by contact with these substances should be no objection to the test, for blood is not blued by guaiacum alone. It is only after the addition of an oxidizing agent that the blue color appears.

The test is made as follows: Two portions of the stained material are taken, and each moistened with a few drops of water. To one of them add a few drops of a freshly prepared tincture of guaiacum and to the other a few drops of fresh solution of hydrogen dioxid. Neither will be changed in color. Then to the first specimen add a few drops of the hydrogen dioxid solution, and to the second add a few drops of the tincture of guaiacum. Both specimens become a distinct, dark blue, and it is evident that neither reagent alone would have produced the change in color. In instances where the stain is on a dark-colored cloth, on which the blue color would not show, add the reagents, and afterwards press the fabric between two pads of white blotting paper, when the color will be absorbed by the paper. A number of impressions may in this way be obtained, and the reaction rendered apparent.

294b. Hemin test.-The formation of hemin crystals (Teichmann's test) has long been a standard for the medical profession and for the legal profession. The crystals are characteristic of blood, and can be obtained from most specimens of blood stains. If the hemoglobin has been decomposed by high temperatures, prolonged exposure to direct sunlight for a long time, or by a few chemicals, the test will not work. It has the great advantage that it requires but a single thread of material stained by blood to give the characteristic crystals.

The least fragment of suspected blood is placed upon a microscope slide. A drop of water and a crystal of salt (sodium chlorid) are put alongside of it, and the three covered with a cover glass, heated, and the water evaporated. Then a drop or two of glacial acetic acid is allowed to run under the cover glass and the preparation again heated till bubbles of gas come off. The preparation is then allowed to cool, and is examined under the microscope. Among the fibers of the thread, or close to it, are seen minute, elongated, rhombic crystals, having a yellow or brownish color. The crystals, as a rule, lie independent of each other, but may lie in pairs, crossed. They are wholly insoluble in water, alcohol, ether, and chloroform, and in acetic, phosphoric, and hydrochloric acids; slightly soluble in ammonia, and in dilute sulphuric or nitric acids. Entirely soluble in potassic hydrate, to which they give a green color. In strong sulphuric and in fuming nitric acids they are soluble; to the nitric acid they give a brownish

red color. In chlorin water they become disintegrated and lose their color. 24

295. Spectroscopic tests. The spectroscopic test for blood is also characteristic of blood, the only disadvantage being that more appa ratus is necessary for its performance. On the other hand the ab sorption spectra of blood and its products is as free from error in its diagnosis as any test that we have. But it is merely a test for blood, and does not tell us from what animal the blood came.

If the blood solution be placed before the spectroscope and examined, even in dilute solutions, it is seen that a portion of the red end of the spectrum as well as a much larger part of the blue end is absorbed; but the most striking fact is the presence of two stronglymarked absorption bands lying between the two solar lines marked "d" and "e." Of these two bands the one towards the red side, sometimes spoken of as band "a" is the thinnest, but the most intense; and, in extremely dilute solutions, the only one visible. Its middle lies a little to the blue side of "d." The other line, called "b," is much broader, lies a little to the red side of “e," its blueward edge, even in moderately dilute solutions, coming close up to that line. In solu tions one centimeter in diameter these two absorption lines may be seen when the solution contains one gram of hemoglobin in ten liters of water. In the concentrated solutions the bands fuse, and more and more of the light is absorbed, till the only rays that pass through the hemoglobin solution are those in the green between the united bands and the blue which has been largely absorbed, and on the other side, between the fused "a" and "b" bands and the slowly advancing absorption of the red end.

If the hemoglobin solution be reduced by the action of a few drops of ammonium sulphid or of an alkaline solution of ferrous sulphate, kept from precipitation by the presence of tartaric acid, the spectrum of reduced hemoglobin as distinguished from that of the previous oxyhemoglobin is obtained. The spectrum of reduced hemoglobin shows a single band in the place of the two characteristic bands of oxyhemoglobin. This single band is fainter than the two bands of the previous spectrum, and has its center a little more to the red. The absorption of the blue end, too, is much less than in the oxyhemo globin. Even in concentrated solutions some light comes through the interval between the central band and the blue end absorption There are some coloring matters besides hemoglobin that give two

24 Buchner and Simon, Virchow's

Archiv, Vol. XV., p. 52.

absorption bands similar to oxyhemoglobin, or others that possibly give an absorption spectrum simulating that of reduced hemoglobin, but if the typical "a" and "b" lines are obtained and are changed on the addition of a reducing agent like ammonium sulphid to the one of reduced hemoglobin, there can be no question but that the solution examined contains hemoglobin. Carbonic acid passed into the solution of hemoglobin produces a further change in the appearance of the spectrum, in that now two bands similar to the oxyhemoglobin bands are present, but they are darker, narrower, and both of them displaced a little to the blue.

If the stain is not a fresh one the spectroscope may show the absorption of methemoglobin, a stable form of oxyhemoglobin. In this case there appears a distinct absorption band between the solar lines "e" and "d" and considerable absorption of the two ends of the spectrum, especially at the blue end. In still older stains, where the blood has been more or less decomposed and hematin formed, the stain is no longer soluble in water, and either acid or alkali must be used to get a solution. The acetic acid solutions give a spectrum similar to that of methemoglobin, though the single band is fainter and broader. The ethereal solution of this acid hematin (Stoke's acid hematin) allows the passage of light through a number of points in the blue region that, in the simple acid solution, is all absorbed. The alkaline solution of hematin gives an absorption spectrum similar to acid hematin, but less of the blue end is absorbed and the single band is moved to such a point that its blueward end coincides with the solar line "d." This alkaline hematin solution, like the hemoglobin solution, changes its characteristics on being treated with reducing agents. When ammonium sulphid is added to this alkaline hematin solution, the spectrum is very much like that of the two bands of oxyhemoglobin, but the two bands are moved somewhat to the blue end of the spectrum. If, in any suspected stain, we obtain this change from the single band of alkaline hematin to the double band of reduced hematin by the action of a reducing agent, the presence of blood can not be questioned.

If there is only a minimuin quantity of blood present, that trace of the stain should be put upon a microscopic slide with a hollow center, and moistened with some physiological salt solution (34 per cent), and examined with a microspectroscope. When the spectrum has been identified the moisture may be rotated away from the stain and the blood corpuscles examined and measured, and then a drop of re

ducing agent added to the stain, if desired, and the reduced hemoglobin spectrum looked for.

Dr. J. G. Richardson, of Philadelphia, who has investigated most thoroughly the size of the corpuscles in different animals, concludes that it is possible to decide with certainty whether the corpuscles in a suspected fragment of blood-clot belong to man or to certain of the lower animals. This can be done only by very high powers of the microscope, those magnifying from 1,200 to 1,800 diameters.

Should the fragment of blood clot to be examined be very small Dr. Richardson has devised the following ingenious method of testing it:

"Procure a glass slide, with a circular excavation in the middle, called by dealers a 'concave centre,' and moisten it around the edges of the cavity with a small drop of diluted glycerin. Thoroughly clean a thin glass cover about 1% of an inch larger than the excavation, lay it on white paper, and upon it place the tiniest visible fragment of a freshly-dried blood clot (this fragment will weigh from one twentyfive thousandth to one fifty-thousandth of a grain). Then, with a cataract-needle, deposit on the centre of the cover, near your blood spot, a drop of glycerin about the size of this period (.), and with a dry needle gently push the blood to the brink of your microscopic pond, so that it may be just moistened by the fluid. Finally, invert your slide upon the thin glass cover in such a manner that the glyc erined edges of the cavity in the former may adhere to the margins of the latter, and, turning the slide face upwards, transfer it to the stage of the microscope.

"By this method, it is obvious, we obtain an extremely minute quantity of strong solution of hemoglobin, whose point of greatest density (generally in the centre of the clot) is readily discovered under a one-fourth-inch objective, and tested by the adjustment of the spectroscopic eye-piece. After a little practice it will be found quite possible to modify the bands by the addition of sulphuret of sodium solution, as advised by Preyer.

"In cases of this kind, where the greatest possible economy or even parsimony of material is needful, I would advise the following mode of procedure for proving and corroborating your proof of the existence of blood, so that its presence in a stain may be affirmed with absolute certainty:

"From a suspected blood spot upon metal, wood, leather, paper. muslin, or cloth, scrape with a fine, sharp knife two or three or more minute particles of the reddish substance, causing them to fall near

the middle of a large, thin glass cover. Apply in close proximity to them a very small drop of 34 per cent salt solution, bring the particles of supposed blood-clot to its edge, and proceed as I have already directed.

"After thus examining the spectrum of the substance, you may generally, by rotating the stage, cause the colored fluid to partly drain away from the portion, wherein, under favorable circumstances, should the specimen be blood, the granular white blood globules become plainly visible, as do also cell-walls of the red discs. Among the latter, if your mental and physical vision is keen enough, you can, by the aid of a one-twenty-fifth immersion lens and an eye-piece micrometer, measure a series of corpuscles accurately enough to discriminate human blood from that of an ox, pig, horse, or sheep.

"Lastly, to make assurance triply sure, lift up the thin glass cover, wipe off the tiny drop of blood-solution and clot you have been examining on the folded edge of a thin piece of moistened blotting-paper, let fall upon it a little fresh tincture of guaiacum, and then a drop of ozonized ether, which will at once strike the dark blue color of the guaiacum-test for blood.

"In this way I have actually obtained these three kinds of evidence to wit, that of spectrum analysis, that of the microscope, and that of a chemical reaction-from one single particle of blood, which, judged by a definite standard (see Handbook of Medical Microscopy, Phila., 1871, p. 283), certainly weighed less than one fifteen-thousandth, and probably less than one twenty-five-thousandth, of a grain."

296. Microscopic test. To determine the kind of animal from which the blood came, research, until within the last few years, has been limited to the microscopic examination of the blood, and the determination of the character and size of the red blood corpuscles. The red blood corpuscles of birds, fish, and reptiles are oval, nucleated cells; those of the camel and the llama are oval non-nucleated discs, and those of all other mammals are circular, biconcave discs. Hence, on the shape and structure of the cells all blood except that of mammals, and some of them, can be excluded. The differentiation between the other various species of mammals depends upon the measurement of the size of the cells. Of all the common mammals the red blood corpuscles of man are the largest,-1/3200 of an inch in diameter. The monkey comes next,-1/3400 of an inch; and next the dog,-1/3500 of an inch. The estimates of a number of observ ers - Gulliver, Wormley, Formad, Richardson, Schmidt, Marson, the