STAINS, ETC. 



1402 



STAINING REAGENTS 



I part of hydrochloric acid ; place the tissues in this 

 mixture for 2 or 3 hours ; wash with water, and keep in 

 the dark in chromic acid, - J ff or T i^ per cent, for 2 or 3 

 days ; then wash thoroughly. Lowit' s Method. Mix 2 

 parts of distilled water and I part of formic acid (sp. gr. 

 1. 16) ; in this mixture place small pieces of fresh tissue, 

 from I to 2 mm. in thickness, for from a y z to I minute, 

 or until they become somewhat transparent. Then trans- 

 fer to a I per cent, solution of gold chlorid, protecting 

 the tissue from the light as much as possible ; in from 15 

 to 20 minutes, or when it is yellow, place it in a solu- 

 tion of formic acid, I : 3, for 24 hours, keeping it in the 

 dark. Then immerse in pure formic acid for 24 hours, 

 in the dark, and, finally, wash thoroughly in water. 

 Pritshard' 's Method. After the tissues have been 

 treated with a gold-solution, the gold is reduced with 

 the folowing mixture: amyl-alcohol I c.c. , formic 

 acid I c.c, water 98 c.c. Remove the tissue from the 

 gold-solution, wash it in water, place in the preceding 

 mixture for 24 hours in the dark, when it will 

 probably have become of a violet color ; if not, 

 place it in a fresh quantity of the fluid for 24 hours 

 longer. Wash in water and harden in alcohol. Ran- 

 vier 1 s Formic-acid Method. Place the tissue in a mix- 

 ture of 4 parts of a I per cent, solution of gold chlorid 

 to I part of formic acid, the mixture having been pre- 

 viously boiled and cooled. Allow muscle to remain in 

 this solution for 20 minutes, epidermis for from 2 to 4 

 hours. The reduction of the gold is accomplished in 

 acidulated water by the action of daylight, or in the dark 

 in I part of formic acid to 4 parts of water. Ranvie/s 

 Lemon-juice Method. Express and filter the juice of a 

 lemon, and place the fresh tissue in it for 5 or 10 min- 

 utes, when it becomes transparent. Quickly rinse in 

 distilled water, and transfer to a I per cent, gold-chlorid 

 solution for from 10 minutes to I hour, the time de- 

 pending on the tissue. Wash with water, and place 

 in 50 c.c. of water acidulated with 2 drops of 

 acetic acid ; reduction occurs on exposure to light. 

 Viallanes 1 Osmic-acid Method. Treat the tissues 

 with a I per cent, solution of osmic acid until they be- 

 gin to turn brown ; then with y£ formic acid for 10 

 minutes ; then place them in a solution of gold chlorid, 

 I : 5000, in the dark, for 24 hours. Reduce in the light 

 in ]^ formic acid. Osmic Acid. Tissues fixed in 

 osmic acid and subsequently treated with weak pyro- 

 gallic acid are stained greenish-black (Lee). A devel- 

 oping mixture of water, alcohol, tannin, and pyrogallic 

 acid or a 5 P er cent: solution of tannin is used by 

 Kolosson. Treatment with oxalic acid 1 part, in water 

 15 parts, gives a Burgundy-red stain to osmium- 

 objects, which should be washed in water before they 

 are put into the acid (Brdsicke). Silver Nitrate. 

 Particularly adapted to the study of epithelial and con- 

 nective tissues. Make a 1 percent, solution in distilled 

 water, and dilute from 2 to 4 times for use. Very thin 

 sections of fresh tissue are washed in distilled water, to 

 remove the chlorids, immersed for )/ z hour in the solu- 

 tion, in the dark, washed in distilled water, and then 

 placed in water and exposed to sunlight until brown. 

 Fix in a solution of sodium hyposulphite, in the dark, 

 and mount in glycerin-jelly. The ffertwig's employ a 

 I \)?.r cent, solution for marine animals. Tourneux an,/ 

 Hermann, in their studies of the epithelia of fiwerte- 

 b rates used a solution of 3 : 1000, in which the tissues 

 were left for 1 hour and then washed in alcohol (36 ) . 

 Dekhuysen treats the tissue with a 1.3 per cent, solution 

 of potassium nitrate, then immerses it in 0.25 per cent, 

 solution of silver nitrate containing 3 per cent, of nitric 

 acid. After from 3 to 6 minutes in the silver-bath, the tis- 

 sue is placed for a few minutes in pure 3 per cent, nitric 

 acid, then in 96 per cent, alcohol, then in clove-oil, in 



which reduction occurs, in diffused light, in a few min- 

 utes. This method is said to give good fixation of tis- 

 sues, and to permit the use of a nuclear after-stain with 

 hematoxylin, safranin, or methyl-green. The process is 

 the same as that employed by Harmer for marine ani- 

 mals. Von Recklinghausen effects reduction by washing 

 the preparation in normal salt-solution before exposing 

 to light in distilled water. Thanhoffer exposes to light 

 for a few minutes in water acidulated with acetic acid. 

 Krause uses, after washing, a light-red solution of potas- 

 sium permanagate, in which reduction occurs very quie!dv 

 even in the dark. Jakimovitch expos2s the tissues to 

 light in a mixture of formic acid I part, amyl-alcohol 1 

 part, water 100 parts; from 5 to 7 days are required, and 

 the mixture must be renewed from time to time. The 

 after-blackening is prevented by washing in sodium- 

 hyposulphite solution (Legros). V. Other Organic 

 Stains. Grenacher's Purpurin. Dissolve from 1 

 to 3 per cent, of powdered alum in 5° c -c of glycerin, 

 add a knife-pointful of purpurin, and boil. No alcohol 

 should be used. The orange-colored solution should 

 stand for 2 or 3 days, and then be filtered. This is a 

 nuclear stain which is stable, from 10 to 30 minutes pro- 

 ducing a good result. Nuclear Black (Kernsc/nvarz). 

 A black liquid of unknown composition, recommended 

 by Platner as a cytologic stain. Dilute the liquid some- 

 what and wash out in dilute ammonia or a saturated so- 

 lution of lithium carbonate diluted with 3 or 4 volumes 

 of water. Mitotic figures stain deeply, resting chro- 

 matin less deeply, cytoplasm faintly gray. Phloroglu- 

 cin. For staining lignified cellulose. Take of phloro- 

 glucin 1 gm., 90 per cent, alcohol 20 c.c, distilled 

 water 8o c.c. ; treat the sections for 1 ■; minutes, and fol- 

 low by strong hydrochloric acid. This gives a stain of 

 cherry-red, varying in proportion to the extent of the 

 lignification. Ranviers Purpurin. Boil in a porce- 

 lain capsule 200 c.c. of water and I c.c. of alum; 

 then add purpurin rubbed up in water, and continue the 

 boiling. A saturated solution of purpurin is secured by 

 having an undissolved excess in the capsule. Filter the 

 hot mixture into a flask containing 6o c.c. of 90 per 

 cent, alcohol. This solution does not keep well. | 

 Wedl's Orseille. French orchilla extract, a sufficient j 

 quantity, from which the excess of ammonia has been 

 removed by warming in a sand-bath, is poured into a , 

 mixture of 20 c.c. of absolute alcohol, 5 c.c of glacial 

 acetic acid, and 40 c.c. of distilled water, the dye being 

 added gradually until a dark-reddish fluid is produced. ; 

 This is a protoplasmic stain, the nuclei remaining color- .' 

 less. VI. Combination-stains. These are of two 

 kinds. In the one class a pure nuclear stain is com- 

 bined with a dye taking effect on the extra nu 

 elements ; in the other, a stain giving a reaction 

 all the elements of one tissue is combined with 

 or more stains taking effect on all the elements 

 the other tissues. Alum-carmin and Osmic Acid 

 (Zo/tdn von Roboz). To 50 or 6o gm. of water 

 alum-carmin until the mixture is nearly rose iv i ; then 

 add IO drops of a 1 : 500 solution of osmic acid. S 

 objects, in the dark, from 24 to 48 hours. A nu 

 double stain; resting chromatin and nucleoli appear pur- 

 ple, kinetic chromatin red, protoplasm brown. Recom- 

 mended for staining Pluteus and similar objects. Alum- l 

 carmin and Picric Acid. Mix 10 volumes of alum- 

 carmin and I of saturated picric-acid solution ( Ta 

 Anilin Blue and Safranin (Garbini). Stain 

 tions for from 2 to 4 minutes in 0.5 per cent, anilin 

 blue solution, wash in water, then place in a 0.5 p*r 

 cent, lithium-carbonate solution, then in 0.5 percent, 

 hydrochloric acid until a clear, blue color app 

 Wash again in water, and stain for IO minutes in 1 

 cent, safranin-solution, dehydrate in methyl -alcohol and 



