 A process for removing siliceous parts from sponges, 

 etc. Place the object in alcohol in a vessel of gutta- 

 percha or glass coated internally with paraffin. Hy- 

 drofluoric acid is then added, drop by drop, care being 

 taken to avoid the fumes, which attack mucous mem- 

 branes with great energy. Contact with the acid causes 

 wounds that heal with difficulty. Small pieces of sponge 

 are freed from silica in a few hours or a day, and the 

 tissues do not suffer. (A dangerous method.) Nitric 

 Acid. An efficacious agent, which causes no swelling, 

 and does not attack the tissue-elements. One per cent, 

 and 10 per cent, solutions are used, the latter for large, 

 hard bones, the former for young bones. The speci- 

 mens should previously have been fixed in absolute 

 alcohol, and the decalcifying fluid changed daily. 

 They must be removed as soon as decalcification is 

 complete, or they will become discolored. They are 

 then washed in running water for two hours, and pre- 

 served in alcohol, which should be renewed in a few r 

 days. Pereny's Fluid, is recommended for rapid 

 action and preservation of the tissue-elements. It is 

 composed of 10 per cent, nitric acid, 4 parts, absolute 

 alcohol and one-half per cent, chromic acid, each, 3 

 parts. Phosphoric Acid. A 10 to 15 per cent, 

 solution is recommended for young bones. Picric 

 Acid. A saturated watery solution is employed, to 

 which a few crystals of the acid are added to keep 

 it saturated. The tissue is suspended in the fluid, 

 and when soft is washed and preserved in alcohol. 

 Young bone is usually decalcified in a fortnight. 

 Thoma's Fluid. This consists of 95 per cent, 

 alcohol and pure nitric acid, in the proportion of one 

 c.c. of the latter to 25 c.c. of the former. Fix the 

 tissue in 95 per cent, alcohol ; then place it for several 

 days in the decalcifying fluid, shaking and renewing it 

 frequently. When this has been done, wash in alcohol, 

 and place it in a jar containing an excess of precipitated 

 calcium carbonate ; renew and shake until every trace 



