STAINS, ETC. 



1382 



FIXING AND HARDENING FLUIDS 



tion is recommended for macerating nerve-tissue and 

 non-striated muscle-tissue, a cube of 5 mm. requiring 

 about 24 hours' immersion in 10 c.c. of the medium 

 (Ranvier). Copper Sulphate. Recommended by 

 Bedot for the preparation of delicate pelagic animals. 

 A large volume of a 15 or 20 per cent, solution is 

 added to the sea-water containing the animals. Fix- 

 ation occurs in a few minutes. A few drops of nitric 

 acid are added, and the whole is left for 4 or 5 hours. 

 The animals are then hardened in a large volume of 

 Flemming's fluid, in which they should remain for 

 24 hours, and are preserved in alcohol. Corrosive 

 Sublimate. A most excellent reagent, gener- 

 ally applicable, useful particularly for fixing glands 

 and glandular structures. Saturated aqueous and 

 alcoholic solutions are usually employed. The tissues 

 turn white when fixed. All the corrosive sublimate 

 must be washed out in alcohol, or the sections will be 

 sprinkled with crystals of the salt. The hardening is 

 completed in alcohol. Glass, wood, or platinum 

 should be used in manipulating objects immersed in this 

 reagent. Sections may be stained with any of the usual 

 reagents. Cox's Sublimate Solution. A combined 

 hardening and impregnating fluid, consisting of 5 per 

 cent, solutions of potassium bichromate and mercuric 

 chlorideach 20 parts .potassium chromate 1 6 parts, water 

 30 or 40 parts. The reaction should be only slightly acid. 

 The pieces are left in this solution two or three months, 

 then cut with a freezing-microtome. The sections are 

 put in 5 per cent, solution of sodium carbonate for an 

 hour or two, washed in water, then in oil, and covered 

 by a rapidly drying resin, like sandarac. This coating 

 may be covered by castor-oil, and the cover-glass pressed 

 down. The preparations are not permanent in balsam 

 or dammar. Davidoff 's Fixing Fluid. This is a 

 mixture of a saturated solution of corrosive sublimate 

 75 c.c. and glacial acetic acid 25 c.c. It is useful 

 for ova, which should remain in it for a half to one 

 hour ; then wash in water and transfer to alcohol, 

 gradually increasing its strength. Delafield's Fluid. 

 A fixing agent suitable for tissues having delicate 

 structural elements. It has the following composi- 

 tion : 1 per cent, osmic acid solution 10 c.c, 0.2 

 per cent, chromic acid solution loo c.c, 95 per cent, 

 alcohol 100 c.c, acetic acid I c.c. Erlicki's Fluid. 

 One of the best hardening agents for large objects, 

 and more rapid in its action than Midler's fluid. It is 

 composed of potassium bichromate 2.5 parts, copper 

 sulphate I part, water loo parts. The formation of 

 mold is prevented by adding a little camphor or 

 naphthalene. Ferric Chlorid. Objects are fixed for 

 a short time in a dilute alcoholic solution, washed in 

 alcohol, and stained in pyrogallol. This process is 

 recommended as a general zoologic method, but 

 chiefly for Infusoria and other ciliated organisms, and 

 for its selective action on nerve end-organs. Fish's 

 [P. A.) Liquid. This is found to preserve the shape, 

 to harden, and to differentiate brain-substance. It con- 

 tains alcohol (95 per cent.) 200 c.c, water 800 c.c, 

 glycerin 75 c.c, zinc chlorid 50 gm. The alcohol 

 and water should be mixed first, so as to get a 

 strength of 22 per cent., as determined by the alco- 

 ometer. More alcohol maybe required, but the entire 

 quantity of dilute alcohol should not exceed 1000 c.c. 

 Then add the glycerin and zinc until the specific 

 gravity of the mixture is 1.04. When the brain is 

 introduced, it should rest just at the surface, and the 

 vessel should be deep enough to prevent it touching 

 the bottom. Cotton may be laid over the top if any 

 part projects. It is well also to inject the fluid into 

 the cavities and blood-vessels daily for a week. 

 After a few days the brain will sink, when, without 



removing it from the liquid, the pia is stripped 

 off. Half of the liquid is now replaced by alcohol 

 and the brain is steadied with cotton. After 2 days 

 half of the lighter liquid is replaced by alcohol, 

 and in 2 or 3 days more pure alcohol is used. 

 Flemming's Fluid. 1st Formula : Chromic acid 

 0.2 gm., glacial acetic acid O.I c.c, water 1 00 c.c. 

 This is especially recommended for fixing the achro- 

 matic spindle-fibers in nuclei. 2d Formula : One per 

 cent, chromic acid 45 c.c, 2 per cent, osmic acid 

 12 c.c, glacial acetic acid 3 c.c. This fixes small 

 pieces (2-3 mm. thick) in from a few to twenty-four 

 hours, and is useful for fixing the figures in cell-division 

 and for many other purposes. A weaker solution is 

 also used : One per cent, osmic and glacial acetic 

 acids each 10 c.c, one per cent, chromic acid 25 

 c. c, water 100 c.c. The second formula is the one 

 generally known as Flemming's Fluid. Fol's Fluid 

 A modification of Flemming's fluid. It contains les: 

 osmic acid. To 2 c.c. of 1 per cent, osmic acid adc 

 25 c.c. of I per cent, chromic acid, 5 c - c - of 2 pe 

 cent, glacial acetic acid, and 68 c.c. of water 

 Giacomini's Method of Preservation of the Brail] 

 consists of two stages. In the first stage the freslj 

 organ, still enveloped in its membranes, is immersed in 

 a saturated solution of zinc chlorid. In this it float 

 with a little of its surface above the fluid ; and sc] 

 while its form is not interfered with by pressure, j 

 must be turned two or three times a day, in order the! 

 all parts maybe uniformly acted on. If the subjra 

 has been dead for some time, 600 grams of the soli | 

 tion may be injected through the carotids under sligl 

 pressure, so as to give a firmness to the somewhat sof 

 ish brain before its removal. After 48 hours, the suj 

 face is hard enough to have the membranes removed 

 Let this be done without taking the organ out of tl 

 solution, or, if it be taken out, let it be put into wat 

 immediately, so that it may the less lose its form 1 1 

 pressure. After having been cleaned, let it remain i 

 the solution till, as the hardening proceeds, it begi 

 to sink no longer, and then remove it. At this sta: 

 it will be firm, slightly diminished in volume, the fi 

 sures a little opened, and the color whitish, unless t 

 membranes have been left on too long, in which ca 

 the course of the large vessels will be stained ot 

 rusty color from the blood pigment. It is now ii 

 mersed in alcohol of commerce for not less than 10 I 

 12 days, but it may be for an indefinite period ; ht 

 it sinks, and so must be often turned, to avoid defor 

 ity by pressure on the bottom of the vessel, and it 

 well to renew the spirit 2 or 3 times — the oftener, t 

 sooner the process is finished. After this in 11 

 the consistence is greater, the size a little less, ami t 

 convolutions somewhat closer together. In tli 

 stage the organ is immersed in glycerin of commer 

 or with I per cent, of carbolic acid added. \\ lun 1 

 put in it floats, with some of its upper surface above 

 surface of the glycerin, but gradually becoming hea\ 

 as the alcohol evaporates, and glycerin is imbibedj 

 sinks more and more deeply till it is just level with b 

 liquid — then it is to be taken out. In this part oi 

 process, neither surface, color, consistence, nor voluj 

 is altered, but it becomes heavier. A brain should gl 

 from 150 to 200 grains in from twenty to thirty d: ■ 

 according to its volume. Now set aside for sev<! 

 days, till the surface is dry, and then covet 

 several layers of gum mastic varnish, or, better i\ 

 marine glue, diluted with a little alcohol. Thi 

 is not to prevent evaporation — the glycerin does thi ' 

 but is simply as a protective against dust and inn 

 Hermann's Fluid. A modification of Flemroii ,> 

 fluid. Platinum chlorid is used instead oi chro* 



