(4) The test should be applied to an unstained portion of the material which is under examination previous to its application to the stain. Bibulous paper, if used, must be tested in the same manner.

Under the conditions we have given the guaiacum test is reliable and certain. Taylor, as the result of experiments made with a large number of substances, including cochineal, red wine, red ink, extract of rose petals, kino, catechu, Brazil-wood, etc., states that "no red coloring matter, animal or vegetable, excepting the red of blood, produces the blue coloration of guaiacum in presence of peroxide of hydrogen."

(Taylor, Guy's Hospital Reports, third series, 1867, vol. xiii., p. 431.) (Lefort, Annales de Hygiene, second series, vol. xxxiv., p. 429.) (Clement, Conferences Pratiques sur Médecine Legale, Paris, 1880.) The Hæmin Test.-To make this test a drop of blood solution is taken up by the end of a glass rod and spread upon a microscope-slide, so as to give it an extended surface. The slide may be set aside in a place free from dust and allowed to evaporate spontaneously, or it may be heated very carefully in the flame of a spirit-lamp, the slide being moved to and fro, so that the liquid will not be raised to a temperature of more than twenty or thirty degrees above the natural heat of the body. The warmth may be ascertained from time to time by applying the slide to the back of the hand. The temperature must not be allowed to reach 140° F., the point at which albumen coagulates. Should the coagulation take place, the test is likely to fail. Some writers recommend the addition of a particle of common salt to the solution during the evaporation. This is unnecessary in the case of fresh blood, because it contains a sufficient amount of this substance as a natural constituent; but with old or dried blood it is absolutely necessary. The amount required is very small. The best plan is to prepare a solution by weighing out two grains of pure sodium chloride and dissolving in eight fluid ounces of distilled water. A drop of this solution is quite sufficient for the test, and is placed upon the center of the film of evaporated blood. The drop is spread over the film so as to cover all parts of it, and dried off with the same precautions as at first. A drop of glacial acetic acid is next added to the film, the latter covered with a thin glass and the slide heated this time to a sufficient degree to produce a slight ebullition. There is no longer any danger of the coagulation of the albumen. If the boiling be too rapid, the cover may be violently projected from the slide by the force of the vapor.

If the blood to be tested is in a dried state so that a small particle may be detached, a minute portion is placed in the center of the slide and warmed with a drop of acetic acid. The acid is evaporated and the residue treated with the salt solution, and afterward with a fresh portion of acid, as previously described. In the case of fresh blood a single heating with the acetic acid is sufficient, but with very old blood it is frequently necessary to add the acetic acid a second time and heat it

When the acid has wholly evaporated the slide is examined in the microscope under a power of three hundred or four hundred diameters. The operation is easy, but it requires a certain degree of skill in manipulation. The attempt to obtain Teichmann's crystals will often fail in unskillful hands. The principal causes of failure are the coagu lation of the albumen and an excess of salt. Coagulation retards the solvent action of the acetic acid, and during the prolonged ebullition

required under such circumstances the salt may be entirely decomposed, and the evolved hydrochloric acid necessary for the formation of the crystals (hæmatin hydrochloride) is expelled before it can enter into combination. The acid employed must be in its highest state of concentration -the so-called glacial acid. Acid of lower strength fails to decompose the salt. If too much salt be employed the experiment fails, and the slide shows only a mass of crystals of sodium chloride. Distilled water must be used. Ordinary water leaves a residue of organic matter and mineral salts-in some cases very considerable in amount-which interfere with the development of the crystals or may produce a crystalline deposit by itself.

Hæmin crystals are minute flat rhomboids. In many cases they lie superimposed in the form of an X or cross. They vary in color, according to their thickness, from a clear yellow to a reddish brown. They exhibit considerable variation in size, according to the amount of hæmin present and the rapidity of the evaporation. The smallest may be no more than 1-6000 of an inch in length, and the largest may reach 1-1200. The crystals obtained as the result of the test may be covered with thin glass and mounted in the usual manner of microscopic objects. Specimens should be preserved as evidence. Teichmann's crystals once seen can scarcely be confounded with any others. Only two substances have been suggested which resemble them-indigotin and murexide. These, it is claimed, may possibly be produced by a solution of the coloring matter of a fabric dyed with indigo or with ammonium purpurate. Crystals of indigotin are blue, and those of murexide are purplish red and appear green by re

flected light.

The hæmin reaction is very delicate. A quantity of dried blood so small as 1-500 of a grain may easily be detected, and with care 1-1000 will give decisive results. (Wormley.)

Time or atmospheric changes, as a general rule, have no effect upon the certainty of the results, provided there be left in the stain a trace of blood-coloring mat

ter.

The celebrated political writer Kotzebue was murdered by an enthusiast named Sand in 1819. He was assassinated in his own house,

Fig. 15. Hæmin Crystals. (450 diameters.)

and the papers upon his desk were stained with his blood. Sixty years afterward, in 1879, some of these stains were submitted to the hæmin test and the crystals readily obtained. (Clement.)

Buchner and Simon detected hæmin in a portion of cloth cut from a butcher's slaughtering-trousers which had been eight years in use, and had not been worn for a year and a half previously. (Casper.)

All authorities agree as to the value and reliability of the hæmin test. Clement asserts that "crystals of hæmin are a certain indication of the presence of blood." A report of a commission appointed by the French Medico-Legal Society declared that "these crystals are so perfectly characteristic, that, should they be found, one may positively assert the presence of blood." (Miahle, Mayet, Lefort, and Cornil, Annales de Hygiene, 1873, vol. xl.)

Wormley says, "Their production is characteristic of blood, there being no other substance known from which they may be produced." (Microchemistry of Poisons, second edition.)

Failures to obtain hæmin crystals may occur even under the most expert manipulation in the case of old washed stains, but it is the experience of the writer that a stain which under proper treatment fails to yield crystals will fail also to give absorption-bands in the microspectroscope, although fragments of corpuscles may still possibly be found under microscopical examination. In brief, crystals of hæmin, if found, furnish conclusive evidence of the presence of blood. Failure to obtain them is not conclusive as to its absence.

(Erdmann, Jour. de Phar. et de Chimie, vol. xli., p. 33.) (Blondlot, Annales de Hygiene, 1868, vol. xxix., p. 130.)

(Clement, Conferences Pratiques de Médecine Legale, Paris, 1880.) (Otto, Der Gifte und Blutflecken, Braunsweig, 1875, p. 162.)

(Wormley, Microchemistry of Poisons, second edition, Philadelphia, 1885.)

THE PRACTICAL TESTING OF BLOOD-STAINS IN THE MICROSPECTROSCOPE.

Different modes of procedure are required in preparing specimens for spectroscopic examination, according to the age of the stain and the amount of material available for the purpose. If the stain be comparatively recent the operation is simple, easily conducted, and absolutely conclusive in its results. In older stains the conclusions reached are reliable when obtained, but specimens require a more complicated treatment.

1. Examination of a Recent Stain.-If the stain is upon cloth, a portion of the fabric with the stain (one-half inch to one inch square, if possible) is cut into small strips and macerated with distilled water. This is best done in small tube-vials holding from one eighth to one fourth of a fluid ounce. Glass stoppers are preferable, but good sound corks may be used for keeping the specimens free from dust. Each vial should be carefully labeled. If a clot can be obtained free from adhering tissue, it may be treated in the same manner. The amount of water should be proportioned to the size of the clot or stain, and such as to yield a reddish or reddish-brown solution of moderate intensity of color. When the water has become colored, which will be the case in from fifteen minutes to an hour, a portion is removed by a capillary pipette made by heating a small glass tube and drawing it out to a point. The vial should be allowed to stand a sufficient time to allow any sediment to deposit, and this should not be disturbed when the liquid is with

drawn. The fluid taken up by the pipette is transferred to a glass cell made as previously described (Fig. 11), and placed on the stage of the microscope. The cell should be covered by a cap with a small opening in the top to cut off extraneous light, or it may be covered on the outside with black paper. The examination may be made either by sunlight or an artificial source of illumination. It is advisable to try both methods. The microscope with a low-power objective (one-half inch to two inches) is focused upon the top of the cell, and the spectrum is ready for observation. The slit of the spectroscope must be narrowed so as to allow only a sufficient light to pass, the amount necessary depending upon the strength of the solution, the degree of illumination, and the depth of the stratum of liquid. Under favorable conditions, the two bands of oxyhæmoglobin will make their appearance, while the blue end of the spectrum will be more or less obscured. In many cases the spectrum will appear like that of No. 4 or No. 5 in Pl. V. This shows that the stain is of greater age (met-hæmoglobin) than if the spectrum is like No. 1. In either case, drop into the cell a minute crystal of ferrous ammonium sulphate (a particle not larger than the head of a small pin), and immediately about the same amount of sodio-potassium tartrate (Rochelle salt). Stir the solution with a platinum wire until both salts. are dissolved; next add a drop or two of ammonia water, and stir once more. The spectrum, if originally that of oxy-hæmoglobin (Pl. V., No. 1), will now change to that of reduced hæmoglobin (No. 2). In case the spectrum originally presented the appearance of No. 4 or No. 5, it will under the action of the added reagents change to that of No. 6 (reduced hæmatin).

If the first spectrum was like No. 1 (oxy-hæmoglobin), a fresh portion of the solution may be tested as follows: To the blood solution in the tube a little citric acid is added, when, if the color be sufficiently intense, a spectrum more or less resembling No. 3 will develop. In most cases the band at the extreme right is not produced, and it frequently happens in dilute solutions that neither of the other bands makes its appearance; but even in this case the addition of the iron salt, etc., and ammonia water until the acid is neutralized will give the spectrum No. 6 (reduced hæmatin).

A blood-stain no larger than one-eighth inch square will show these bands in a satisfactory manner, provided that all the conditions are carefully observed. If the solution be very strong in color, a thinner stratum may be examined or the intensity of the light increased. Sorby remarks that if all the characteristic results here given are obtained, "no one can hesitate in giving evidence that the mark is blood."

2. Examination of Older Stains.-As we have already observed, oxyhæmoglobin becomes gradually converted into hæmatin, and when this change is complete the coloring matter is wholly insoluble in pure water. For such cases, therefore, a different procedure is necessary. If the particles or portions of fabric in the macerating vial do not readily yield their color to water, a few drops of acetic or a grain or two of citric acid may be added, and the bottle with its contents allowed to stand in a moderately warm place for several hours, or longer, if necessary. The solution is examined in the same manner as before, but the absorptionbands produced will be those of acid hæmatin (No. 3). The iron salt, etc., and ammonia may next be added to the liquid in the tube-cell, as

in the former tests, with the effect of developing the spectrum of reduced hæmatin (No. 6).

Sorby states that he has been able to discover hæmatin by the microspectroscope in a stain forty-four years old. Tidy obtained excellent spectra from stains on a garment preserved by the relatives of an officer who died in battle in 1771, and consequently more than a hundred years old.

Letheby, from a portion of a fabric one-fourth inch square, having upon it a blood-stain seventeen years old, and which had been completely changed to hæmatin, obtained as well marked spectra as from comparatively recent blood. (London Hospital Reports, vol. iii., p. 41.) Richardson, in a stain five months old, obtained satisfactory tests by the microspectroscope from a portion one fiftieth of an inch in diameter.

If there is reason to believe that stains have been washed with hot water or heated, whereby the albumen becomes insoluble, it will be necessary to use ammonia water to dissolve the coloring matter, as dilute acids under these circumstances have very little action. If a bloodstained fabric has been washed, only a slight discoloration may be observed, but it will be spread over a greater surface. In such a case cut the stained portion into small pieces and macerate with a little ammonia water, the tube-vials being set aside for several days in a warm place. Should the solution thus obtained be of too faint color, it must be concentrated by evaporation on the water-bath, care being taken that the liquid is kept alkaline during the process by the addition, from time to time, of small additional quantities of ammonia.

The spectrum of alkaline hæmatin may possibly be obtained from this solution, but, in any case, the test should be made for reduced hæmatin as in the previous trials. When the stain is upon a dyed fabric, the addition of ammonia may cause the solution of more or less of the dye color. In such a case tests with the spectroscope are not very satisfactory, as the dye color may completely mask the absorption-bands of blood. Sodium bisulphite will generally bleach these colors, while the coloring matter of the blood is not affected. Citric acid acts less upon a dyed fabric than ammonia, and should be used if possible. Stains upon oak-wood or leather are difficult of detection by the spectroscope, owing to the action of the tannin upon the blood-coloring matter, whereby it is rendered insoluble and not easily converted into a form capable of satisfactory examination. A trial, however, should always be made, even upon this sort of stain.

Spectroscopic tests may be obtained from the red blood of all animals. "Oxy-hæmoglobin from any source is universally the same in its spectroscopic properties, the compounds it forms, and the products of its decomposition, hæmatin, hæmin, etc." (Halliburton.)

SPECTRA OF COLORED SUBSTANCES SOMEWHAT RESEMBLING BLOOD.

Under the action of ammonia, citric acid, and salts of iron, as previously described, stains due to blood may be absolutely distinguished from all other red coloring matters. Quite a number of these colors give absorption-bands which resemble those of oxy-hæmoglobin, but their appearance under the action of reagents is wholly different.