tion, the writer gives an historical review of the application of microchemical methods of staining, giving special attention to the carminepigments. The earliest experiments on microscopic staining with carmine for the purpose of a ready differentiation of tissues were made by Goeppert and Cohn. More extended investigations on the capability of the various elements of vegetable tissues to fix carmine shortly followed by R. Hartig. In animal histology, carmine staining was first employed by Gerlach (1858). Further contributions to its application were made especially by Maschke, Thiersch, Beale, Rollen, Gwancher, Hoyer, Czokor, Ranvier, and others. Reference is further made to the cultivation of cochineal, and to the most convenient methods of obtaining carmine for technological purposes, and its application as a staining material in the form of ammonium carminate, and carmine acetate. The author convinced himself by experiments that old preparations of ammonium carminate, which contain a certain quantity of ammonium carbonate, stain better than fresh solutions. Finally, a shorter reference is made to the aniline-dyes, hæmatoxylin, indigo-carmine, and picro-carmine. The second part includes a chronological and tabular account of the literature of the subject, especially with regard to the following staining materials:-(1) carmine; (2) hæmatoxylin; (3) ammonium molybdate; (4) alizarin and purpurin; (5) alcanna and lakmus; (6) sodium indigo-sulphate (indigo-carmine). Mode of announcing new Methods of Reaction and Staining.* -E. Giltay calls attention to the fact that the publication of new methods of reaction is often made without sufficient precision for others to be able readily to form a judgment on their applicability for the special purpose. In the description of the application of a reagent, at least one mode of preparing it ought to be accurately described, such expressions as "somewhat," "a little," "a short time," and such like, should be avoided, and replaced by exact statements of weight and time. In the case of little known substances, the chemical formula-intelligible in all languages—should be appended. The descriptions of colours should be as correct as possible, with reference to all influencing circumstances, and should be based on some definite colour-scale, such as that of Chevreul's 'Des Couleurs.' Pure Carminic Acid for Staining.t-G. Dimmock has often wondered why naturalists use carmine solutions in which water, with some caustic or destructive material added, is the principal solvent. Carmine of commerce, it is true, is not readily soluble, even in water, until ammonia, borax, or some other aid to solution is added; but carminic acid, the basis of the colouring matter of carmine, has long been stated in the leading chemical dictionaries and handbooks to be readily soluble in water and in alcohol. Watts (Dict. Chem., 1872, 1st suppl., p. 413) says of carminic acid:-" This acid forms a purple * Zeitschr. f. Wiss. Mikroskopie, i. (1884) pp. 101-2. mass, fusible and soluble in all proportions in water and in alcohol. Sulphuric and hydrochloric acid dissolve it without alteration. It bears a heat of 136° C. without decomposition." Earlier still Watts (Dict. Chem., i. 1863, p. 804) says:-"The fine red pigment known in commerce as carmine is prepared by treating a solution of cochineal with cream of tartar, alum, or acid oxalate of potassium. The fatty and albuminous matters then coagulate and carry down the colouring matter with them." Now in preparing most carmine solutions this precipitation takes place, and the carmine, having greater cohesive (not chemical) affinity for impurities of animal origin than for alcohol, its solution is not readily accomplished by that medium, nor indeed by water. In preparing carmine solution for histological purposes by some of the published recipes, more than one-half of the colouring matter of the carmine is lost in the refuse left upon the filter paper. There are two ways commonly in use for preparing carminic acid. The first mode is that of De la Rue, which Watts (Dict. Chem., i. 1863, p. 804) gives as follows:-" To separate carminic acid, cochineal is exhausted with boiling water; the extract is precipitated by subacetate of lead slightly acidulated, care being taken not to add the lead-solution in excess; the precipitate is washed with distilled water till the wash-water no longer gives a precipitate with a solution of mercuric chloride, then decomposed by sulphuretted hydrogen; the filtrate is evaporated to a syrupy consistence and dried over the water-bath; and the dark purple product thus obtained is treated with alcohol, which extracts the carminic acid." The second mode is that of C. Schaller and is given by Watts (Dict. Chem., 1st suppl., 1872, p. 413) as follows:"Schaller prepares this acid by precipitating the aqueous extract of cochineal with neutral lead acetate slightly acidulated with acetic acid; decomposing the washed precipitate with sulphuric acid; again precipitating the filtrate with lead acetate, and decomposing the precipitate with hydrogen sulphide. The filtered solution is evaporated to dryness; the residue dissolved in absolute alcohol; the crystalline nodules of carminic acid obtained on leaving this solution to evaporate are freed from a yellow substance by washing with cold water, which dissolves only the carminic acid; and the residue left on evaporating the aqueous solution is recrystallized from absolute alcohol or from ether." Schaller's mode of preparation gives purer carminic acid than De la Rue's, but either kind is sufficiently pure for histological purposes. The precipitation by lead acetate and the dissolving in alcohol free the carminic acid from animal impurities, and the consequence is a purer form of pigment than can be extracted by any process hitherto employed for the preparation of carmine for histological purposes. It is unnecessary to explain to naturalists the advantages of alcoholic solutions of carmine over aqueous ones. The alcoholic solution colours preparations much quicker than the aqueous solution does; for colouring sections, the author employs a solution of 0.25 gr. carminic acid to 100 gr. of 80 per cent. alcohol, and leaves sections in the solution from two to five minutes. A solution of equal carmine strength but in absolute alcohol can be employed; it has, however, no special advantages, since with the 80 per cent. alcoholic solution the sections can be washed directly in absolute alcohol, and then put into oil of cloves or turpentine. Colouring in the piece before sectioning never takes as long with alcoholic carminic acid as it does with ordinary carmine solutions, and if it did take long the strong alcohol would preserve the tissue from maceration. In colouring pieces of mollusca, or of other equally slimy animals, the slime should be removed beforehand, or the staining will be unsatisfactory, because the slime congealing in the alcohol takes up the colouring matter, forming an almost impervious coloured layer on the outside and leaving the inside of the piece nearly uncoloured. Some preparations coloured in alcoholic carminic acid and then put up in glycerine lost their colour in a few months, the colour seeming to be entirely diffused in the glycerine, while similar preparations mounted in Canada balsam retained their colour perfectly. The author does not know if this fading would occur with preparations coloured with alcoholic ammonic carminate, or even if this diffusion was not due to some impurity of the glycerine (of the purity of which he was doubtful); time to test this matter further failed. An alcoholic ammonic carminate, or ammonia carmine, can be prepared, at a moment's notice, from alcoholic carminic acid, by adding ammonia drop by drop, and stirring until the entire solution changes from its bright red to purple red. By this mode pure alcoholic ammonic carminate can be produced with no excess of ammonia, and at any time. As the carminic acid can be preserved dry without decomposition, and dissolves quickly in alcohol, one can carry the ingredients of a carmine solution in the vest pocket without incon venience. In making and using alcoholic carminic acid pure alcohol and distilled water give the best results, because a portion of the carminic acid is converted to carminates by the salts of impure water. In making alcoholic ammonic carminate this precaution is not as necessary, because the colour of the carminates produced by the impurities of the water is so nearly like that of ammonic carminate. Alcoholic carminic acid may be used, as Grenacher's carmine solution is used, to colour sections from which the colour is to be afterwards partly extracted by very dilute hydrochloric acid, leaving nuclei red. Another way to use carmine solutions, which is especially applicable to alcoholic carminic acid, is to precipitate the carmine in the tissues by some salt, the carminate of the base of which gives a desired coloration. For example, specimens hardened for a moment under the cover-glass with an alcoholic solution of corrosive sublimate (mercuric chloride) and, after washing with alcohol, coloured in alcoholic carminic acid, take a fine colour of mercuric carminate. So, too, specimens coloured in alcoholic carminic acid can be changed by a few moments' treatment with a very dilute alcoholic solution of lead acetate or cobalt nitrate to a beautiful purple. Sometimes salts in the issues of the animals change portions of the carminic acid to purple carminates, giving a double coloration without further treatment. Picric acid added to alcoholic carminic acid in extremely small quantities (best in a dilute alcoholic solution, testing the solution on specimens after each addition) makes a double alcoholic colouring fluid (a so-called picro-carmine). The author has been unable thus far to determine the proportion of picric acid required for this solution, having in every case added an excess. All different kinds of carmine solutions can be made from carminic acid with the advantage of having always uniform strength, of being definite mixtures, and of not spoiling as readily as those made directly from cochineal. Incompatible reagents with carminic acid are, of course, all alkaline solutions and nearly all metallic salts; with ammonic carminate, are naturally all acids; with all carmine solutions, are bromine and chlorine. Hoyer's Picro-Carmine, Carmine Solution, and Carmine Powder and Paste.*-Hoyer proposed + an improved picro-carmine made by dissolving his carmine powder in a concentrated solution of neutral picrate of ammonia. P. Francotte points out that picrate of ammonia is a substance which it is not possible to have constantly at hand, and he has therefore modified Hoyer's preparation in the following manner :-Dissolve 1 gr. of carmine in from 5 to 7 c.cm. of concentrated ammonia, diluted with the same amount of water; in 50 c.cm. of distilled water dissolve (warm) 1/2 gr. of picric acid; mix the two solutions and dilute so as to make 100 c.cm. Then add to the liquid thus obtained 1 gr. of chloral hydrate. If any free ammonia remains, gently warm in a water-bath to drive away the excess, or allow the alkali to volatilize by exposing the liquid to the open air. This solution lasts a long time without changing. M. Francotte also supplements Prof. Hoyer's description of his process for obtaining carmine solution. The latter directs chloral hydrate to be added to the neutral liquid to keep it, but does not state the quantity to be used. M. Francotte forms a carmine solution of 10 c.cm. by the addition of distilled water, to which is added 1 gr. of chloral hydrate. If a paste is required instead of a powder, Prof. Hoyer directs it to be made with alcohol, glycerine, and chloral, but does not give the quantities. M. Francotte uses to 1 gr. of carmine, 2 c.cm. of alcohol, 2 c.cm. of glycerine, and 1 gr. of chloral. Dry Injection-masses.-Prof. H. Fol writes that the red gelatine vermicelli mentioned at p. 312 (carmine emulsions) should be pressed out into slightly acidulated water (1 part acetic acid to 1000 parts water). The carmine will otherwise be washed out. Imbedding Diatoms.§-R. Hitchcock suggests a plan for imbedding diatoms from fresh gatherings. It is to prepare an artificial *Bull. Soc. Belg. Micr., x. (1884) pp. 75-7. † See this Journal, iii. (1883) p. 142. Ibid., p. 141. S Amer. Mon. Micr. Journ., v. (1884) pp. 54-5. calcareous rock from a mixture of finely-ground lime and clay, making a kind of hydraulic cement, with which the diatoms may be mingled. When this hardens, the sections may be cut, and isolated by treatment with diluted hydrochloric acid. The large Pinnularia is a good species to begin with. Zentmayer's New Centering Turn-table.-The turn-table represented in fig. 75 is the invention of Mr. J. Zentmayer. The plan of centering the slide is, it is claimed, quite original and perfect in its results. The slide is placed so that its edges are in contact with the two pins projecting from the face of the plate. A ring with an oval inner edge is fitted to the periphery of the disk, in such a way that by turning it the slide is grasped at the diagonally opposite corners by the inner edge of the ring, and is thus centered longitudinally. The two pins centre it the other way. The ring may be easily removed, and spring clips substituted when desirable. Phosphorus Mounts. It was recently stated that diatoms mounted in phosphorus solution cannot be kept for any time. This is not so. Mr. J. W. Stephenson has slides mounted several years ago (one in 1873), which are as good now as at first. All that is necessary is to avoid long exposure to daylight which turns the diatoms an opaque red. Styrax. On testing this medium (as supplied by Allen and Hanbury) with the refractometer, its refractive index is found to be 1.585 very nearly. It has so much colour that it is difficult to determine the third decimal with accuracy. If we take the index of diatomaceous silex to be 1.43, and of Canada balsam 1.52, it is seen that styrax gives a marked increase of visibility over balsam, for while balsam is only 9, styrax is more than 15. *Amer. Mon. Micr. Journ., v. (1884) p. 23 (1 fig.). |