B. H. Stieda's Method for Permanent Mounts with Nuclear Stains.-1. Stain several hours in lithium carmine. 2. Wash off quickly in water. 3. Place from four to six hours in a 2 per cent. aqueous solution of ferrocyanid of potassium. 4. Transfer to acid alcohol for six to twelve hours. 5. Wash quickly in water. 6. Alcohol, oil, Canada balsam. II. The reaction for the ferrous salts is performed in the same way as for ferric salts, with the exception that ferricyanid of potassium is used instead of the ferro- compound. III. Reactions for Ferric and Ferrous Salts.—A. Use a mixture of ferro- and ferricyanid of potassium (1 gram each to 100 c.c. of water), followed by acid alcohol. Nuclear stains and permanent mounts may be made as above. B. 1. Place sections in a freshly prepared solution of sulphate of ammonium for five to twenty minutes, until they are dark or black-green in color. 2. Wash quickly in water. 3. Examine in glycerin or pass through alcohol and oil to Canada balsam. The iron appears in the form of black or dark-greenish granules. Sulphate of ammonium causes similar precipitates with other metal salts, such as the nitrates of silver, lead, and mercury. Petrifaction.-Calcification, the more common form of petrifaction, is the term applied to the infiltration of tissues with phosphate and carbonate of calcium. The salts appear microscopically as small, very refractive granules which may be mistaken for fat, or as large masses due to the fusion of granules. They are dissolved by hydrochloric or nitric acid (5 per cent. solution). If carbonate of lime is present, bubbles of carbon dioxid are set free. Phosphate of lime dissolves without effervescence. To differentiate between lime-salts and other substances soluble in hydrochloric acid use concentrated sulphuric acid to form sulphate of lime (gypsum), which appears as fine, short, radiating needles. On dissolving out the lime-salts a matrix of dead tissue or of hyaline material will usually be found left behind. As a rule, this hyaline material stains deep blue in alum-hematoxylin or red in Van Gieson's mixture. The deposits of calcium salts themselves also stain with hematoxylin, so that it can be used to demonstrate the masses and coarser granules of them. The tissue must, however, first be freed of certain iron combinations, which are often associated with deposits of lime and also stain with hematoxylin. The following method is recommended by Roehl: I. Fix in alcohol or formaldehyde. 2. Place sections in a half-concentrated solution of oxalic acid for fifteen to thirty minutes to remove the iron. 3. Wash thoroughly in water. 4. Stain in a 1 per cent. aqueous solution of hematoxylin (which must be neither too fresh nor too old) for five to ten minutes. 5. Differentiate in water, to which a few drops of ammonia water are added, until the section is colorless and only the lime deposits remain stained. 6. Wash in water. 7. Counterstain with safranin. Alcohol; xylol; balsam. Lime-salts deep violet; nuclei red. Von Kossa has shown that phosphate of calcium can be demonstrated by means of nitrate of silver, which forms silver phosphate on the surface of the granules and blackens in the presence of light. It gives an exaggerated picture of the amount of lime-salts present. Klotz has shown that the nitrate of silver acting for many hours affects calcium carbonate also: the granules become coated with silver carbonate, which in sunlight gives off carbon dioxid, leaving the black silver oxid. This process can be hastened by putting the sections, after staining and thorough washing, into a dilute soluble sulphide. Von Kossa's Method.-I. Fix in alcohol, formaldehyde, or corrosive sublimate. 2. Place sections (frozen, celloidin, paraffin) in a 1 to 5 per cent. aqueous solution of nitrate of silver for thirty to sixty minutes (von Kossa), three to twelve hours (Klotz). 3. Wash thoroughly in distilled water. 4. Mount in glycerin or balsam. The lime is stained deep black. The nuclei can be counterstained with alum carmine or safranin after the silver staining. Another form of petrifaction is that found in gout, due to the infiltration of certain tissues with uric-acid salts, of which urate of sodium is the most common. The crystals are sol uble with difficulty in cold water, insoluble in alcohol and ether. Therefore, to study the deposits in connection with the lesions, fix in 95 per cent. alcohol and imbed in celloidin; stain sections quickly in a cold solution of alum-hematoxylin. Wash quickly in cold water and transfer to alcohol. Clear and mount in balsam. THE STAINING OF BACTERIA IN TISSUES. Bacteria are demonstrated in sections of tissues almost entirely by means of the aniline dyes, of which three have thus far proved themselves to be particularly valuable— namely, methylene-blue, gentian-violet, and fuchsin. These dyes are employed in aqueous or dilute alcoholic solutions, of which the effective staining power is greatly increased by means of heat and by the addition to the solutions of certain chemical substances. The effect of moderate heat is obtained by placing the sections in the incubator for several hours, or greater heat for a short time is utilized by warming the staining solution on the slide over a small flame for a few seconds or minutes, keeping the fluid steaming, but not allowing it to boil. Of the various methods employed to increase the staining power of aniline dyes by means of chemical substances, the most successful have been the use of caustic potash with methylene-blue, of aniline oil with gentian-violet and fuchsin, and of carbolic acid with fuchsin and methylene-blue. For decolorizing sections after they have been stained, the most commonly employed reagents are 1. Acetic acid in dilute aqueous solutions, I: 100, 1:1000. 2. Alcohol. 3. Iodin in iodid-of-potash solution (with certain dyes only). 4. Mineral acids in various strengths. 5. Chlorid of aniline. 6. Acid aniline colors added to the alcohol to increase its extractive power. 7. Aniline and ethereal oils. The choice of a decolorizer varies with the staining solution employed and with the organism that is to be stained. Sections which are to be stained for bacteria may be divided into two classes: 1. Sections free from celloidin, subdivided into a. Sections cut without an imbedding mass; c. Sections from which the celloidin has been removed. 2. Sections infiltrated with celloidin. Celloidin imbedding is to some extent a drawback to the stains for certain organisms, because the celloidin tends to hold the color, so that the bacteria are not so distinct as they otherwise would be. Still, it is so important to be able to stain bacteria in celloidin sections that particular care is devoted in the following pages to methods which obviate most of the difficulties. Paraffin sections should, as a rule, be attached to the slide by means of Mayer's glycerin-albumin mixture. It will usually be found advisable to attach celloidin sections to the slide by means of ether-vapor. They will then keep perfectly flat in any staining solution, and may be heated without danger of wrinkling or contracting. The heat should never be applied directly under a section, but at one end of the slide. All bacteria yet known will stain when placed in appropriate staining solutions. Some, however, are stained quickly, while others are stained with difficulty; some give up the stain readily to decolorizers, while others retain it tenaciously. In consequence of their reactions to certain dyes and to certain decolorizers, bacteria, from the point of view of staining, may be divided into three groups: 1. Bacteria which do not stain by Gram; 2. Bacteria which stain by Gram; 3. Bacteria which stain by the tubercle bacillus method. Two at least of the organisms in the third group will also stain by Gram. The organisms of the second and third groups are much more easily demonstrated in tissues than those in the first group, because it is possible to stain them of one color and the nuclei of the cells of another color. In other words, it is possible to stain them so that they are differentiated from the tissue in which they lie, and hence stand out prominently. The organisms of the first group have no differential stain; they take the same color as the nuclei of the tissue. Moreover, although they stain easily, most of them do not stain. deeply, and readily part with the color they have taken up. Pathogenic Bacteria which do not Stain by Gram. (See page 94.) Of these organisms certain ones deserve special mention on account of their frequent occurrence or on account of the difficulty of demonstrating them in tissues, and certain variations in staining methods which have proved serviceable will be given. Löffler's methylene-blue solution is generally considered the most useful stain for this class of bacteria, but excellent results can also be obtained with Unna's alkaline methylene-blue solution preceded by eosin, especially after fixation in Zenker's fluid. Löffler's Methylene-blue Stain for Bacteria.-I. Stain paraffin sections twenty minutes to twenty-four hours. 2. Wash in weak acetic acid, 1: 1000, for ten to twenty seconds. 3. Absolute alcohol, two or three changes, to differentiate and dehydrate (as a rule, only a few seconds are required for this step). 4. Xylol. 5. Xylol balsam. For celloidin in sections use 95 per cent. alcohol; blot, and treat with xylol; repeat until sections are clear; mount in xylol balsam. |