in which the Sponge was developing. Numerous larvæ attached themselves to these, which could be easily taken out of the water and examined. With the aid of silver nitrate they formed excellent permanent preparations, which may be set up between two cover-glasses in Canada balsam. The sponges were hardened by absolute alcohol; for the larvæ the best preparation was found to be chrom-osmic-acetic acid. Borax-carmine and hæmatoxylin were the staining reagents. The anilin colours, Lyons blue and malachite green, were the best for staining sections in which it was desired to differentiate the yolk. Imbedding was effected with paraffin; when the larvæ had attached themselves to Elodea-leaves this was quite easy, but when free they must first be fixed to a bit of liver by albumen, on account of their very small size -scarcely larger than a large Infusorian. Very high magnification is necessary to make out the component cells. Preparing Fungus-spores.*-Herr P. Hennings recommends a modification of Herpell's plan † for fixing and preserving the spores of fungi. The discoloration of white spores which frequently takes place when this method is employed, can be prevented by saturating with alcohol. Coloured spores can be best preserved by making the paper absorb from below a solution of colophone in alcohol. Study of Saprolegniaceæ.-Prof. M. Hartog recommends the following processes for fixing and staining this family of fungi. The reagent used for fixing is a saturated solution of corrosive sublimate; the preparation is then washed with water and placed in absolute alcohol. The best staining reagent is a solution of the Naples boracic carmine, and the excess of colour is removed by an alcoholic solution of crystallized acetic acid. The staining succeeds best after the objects have been slightly acted on by a very slightly acidulated alcoholic solution of nigrosine, and is completed by a second more complete staining with nigrosine. The preparation may be mounted either in a solution of equal parts of sulphophenate of zinc and glycerin; in Canada balsam, after placing in absolute alcohol, to which is added, drop by drop, phenicated xylol in the proportion of 3 parts of xylol to one of phenic acid; or in essence of sandal-wood oil. Preparation of the Lower Algæ.§-For cultures of the lower Alge, Chlamydococcus, Eudorina, Gonium, &c., M. P. A. Dangeard uses Van Tieghem's moist chamber, consisting of a ring of glass fixed to the slide, and covered by a cover-glass, on the lower face of which is a drop of water containing the objects to be cultivated. The chambers are kept in a constantly moist atmosphere. The fixation may be effected by concentrated picric acid, 1 per cent. chromic acid, absolute alcohol, or 1 per cent. osmic acid. For studying the vibratile cilia or flagels, chromo-osmic acid is best employed, which admits of an immediate observation, or the object may be fixed on the slide by concentrated osmic acid; it is then covered by a cover-glass and stained by a trace of methyl-green or by hæmatoxylin. To study the internal structure it * Verhandl. Bot. Vereins Brandenburg, xxx. (1889) pp. 136–7. + Cf. this Journal, 1882, p. 122. ccix. Bull. Soc. Bot. France, xxxvi. (1889), Actes du Congrès de Bot., pp. ccviii.§ Notarisia, v. (1890) pp. 1001-6 (16 figs.). is often advisable to fix with absolute alcohol for 24 hours, and stain with picro-carmine or aqueous hæmatoxylin. For spores or cells, the walls of which are not penetrated by these reagents, Tschirch's borated carmine may be used. When coloured to the right extent, the preparations should be studied in glycerin, Canada balsam, or essence of clove, after dehydrating by alcohol of about 80 per cent. Preparing Sections with Elder-pith.*-Herr J. W. C. Goethart recommends the following method, where very thin sections are not required. A vertical slit is made in a cylindrical piece of pith, the pith being left much longer on one side of the slit than on the other, and the longer side is thoroughly soaked with alcohol. The object of which sections are to be made is then placed in the slit, and a thin platinum wire firmly bound round the whole. The whole is now moistened with alcohol, and placed in the microtome. The sections are then placed and examined in glycerin. Mounting Algæ and Fungi.t-From practical experience, Mr. J. E. Humphrey strongly recommends, in the preparation of slides of Algae and Fungi, the discarding of all fluids and cements, and the use of glycerin-jelly as recommended by Dr. L. Klein,‡ which he finds applicable to all classes of Thallophytes, after hardening with osmic acid. Even the colour of the pigments is, in most cases, perfectly preserved by this process. 66 The Preparation of Vegetable Tissues for Sectioning on the Microtome.§-Mr. A. J. M'Clatchie says, Vegetable tissues vary so much as to the amount of protoplasm, cellulose, and other substances contained, that the methods used for obtaining good sections from them must vary greatly. I have prepared and sectioned fungi, lichens, the cotyledons, plumules, hypocotyledonary stems, roots, root-tips of the cucumber, young pine-cones, young wheat-blades, lilac-buds, and beanstems, with varying degrees of success. Lichens and the young firm cotyledons of the cucumber could be dehydrated, and permeated with paraffin much more rapidly than young meristemic tissue, or tissue composed largely of cellulose and water. The former may be placed in 50 per cent., 75 per cent., 90 per cent., and 100 per cent. alcohol, chloroform, chloroform and paraffin, and finally in paraffin at a temperature of 55° C., remaining in each from two to twelve hours, and good results will be obtained. But the meristemic and the thin-walled watery tissue must be treated differently, or the tissue will come through very much shrunken and distorted, worthless biologically. I have had the most success following the method described by Dr. J. W. Moll, in the 'Botanical Gazette for January 1888. I have obtained good sections from all the material that I have treated in this way. I used a 1 per cent. solution of chromic acid and 20 per cent., 35 per cent., 50 per cent., 75 per cent., and 90 per cent. alcohols for * Bot. Ztg., xlviii. (1890) p. 354 (1 fig.). Bot. Gazette (Crawfordsville), xv. (1890) pp. 168-71. § Amer. Mon. Micr. Journ., xi. (1890) pp. 190-1. July 1890. From Amer. Naturalist, dehydrating. The chromic acid seems to fix the protoplasm, and macerate the cellulose, allowing the alcohols to pass more freely. I allowed the specimens to remain in the several per cents. of alcohol from two to twenty-four hours, according to their size and texture. As a rule, I found that the more gradually the specimens were dehydrated the better. From absolute alcohol, the specimens were placed in a solution of equal parts turpentine and paraffin. The solution containing the specimens was then raised gradually from a temperature of 20° C. to about 45° C. They were then placed in melted paraffin, kept as nearly at 50° C. as possible. Small specimens will be permeated in one or two hours, but large specimens require from four to six hours. From the 75 per cent. alcohol I placed the specimens in a stain. The stains I tried were alum-cochineal, hæmatoxylin, fuchsin, methylgreen, methyl-blue, methyl-violet, and ammonia-carmine. I found alum-cochineal a good stain for fungi, plumules, stems, roots, and roottips, but it would not penetrate the cucumber cotyledons. Fuchsin would penetrate anything I tried; but as it is soluble in alcohol, it is necessary to overstain the specimens, and then allow the colouring to come out until it is about right. Hæmatoxylin stained all the tissue that I tried except the young cucumber cotyledons. This stain gives large specimens a dark blue colour on the outside, and a purplish-pink colour on the interior. The nuclei and the cell-walls are brought out clearly. I did not have good success with the methyl colours, as they were easily dissolved out by the alcohol. If specimens have not taken sufficient colour, or if the alcohol has removed too much of the colour, sections can be stained upon the slide, after they are cut. Any stain can be used, but none that I tried differentiated the parts sufficiently. Fuchsin will give enough colour in a few seconds. The sections must stand in hæmatoxylin from two to ten minutes, and in alum-cochineal from ten to twenty minutes. If it is intended to stain upon the slide, an alum fixative will be found better than collodion. If I heated the slides in the gas-flame to melt the paraffin, and poured on turpentine to wash it out. The specimens were then mounted in balsam dissolve in chloroform. Air-bubbles that appear when sections are first mounted, will disappear after the slides stand a few hours. the razor or knife used for cutting is very sharp, small specimens may be cut 1/2500 or even 1/5000 in. in thickness. But larger specimens cannot be cut more than 1/600 to 1/1500 in. thick without crowding the tissues together and giving them the appearance of being shrunken." Preparing, Preserving, and Mounting Objects of Natural History for the Microscope.*-Mr. N. Pike says his own method of procedure in selecting, preparing, and preserving small delicate specimens (excepting eggs) is as follows: "I first procure the most perfect live specimens and drop them in strong alcohol, and let them remain about twenty-four hours. This not only instantly destroys life without injuring the objects, but also hardens them a little. They are then taken from the alcohol and placed in small narrow tubes, which I have for this purpose, just large *The Microscope, x. (1890) pp. 266 8. enough to receive them, and are then covered with the following solution:-Chloral, in crystals, 1 oz., dissolve in 5 oz. of distilled water; alcohol, 1 oz.; glycerin, 1 dr.; rock salt, 15 gr.; saltpetre, 30 gr. Dilute the glycerin, salt, and saltpetre in the alcohol, and when well mixed add to the chloral solution. Shake well till thoroughly incorporated, filter, and it is ready for use. The liquid, if properly and carefully made should be bright and sparkling. Larvæ, spiders, &c., when prepared according to the above formula, are really beautiful objects, and can be examined with a low power of the Microscope. If wanted for dissection, they can be removed from the tube and be returned to it without any difficulty. I have thousands of specimens of soft-bodied animals now preserved in this solution, as fresh as the day I collected them. If the objects are required for immediate anatomical examination they can be preserved for an indefinite time, and brought to the dissecting table as fresh and flaccid as possible, by omitting the alcoholic bath. I have always by me a jar filled with the above-mentioned fluid, in which I place specimens I intend for dissection and minute microscopical examination. This jar is always very carefully corked, as the preparation deteriorates when allowed to evaporate. The preserving of small objects in these tubes of pure white flint glass is far preferable to the building of glass cells, which are often leaky and easily get out of order. When very small objects are required I make a slight difference in the solution, but only long practice can give the precise methods for each article, as different specimens require different manipulation. Goadby's solution makes fine preparations, but in time the corrosive sublimate in it produces white deposit on the specimen and spoils it. All solutions containing much glycerin are apt to affect calcareous substances when present. Among many of the freshwater and marine Algæ I have succeeded in preserving specimens, to my perfect satisfaction, in the following solution --Distilled water, 1 oz.; rock salt, 2 gr.; alum, calcined, 1 gr.; carbolic acid, 1 drop. Some specimens of Alge, now twelve years in this solution, are as fresh and bright as when first prepared. Chloride of zinc solution is very useful, and has proved satisfactory in the preservation of animal tissues; it must be made of varying strengths, according to the softness of the parts to be preserved. It is recommended to use twenty to twenty-five grains of the fused chloride to one ounce of distilled water, and ten drops of phenic acid added to it. This is a capital solution for the larvae of insects, and if stored in an air-tight tube or cell, will keep perfectly for years without deterioration." BACHMANN, O.-Leitfaden zur Anfertigung mikroskopischer Dauerpräparate. (Instructions for making permanent microscopic preparations.) München und Leipzig, 1890. LOEWENTHAL, N.-Zur Frage über die Anwendung von Terpentinöl in der histologischen Technik. (On the use of turpentine-oil in histological work.) Centralbl. f. Physiol., XXV. (1889) 2 pp. |