In cases where the blood-stain is small, the test may be applied as follows. Moisten a pure white filter paper with a drop of distilled water, or one of the solutions recommended in the section on physical examination, and touch the stain with the moistened portion. On adding a drop of tincture of guaiacum followed by a drop of ozonic ether to the wet filter paper the blue colour will be produced and easily recognised on the white surface. The guaiacum test, although extremely delicate, can only be accepted as providing negative evidence. The absence of reaction proves the absence of blood, except in _very old_ blood-stains, which may not respond to the test. The blue colour produced indicates that the substance _may_ be blood, but it cannot be accepted without corroboration. Gluten, raw potato, milk, bile, sweat (Ogston), and other oxidising substances give a blue colour with guaiacum and ozonic ether; some substances give the blue colour with guaiacum alone. With blood, however, the test is sufficiently delicate to detect one drop in six ounces of water. 4. _Nitric acid_ added to a portion of the solution of blood in distilled water produces a whitish-grey precipitate. [Illustration: Fig. 13.--Photo-micrograph of frog‘s blood showing oval nucleated red corpuscles, × 250. (R. J. M. Buchanan.)] 5. _Hæmin Crystals._--Concentrate a portion of the solution upon a microscope slide, add to it a minute crystal of chloride of sodium and a few drops of glacial acetic acid. Heat gently to dryness or to a lesser degree under a coverslip; examine with the microscope; if blood be present, crystals of hæmin, or the hydrochloride of hæmatin, will be found. They are of a yellowish-red to brownish-black colour with a metallic lustre. They occur in rhomboidal prisms, or six-sided in shape, or in the form of “whetstones,” often in clusters; many of the crystals exhibit a lipped projection on one side. They are known as Teichmann‘s crystals. It is well to verify their origin from blood by placing upon them a drop of hydrogen peroxide, when they will give off bubbles of oxygen gas. They are insoluble in water, alcohol, and dilute acetic and hydrochloric acids. They dissolve in boiling acetic or hydrochloric acids, and the caustic alkalies. They respond to the guaiacum test, and the ash produced by incineration shows the presence of iron by the red colour produced on the addition of a drop of hydrochloric acid and a solution of potassium sulphocyanide. The production, by the methods described, of such crystals affords conclusive proof of the presence of blood. Spectroscopic Examination To a portion of the coloured solution, filtered if necessary, the spectroscopic tests should be applied. The following points must be remembered in carrying out a spectroscopic examination: (_a_) The colouring matter of fresh blood is hæmoglobin, and it may exist in two states, according to the degree of its combination with oxygen.