bility to the injection. Some thrive on the treatment; others are easily made sick and die. Further, the animals vary a great deal in the amount of precipitin contained in their sera. Some produce a strong reaction, others only a weak one, and occasionally one is met with which does not seem to react after numerous injections. Of course. the injection must be done under strict asepsis, and the serum that is used must be sterile. If the rabbit develops an abscess it is better to use another rabbit rather than keep on hoping that the animal will recover from the abscess. Any pleuritic effusion or exudate or hydrocele fluid even may be used, but the blood serum itself is the best. After the animal has received a certain number of injections, the rabbit's serum can easily be tested as to its efficiency by withdrawing a little blood from the central vein at the base of the ear.

If found that it produces a reaction of precipitin, as described later, the animal can either be etherized and bled from the carotid arteries or, if it is desired to save the animal, it can be bled from the central vein at the base of the ear. Once the serum of the rabbit is active it can be kept so by the occasional (once or twice a week) injection of human. serum. The blood, after being withdrawn, is allowed to coagulate, and, if necessary, centrifugalized, in order to obtain the serum as clear as possible. By this latter means the serum can often be obtained perfectly clear and almost colorless.

Method of Examination.—If we desire to examine fresh blood, it can be diluted with 100 parts of normal salt solution and, if necessary, allowed to settle, so that the solution is absolutely clear. If a stain is to be tested and it has not been absorbed by the material, a few bits of the cloth can be scraped onto a slide or into a small test-tube, treated with a few drops of water and allowed to dissolve. If the stain has soaked into the fabric so that it is firmly imbedded in the meshes of the material, a few shreds can be soaked out in a few drops of sterile water. In either case the material should be diluted in salt solution. The degree of dilution is not of so much importance as is the degree of dilution of the rabbit's serum. A dilution of 1 part in 100 of the fluid to be tested seems to be the most favorable for the reaction. The dilution of rabbit's serum is of much more importance, as it is upon this dilution that the most accurate results are to be obtained.

Positive reactions have often been obtained with humanized rabbit serum and the sera of lower animals when the rabbit serum was used in too strong solution. It has happened that when the rabbit serum is used in the proportion of 1 part in 10 or 1 part in 20 of blood solution, precipitates have been formed which might be considered as positive. On the other hand, humanized rabbit serum used in the proportion of 1 to 10 or 1 to 20 of solution of human blood has given no precipitate,

while if the rabbit serum was used diluted 1 to 50, or even 1 to 100, a positive reaction was obtained in the same blood. A dilution of 1 of the test serum to 100 of the blood solution will be found to give the most accurate results. If the amount to be tested is small, it may, perhaps, occasionally be better to use a solution of 1 to 50. The second point that is of importance is the length of time that is to be allowed the precipitate to form. In using a dilution of 1 to 100, it often happens that the precipitate does not occur for an hour and a half or two hours; and where the material available for the test has been small and old, even this length of time is not sufficient, and it may be necessary to allow as much as six or twelve hours at a temperature of 37° C. In dealing with small stains, and especially with old ones, the results must be interpreted according to the experience of the investigator, and at best can only be expressed as an opinion.

A detailed account of this test and its application to medico-legal practice will be found in the resumé of Ewing and also in the recent work of Nuttall.

SUMMARY.

We may, therefore, say that under favorable circumstances it can always be determined whether the blood is mammalian or oviparous; it is often possible to distinguish between stains caused by human blood and those caused by the blood of domestic animals: that, if the blood is mammalian blood, and the cells have an average diameter of 33 to 1 of an inch, that the blood is not that of a cat, goat, sheep, horse, ox, or goat, that it may be the blood of a dog, guinea-pig, rabbit, or man. If the cells have a diameter of 3200 of an inch, slightly more or less, then these measurements are consistent with the blood being human blood; and finally, if the biological test, under favorable conditions and properly performed by a recognized competent man, gives the characteristic reactions, then the blood may be said to be human blood.

Graham-Smith and Sanger, referring to the biological test, say: "These experiments have led us to the conclusion that, with sufficient material and due precaution to exclude the various sources of error, there are but few conditions met with in forensic practice under which human could not be readily differentiated from other bloods. By this, however, we do not mean to imply that a considerable acquaintance with the action of precipitating antisera on blood solutions is not necessary in the successful application of this test."

Nuttall, in speaking of the use of this test in forensic questions, says: "That this conclusion is fully justified is proved by the official recogniNuttall. "Blood Immunity and Blood Relationship."

tion of the precipitin method in forensic practice in foreign governments. Prof. Uhlenhuth informs me that the method has been recommended by the Ministers of Justice in Germany and Austria, and that it has been officially recognized by the governments of Egypt and Roumania."

The late Prof. E. S. Wood used it in testifying in the Greenleaf case in New Hampshire and in the Blondin case in Massachusetts. It has also been used in a case in New Jersey and elsewhere.

STAINS CONTAINING BLOOD IN VARIOUS DISCHARGES

It is often possible to determine the source of blood when it has not come from a wound or from a blood-vessel; but this can only be determined by finding, associated with the blood, other formed elements, such as epithelial cells, mucus, spermatozoa, pus, etc.

In cases of nasal hemorrhage the source of the blood can be determined by the recognition of the cylindrical or ciliated epithelium from the mucous membrane and of dried mucus mixed with the blood. Often the hemorrhage is so severe that the stain consists entirely of blood without mucus or epithelium. Of course, where we find mucus and cells we can express an opinion that the blood is consistent with having come from the nose. But we are not justified by finding an absence of mucus or cells to say that the hemorrhage may not have been from the nose. The position and other characteristics of the stain may help us in forming an opinion and enable us to decide whether or not the blood was of nasal origin. If the blood was caused by forced expiration through the nose, the stain is apt to be pale and large, as the amount of mucus is considerable, and when dried we have a very different appearance from ordinary blood-stains.

Generally speaking, menstrual blood will be found to have numerous vaginal cells mixed with it, especially if the blood that is examined was that at the beginning or end of a period. When the hemorrhage is severe we may not be able to find any cells at all, but as with nasal stains, we are not justified in saying that the blood was not of menstrual origin if we fail to find vaginal cells mixed with it. The location of the stain may be of importance in helping to recognize it as being of menstrual or lochial origin. The cells from the vagina are large, polygonal, squamous epithelial cells, similar to those of the mouth and often arranged in small clumps. If there was coexistent with the menstrual flow a discharge of gonorrheal or leukorrheal origin, then the blood and cells will be found mixed with pus. Rarely a ciliated cell from the lining membrane of the uterus may be found mixed with the blood of menstrual origin.

CHAPTER VIII.

SEMINAL STAINS

As a rule, examination of seminal stains does not include the large variety of articles that examination for blood-stains does. The common articles submitted for examination are bedclothes and underclothes; occasionally other articles of wearing apparel; less often bits of wood or leaves, and occasionally scrapings from the skin or genitals of the victim. The stains may exist as dried masses of seminal fluid, or of this mixed with other fluids, such as blood or discharges from the vagina. The seminal fluid itself varies according to circumstances. Its specific gravity varies from 1.027 to 1.037. It has a slightly alkaline reaction, is more or less viscid, with an opalescent appearance, sometimes grayish, occasionally yellowish, or even reddish. It has a peculiar odor which may be considered more or less diagnostic. When such a stain has dried upon a non-absorbent surface, it forms a scale, more or less grayish in color, which can be easily removed by the point of a knife. If the material is absorbent, it may be very difficult to see the stain at all. It will have a more or less irregular contour and a peculiar feel which is more or less stiff, similar to that caused by blood or other albuminous fluids. It may often be seen by holding it against transmitted light, when it will appear more or less translucent. In cases of rape it is more apt to be found on the posterior portion of the victim's garments or upon the anterior part of the assailant's clothing. A body called "spermin", which gives the general reaction of alkaloids, has been isolated from this fluid.

Florence has isolated from the seminal fluid of man an alkaloidal substance which he claims can be obtained only from human seminal fluid. This body, which he calls "virispermin", gives characteristic crystals with a concentrated solution of iodin in potassium iodid. The test is extremely delicate. Other investigators have claimed that any tissue containing cholin will yield the same crystals.

Florence's test is as follows: The reagent is potassium iodid, 1.65 gms.; iodin, 2.54 gms., and water, 30 c.c. A small bit of the clothing or material containing the stain is cut away with scissors and deposited upon a glass slide. A drop of water is then added and the stain allowed to soak for a few minutes. A drop of the above reagent is then placed upon the glass slide near the first drop, and the two drops are allowed

to mix and are immediately covered with a cover slip. The appearance of the crystals is almost immediate if the stain contains seminal fluid.

For the absolute determination that the suspected stain is a seminal stain it is necessary to obtain the spermatozoa. Although the Florence test is a good preliminary test, still it is not conclusive. Spermatozoa are bodies with an oval head about of an inch in length and a tail about ten times as long. The head is less dense in its first third than it is in its posterior two-thirds. Spermatozoa are fairly stable and resist decomposition, but when dry they are very fragile and the head is easily broken from the tail. The recognition, especially in an old stain, is often difficult.

In staining slides Florence sometimes uses crocein, and Whitney has suggested methyl green. In the preparation of this stain for microscopic examination, a small bit of the cloth containing the stain is soaked in distilled water on a glass slide, and then carefully teased apart with a needle, covered with a cover-glass, stained or not, as preferred, and then examined with the microscope. If clothing which is absorbent is to be examined, the Florence reaction may often be obtained from any portion of the stain, but the spermatozoa will be found, as a rule, only in the middle portion.

Farnum has suggested that the biological test similar to that described under Blood be employed in the examination of suspected seminal stains. Instead of blood, he uses 5 to 10 c.c. of semen at intervals of three or four days until the rabbit has received five to eight injections. He has found the test specific for human semen when human semen was used for injection. According to him, blood serum does not give a positive reaction.