STAINS, ETC. 



1381 



FIXING AND HARDENING FLUIDS 



rcerin, and add a little thymol as a preservative. 

 Paint a thin, even film on the slide ; on this arrange 

 the sections, and warm just enough to coagulate the 

 albumen (70 C). A very useful fixative for sections 

 that are to be stained on the slide. Acids, alkalies, 

 and picrocarmin must be avoided, as they dissolve the 

 albumen. Ohlmacher s Modification. Apply a thin, 

 smooth layer of Mayer's albumen to the slide, and on 

 this' drop a little distilled water, which will diffuse 

 evenly over the albumen. Arrange the sections in 

 position, and heat gently over a spirit-flame until they 

 flatten out, being careful not to melt the paraffin. 

 Drain off the water, dry the slide with bibulous paper, 

 and evaporate the remaining moisture in an oven at 

 a temperature below the melting-point of paraffin. 

 Obregia's Method. Coat slides with a solution pre- 

 pared as follows : Mix 30 c. c. of a syrupy solution 

 of confectioners' sugar, made with distilled water, 

 20 c. c. of absolute alcohol, and 10 c. c. of a clear, 

 syrupy solution of pure dextrin in distilled water ; 

 put them aside to dry. After 2 or 3 days, when the 

 surface is just sticky to the moistened finger, arrange 

 the sections in position, and heat for a few minutes; 

 remove the paraffin with xylol, wash with alcohol, and 

 then cover with a 3 per cent, solution of photoxylin in 

 a mixture of equal parts of absolute alcohol and ether. 

 Leave the slides to evaporate in the horizontal posi- 

 tion, cut the sheet of collodion into ribbons, float them 

 off in water, and proceed as in Weigert's method. 

 Schallibaum's Collodion Method. Shake up one 

 part of collodion with 3 to 4 parts of clove-oil. Ap- 

 ply this mixture thinly on the slide ; place the sections 

 in position, warm over a spirit-lamp until the oil col- 

 lects in drops between the sections and the paraffin is 

 melted ; then hold the slide to the lips, blow upon it 

 vigorously, and the paraffin and oil will be scattered, 

 leaving the sections dry and securely fixed. This 

 method is recommended for stained sections. The 

 Shellac Method. A filtered solution of bleached, 

 white shellac in absolute alcohol is spread over the 

 i slide with a glass rod. When dry, apply a very thin 

 layer of creasote, which gives a sticky surface for the 

 sections, then heat on a water- bath for 1 5 minutes at 

 the melting-point of paraffin, allow the slide to cool, 

 and the sections will be fixed. 



FIXING AND HARDENING FLUIDS. 

 eagents used to preserve the structural elements of 



i tissues. Their action consists in the rapid coagulation 

 of certain constituents of tissue — albuminoids, gelatin, 

 mucin — by which their histologic and cytologic details 

 are fixed in the form and attitude they normally had 

 in life, and are also hardened so as to resist any change 

 of form from the action of other reagents with which 

 they may subsequently be treated. Acetic Acid. A 

 valuable fixative of wide application, and especially 

 useful for fixing very contractile objects, such as are 

 found in the Vermes and Calenterata. Pour a liberal 

 quantity of glacial acetic acid over the organism, and 

 in five or six minutes wash in 50 per cent, alcohol, 

 changing it frequently and gradually increasing the 

 trength. Acidulated Alcohol. Used for the pre- 



' paration of marine animals which are to be preserved 

 in alcohol. It is a mixture of 97 volumes of 90 per 

 cent, alcohol (in which is dissolved a little picric acid) 

 and 3 volumes of pure hydrochloric acid. The acid 

 facilitates penetration by preventing the precipitation 

 of the salts of sea- water. After penetration, the or- 

 ganism is washed in 00 per cent, alcohol until the 

 stain of the picric acid no longer appears. 



£An excellent fixing medium, suitable for 

 5, except those of the central nervous 



system and those undergoing fatty infiltration or 

 degeneration. It is also used to harden and preserve 

 objects that have been fixed in other fluids. . As a 

 preservative it is not without defects, as it alters the 

 structure of tissues by continuously dehydrating their 

 albuminoids. Toluol, ether, and xylol are recom- 

 mended as substitutes (A'ultschitzhv). As a fixing 

 agent alcohol is usually employed in gradually increas- 

 ing strengths, beginning with 50 or 70 per cent. Abso- 

 lute Alcohol. One of the most penetrating fixing 

 agents, which has the advantage of preserving the 

 structure of glands and of nuclei. It should be em- 

 ployed in large quantities. Hydration may be pre- 

 vented by suspending in the alcohol strips of gelatin 

 (Lowrie). After fixation, preserve the object in 90 

 per cent, alcohol. Mayer recommends boiling abso- 

 lute alcohol for fixing certain Arthropoda. Absolute 

 alcohol is prepared in Ranvier's laboratory by add- 

 ing anhydrous copper sulphate to 95 per cent, alcohol. 

 Pulverized copper sulphate is heated to red heat, in 

 order to drive off the water of crystallization ; when 

 cool, the white powder is placed in a wide-mouthed 

 bottle, holding about a liter, and three-fourths full of 

 alcohol. The bottle is quickly closed and the whole 

 shaken. After standing a day or more — with occasional 

 shakings — it is decanted, treated with fresh copper sul- 

 phate, and the operation repeated until the copper sul- 

 phate no longer shows a blue color on contact with the 

 alcohol. As a test, a drop of the alcohol thus dehy- 

 drated may be mixed with a drop of turpentine on a glass 

 slide and examined under the microscope; if no particles 

 of water are to be seen, the alcohol is absolute enough 

 for all practical purposes. Methylated Spirit contains 

 a little methyl-alcohol, and is nearly as strong as abso- 

 lute (ethyl) alcohol, and its strength may be increased 

 by placing in it some well dried potassium carbonate, 

 which absorbs any water present in it. •' One-third 

 Alcohol" (Han-trier's Alcohol). This classic reagent 

 consists of two parts of water and one part of alcohol 

 of 36 Beaume, which contains nearly 89.6 per cent, 

 absolute alcohol. It is a very mild fixative, and is 

 chiefly used for extemporaneous and dissociation pre- 

 parations. Rectified Spirit contains 84 per cent, of 

 alcohol and 16 per cent, of water. Altmann's 

 Nitric Acid. Particularly useful for preserving 

 mitotic figures, nuclei, embryonic tissues, and the 

 retina. A 3 per cent, pure acid is used, the solution 

 having a sp. gr. of 1. 02. Immersion for a quarter to 

 half an hour is sufficient for blastoderms and small 

 embryos ; two to four hours are required for fixing 

 larger objects. Wash out thoroughly and harden in 

 strong alcohol. Any staining process may be used. 

 Broca's Fluid. Used for the preservation of brain- 

 tissue. It consists of zinc chlorid, 10 grams, and 

 alcohol, 90 per cent., 100 c.c. After two or three 

 days, transfer the tissue to 95 per cent, alcohol. 

 Carnoy's Fluid. One of the most penetrating and 

 rapid of fixatives, applicable to tissues in general, and 

 useful for the study of karyokinesis in the ova of 

 Ascaris. First formula : glacial acetic acid I part, abso- 

 lute alcohol 3 parts. Second formula : glacial acetic acid 

 I part, absolute alcohol 6 parts, chloroform 3 parts. 

 The chloroform is said to render the action of the mix- 

 ture more rapid. Chromic Acid. Used in aqueous or 

 alcoholic solution in strengths varying from 0. 1 to 2 per 

 cent. Weaker solutions are used for nerve-tissue. 

 Wash out several hours in running water. If the 

 object be preserved in alcohol, it must be protected 

 from the light, or a precipitate will be thrown down on 

 its surface. The brownish-green color of objects 

 fixed in chromic acid may be removed by treating 

 them with hydrogen dioxid. A 0.02 per cent, solu- 



