The various methods now employed to produce haemin crystals were proposed by Hoppe-Seyler, by Brücke and by Erdman. Whichever process is used, the suspected stains are at first carefully separated from the material upon which they are deposited. If they are present on linen, or other fabrics, the stained portions, which always remain somewhat stiff, are cut off: they will present a reddish-brown color, in case the cloth is not dyed: if the stains are on wood, they are removed by means of a sharp knife; if on stone or iron, they are detached by scraping.
In case Hoppe-Seyler's method is used, the stains, separated as directed above, are macerated with a little cold water (warm water would coagulate the albumen present, and consequently prevent solution taking place): the stains become soft, striae and brown or reddish clouds are observed, especially when the dried blood is fresh, and, at the same time, the objects upon which the stains were deposited are decolorized. Upon allowing the fluid obtained in this way to spontaneously evaporate on a watch-glass, a reddish brown or brownish residue is left, from which the crystals of haemin are prepared in the following manner: An almost imperceptible amount of common salt is added to the residue, then, six to eight drops of concentrated acetic acid, and the mass thoroughly mixed by stirring with a small glass rod. The mixture is at first heated over a small gas flame, then evaporated to dryness by the heat of a water-bath. If the stains were produced by blood, a microscopic examination of the residue will reveal the presence of haemin crystals. This method presents an objection: if the stained objects have been washed with warm water previously to the examination, the albumen will be coagulated, and the blood rendered insoluble; in this case, cold water will fail to dissolve anything, and the residue will not produce crystals when treated with acetic acid.
In order to remedy this difficulty Brücke operates directly upon the stained woven or ligneous fibre, or the matter removed from the stone or iron: The materials are boiled in a test-tube with glacial acetic acid, the fluid decanted or filtered, a trace of common salt added, and the liquid then evaporated on a watch-glass at a temperature between 40 and 80°. If the stains really originated from blood, haemin crystals will now be easily perceptible upon examining the residue obtained under the microscope.
The stained fabric, the matter removed from the stone or iron, or the residue left by the solution with which the stains have been treated, is placed on the glass, a trace of chloride of sodium added, and the whole covered with a thin glass plate. A drop of acetic acid is then placed at the edge of the plates—between which it is soon introduced by capillary attraction—and the mixture allowed to rest in the cold for a few moments. The mass is next brought into solution by slightly heating, and is then evaporated by holding the plate at a considerable distance above a gas burner. The fluid is examined from time to time under the microscope: when it is sufficiently concentrated, crystals, presenting the appearance represented in Figs. 21 or 22, will be observed. These are especially well-defined, if an insoluble substance is also present between the plates—which prevents their adhering. The fluid collects by capillary attraction at the points of contact of the plates as a more or less colored layer, in which the crystals are deposited.
Should the above test fail to present distinctive indications at first, one or two fresh drops of acetic acid are introduced between the plates, and the examination is repeated. The result is not to be regarded as negative, until several trials have proved fruitless, as the stained portions are but slowly soluble, and crystallization may have been prevented by the too rapid evaporation of the acetic solution.
Haemin crystals, once seen, can hardly be confounded with other substances; still, it is well to identify them by confirming their insolubility in water, alcohol, and cold acetic acid, as well as their instantaneous solubility in soda lye.
The addition of common salt is ordinarily superfluous, as it is normally contained in the blood; but it is possible, if the stains were washed with warm water, that, in addition to the coagulation of the albumen, the solution of the salt may have taken place, in which case crystals will fail to form. The addition of salt is to remedy this possible contingency; albeit, the delicacy of the test is not affected, even if crystals of chloride of sodium are produced, as these are easily soluble in water, and are readily distinguished from those of haemin by aid of the microscope.
The indications furnished by means of the spectroscope are less reliable than those given by the production of haemin crystals; moreover, the spectroscopic examination requires favorable weather for its execution. Still, the test should be employed in all possible instances. The course pursued is the following:
The aqueous fluid, with which the stains have been treated, is placed in a watch glass, and evaporated in vacuo over sulphuric acid; the last remaining portion of the fluid being united in the bottom of the glass by causing it to collect in a single drop. When the evaporation of fluid is completed, the watch-glass is placed before the narrowed slit of a spectroscope, and a ray of diffused light (or better, light reflected from a heliostat) made to pass through the part of the glass containing the residue. If the stains originate from blood, the absorption lines of haemoglobin, consisting of two large dark bands, to the right of the sodium line (Frauenhofer's line D), will be observed in the spectrum. In case both of the above tests fail to give positive results, it is almost certain that the stains examined were not caused by blood. If, on the contrary, the reactions were produced, scarcely any doubt exists as to the presence of blood. Under these circumstances it is advisable to confirm the results by means of the tests that have been previously spoken of as being formerly exclusively employed; these are the following:
a. 1/2 to 1 c. c. of ozonized oil of turpentine, i. e. turpentine which has been exposed to the air sufficiently long to acquire the property of decolorizing water that is slightly tinted with indigo—is introduced in a test-tube, and an equal volume of tincture of guaiacum added (the latter tincture is prepared by treating an inner portion of the resin with alcohol, until its brownish color is changed to a brownish-yellow).