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ammonia, but not by citric acid, which ought therefore to be used, whereas in other cases ammonia is the best solvent.
Unless the stain is faint, a portion should be soaked in a few drops of water in a watch-glass, the liquid squeezed out, allowed to stand a short time in the glass, so as to deposit any small portions of the fabric, and poured into one of the experiment cells. If the stain had been recently made, and had not been changed by any special action, a solution of hæmoglobin would be obtained, and the various spectra could be seen one after the other, as already described. If, however, the stain were a few days or a few weeks old, we should obtain a mixture of hæmoglobin and methæmoglobin, or the latter alone. The various spectra could then be developed, and compared side by side with those from fresh blood, to be sure that there is complete correspondence in the position and relative intensity of the bands. The residue insoluble in water should then be dissolved in dilute citric acid or ammonia, according to the nature of the fabric, and the spectrum of deoxidized hæmatin developed. If insoluble in cold citric acid or ammonia, hot ammonia should be tried, since the stain might have been so heated as to coagulate the albumen. If it be desirable to keep the specimen of deoxidized hæmatin for subsequent reference, the cell may be covered with a piece of thin glass, and, after removing the excess of liquid, the edge of the cover painted round with gold-size. When properly managed, such an object will show a perfectly good spectrum, even after many weeks.
If therefore we have a sufficient amount of a moderately old stain, we may easily see in succession the seven very different spectra of the following solutions : 1. Neutral methæmoglobin, 2. Alkaline methæmoglobin. 3. Deoxidized hæmoglobin, 4. Oxidized hæmoglobin. 5. Acid hæmatin. 6. Alkaline hæmatin. 7. Deoxidized hæmatin. If the amount was very small, only Nos. 4 and 7 would show distinct bands, and the rest would be characterized rather by their comparative absence; and it must always be borne in mind that Nos. 1 and 2 may be modified by the presence of unaltered hæmoglobin, No. 3 by that of dissolved hæmatin, and Nos. 5, 6, and 7 by that of undecomposed hæmoglobin or methæmoglobin.
It would be easy to obtain other preparations, and to see several other spectra derived from blood, but it appears to me unnecessary, since the above are so remarkable and unique in the manner in which they are produced, one after the other, especially by deoxidization and reoxidization on stirring, which seldom occurs in other colouring matters, that they afford as satisfactory a test for blood as could be desired, and still more so when we consider not only the general character of the spectra, but also the exact position of the absorption-bands, and that some are so most unusually distinct.
The above directions apply to simple cases, where the amount of material at command is amply sufficient, and the fabric on which the stain is found does not contain anything that makes the blood insoluble, or interferes with the various tests. I shall, however, now describe what should be done in cases which are made specially difficult by various causes. If the stain were very faint, from the presence of very little blood, or if the greater part had been removed by washing with water, it might be desirable not to divide the material, but to examine the whole at once. The stained portion should therefore be digested in a few drops of dilute citric acid or ammonia, and the presence of hæmatin determined, as already described. If faint and spread over a considerable surface, it might be well to digest in citric acid or ammonia diluted with much more water than would fill the experiment cell, and afterwards concentrate the solution by gentle evaporation. By this means blood could be detected, even when considerable effort had been made to remove it, and only a faint brown tinge left, just visible on white linen. There would, generally be no difficulty in the case of a stain on cloth which had been sponged, for enough blood solution would be left in the fabric.
The presence of mordants in cloth or prints may require us to somewhat modify our proceedings, especially if the stain had been made wet, and to a great extent removed, so that we have only the dried-up solution of blood, thoroughly incorporated with the mordant. Certain kinds of brown cloth are of such a character, and about seven years ago portions of a wetted stain were sent by me to a number of the highest authorities in the detection of blood, and they said that neither they nor anyone else could recognize it. However, by proper care, I found that after a lapse of six years it could be detected by the spectrum method. The best plan was to digest a portion of the cloth in dilute ammonia, and to squeeze it well over and over again, with a pair of forceps, and finally with the finger and thumb, so as to obtain as much of the solution as possible. This was very turbid, but when deoxidized in the usual manner, and illuminated by concentrated light direct from the sun itself, the band of deoxidized hæmatin was quite distinct. When the cell was kept for a while, so that the insoluble part settled to the side, no band was visible, and therefore the hæmatin was evidently combined with the mordant. It will thus be seen that it may be most important not to filter or allow the insoluble matter to subside, but to overcome the opacity by means of a sufficiently intense light. If the sun could not be made use of, the lime or electric light would no doubt be the best substitute.
When fresh blood solution is agitated in a test tube with vegetable soil, and left until quite clear, the colouring matter is completely carried down with the earth. Dilute ammonia, however, dissolves out hæmatin, and therefore, in testing portions of soil, they should be digested in considerably more of that solvent than will fill an experiment cell, and after the solution has become quite clear it should be concentrated by evaporation. The spectrum of deoxidized hæmatin may then be seen by following the ordinary method. The same process should be adopted in examining stains on clothes impregnated with earth or earthy dust, and marks on iron contaminated with much rust, if water will not dissolve ont unaltered blood or methæmoglobin."
The importance of being able to detect blood-stains on leather was prominently brought before me by a case in which the trial of a suspected person depended on the nature of certain dark marks on his gaiters. The presence of tannic acid so completely mordants the blood, that neither water nor citric acid will dissolve it, and ammonia gives rise to a most inconveniently dark solution. If the stain is on the surface, and has never been wetted, a thin shaving should be cut off, so as to have as much blood and as little leather as possible, and the blood should be dissolved off without exposing the solution to the action of the leather itself. This may be accomplished by taking one of the experiment cells, nearly filled with water, bending the shaving, and inserting it into the upper part of the tube, so as to touch the water, being careful to arrange it so that the stain may be on the convex side of the leather, and in contact with the water. When a drop of blood falls on leather, many red globules are filtered out from the serum and left on the surface, and, when thus treated, they dissolve, and the coloured solution sinks at once to the bottom of the cell, without coming in contact with the leather. The various spectra may then be observed in the usual manner. This method would be of little or no use if the stain had been wetted, and for a long time I concluded that after such treatment it would be impossible to recognize blood. However, after many experiments, and after having again and again almost given up the inquiry in despair, I found that the difficulty could be overcome in a very simple manner. The best solvent for the insoluble compound of the colouring matter of the blood with tannic acid, is hydrochloric acid diluted with about fifty times its bulk of water. If stronger or weaker, the result is not so good. When a portion of unstained common brown leather is digested in this dilute acid, the solution is scarcely tinged yellow. On adding excess of ammonia, the colour becomes pale purple, or neutral tint, made deeper when the double tartrate and the ferrous salt are added, but remaining nearly clear. This gives a spectrum very dull all over, but without any trace of definite bands in any part. The depth of colour varies much with different specimens of leather. A portion of similar material soaked with wetted blood, gives a yellow solution, made brown-purple and turbid by the
double tartrate and ammonia, and remains so when deoxidized. The band of deoxidized hæmatin can however be distinctly seen with a light sufficiently strong to penetrate the turbid and dark solution. Before examining the suspected stain, it would be well to make out how much of the unstained leather could be used without giving too dark a solution, and to use no more of the stained. If the deoxidized solution be too turbid, the cell may be kept for a while horizontal, until the deposit has subsided sufficiently to allow the principal absorption-band to be seen ; but it is not so distinct, when all has subsided, as though the greater part of the hæmatin still existed as a compound insoluble in dilute ammonia.
The presence of tannic acid in wood and other substances might make it necessary to employ a similar process, if the relative amount of blood were so small, that none could be dissolved out by water, or dilute citric acid.
Cases might occur when it would be necessary to decide whether blood were present, along with some other coloured substance, soluble in water. The method to be employed would depend much on the nature of this impurity. If it were a colouring matter, belonging to what I have described in former papers as group A, in which the absorption is removed by sulphite of soda, in an alkaline solution, there would be no difficulty in seeing all the spectra. Thus, for example, it is easy to add so much magenta to the solution of a little blood, that its absorption-bands are entirely hid; but a small quantity of sulphite of soda so completely removes the colour of the magenta, that the various spectra of the blood may be seen almost as well as if it had been pure.
The colouring matters of my group B that are most likely to occur, are those of fruits, and in them the presence of the free acid would be almost certain to have changed the hæmoglobin into hæmatin. The best plan would then be to add excess of ammonia, and, if the solution were made too dark, to dilute it with so much water that the strongest light at our command would show the green part of the spectrum sufficiently bright to prove that no absorption-band occurred there. On deoxidizing in the usual manner, the solution may be made somewhat darker by the presence of tannic acid, but the darker band of deoxidized hæmatin could be recognized without material difficulty.
By far the greater number of the colouring matters belonging to my group C are yellow and orange-coloured ; and, since these chiefly absorb the blue rays, they do not interfere with our seeing the bands of the blood spectra, which occur in the green. Cochineal is one that requires special attention. The addition of ammonia to its solution in water gives rise to two bands in the green, which, though differing materially from those of blood, are yet so nearly in the same situation, that they completely disguise the presence of
a small amount of blood. However, on adding a small excess of boric acid, the bands of the cochineal are made more faint, and very considerably raised towards the blue end, so as to leave the red end of the green clear, whilst those of oxidized hæmoglobin are not changed, and that nearer the red end, if not both, can be seen perfectly well. By proceeding in the usual manner, there is no great difficulty in recognizing the darker band of deoxidized hæmatin.
Other special difficulties might occur in particular instances, but I trust that these examples will suffice to show how they may be overcome. I do not now know of any that require special remarks ; and, as far as I am able to judge, we need never despair of detecting blood, so long as any hæmatin remains undecomposed. Fortunately it resists decomposition so well, that this would rarely happen in ordinary circumstances; but yet there are cases in which it does occur, as, for example, when acted upon by strong ozone, or other powerful oxidizing reagents.
It is quite possible that stained garments might have been washed, and some of the water employed might be obtained. If no soap had been used, this water could be examined in a long tube of thick glass, ten inches or more in length, and a quarter of an inch in internal diameter, permanently closed at one end with a circular piece of plate glass, and, when filled, covered over at the other with another glass. For examining solutions in such tubes a small pocket spectroscope, such as recently made for me by Mr. Browning, is extremely convenient, and suitable in every respect. If only two or three days old, the bands of oxidized hæmoglobin might be seen ; but if the solution had been kept longer, and they could not be detected, it should be concentrated by evaporation at a gentle heat, and tested for hæmatin. If during evaporation any deposit be formed, insoluble in cold dilute ammonia, it should be dissolved by the aid of heat. When soap is used in washing off stains, the alkali soon changes the hæmoglobin into hæmatin, and the soap makes the solution inconveniently turbid and opaque. It is best in such a case to agitate the suspected soap and water with ether, remove it with a pipette, after the two liquids have completely separated, and repeat the process over and over again, with fresh ether, until the aqueous solution at the bottom has become quite clear and free from soap. It should then be concentrated by evaporation, and examined for hæmatin, as usual. Of course in all such cases it would be desirable to test the solution as soon as possible, lest decomposition should occur, but by these means a very small quantity of blood, that would show no colour, might be recognized within a week or two, but probably not after.
For the detection of blood in urine, a tube about ten inches long is very suitable. If turbid it should be filtered ; but, since a
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