Chapter VII: Appendix: 163 (5)

Good milk contains, on an average, 3.7 per cent. of butter; 5.7 per cent. of lactose, and leaves upon evaporation 12 to 14 per cent. of solid matters.[T] The most common adulteration of milk consists in the addition of water. This fraud is detected by means of an areometer (_lactodensimeter_) which gives directly the specific gravity of the fluid under examination. Should the density be much below 1.030, it is certain that water has been added. It does not, however, necessarily follow if it is about 1.030 that the milk is pure, since the gravity of the fluid, which would be increased upon skimming, could be subsequently reduced to 1.030 by the addition of water. The lactodensimeter, therefore, although useful in the detection of a simple admixture, fails to give reliable results if the fraud perpetrated is a double one; and a determination of the proportion of butter present is also usually necessary. Numerous methods have been proposed to accomplish this estimation. The most preferable of these, owing to the rapidity with which the operation is executed, is the use of the lactoscope (_galactoscope_). This instrument consists of a tube provided with a glass plate fitted at one end, and with a movable glass plate at the other extremity. A few drops of the milk to be tested are placed between the two plates, and the tube lengthened, by screwing out the movable plate, until the fluid no longer transmits the light of a candle placed at a distance of one metre. As the opacity of milk is due to the butter present, it is evident that the proportion of this substance contained in the sample can be estimated by the relative distance which the plates have been separated.

[T] The British Society of Public Analysts regard the following as the
_minimum_ proportions of constituents in unadulterated milk:

Fat 2.5 per cent.
Solids, not fat 9. " "
----
Total 11.5 " "
Water 88.5 " "

--_Trans._

The lactoscope possesses, however, but a limited degree of precision. _M. Marchand_ substitutes to its use the following tests: A test-tube is graduated in three equal divisions, the upper one being subdivided into hundredths extending above, in order to determine accurately the correct volume of the fluid, expanded, as it is, by the temperature of 40°, at which the examination is executed. The first division of the tube is filled with milk, a drop, or two of strong potassa lye added, and the mixture well shaken: the second portion is then filled with ether, and the third with alcohol. The mixture is next again thoroughly agitated, and then exposed to a temperature of 40° in a water-bath. After standing for several hours, a layer of fatty matter becomes sufficiently separated to allow of measurement: but, as it contains some ether and as a small amount of butter may still be retained in the lower aqueous fluid, a correction of the results obtained is necessary. M. Marchand has compiled a table, which facilitates this correction (_vide_: _Journ. de Pharm., Novembre 1854_, and _Bulletin de l'Académie de Médecine, Paris, 1854_, xix., p. 1101).

Previously to the introduction of Marchand's apparatus, use was made of the _lactometer_, which consists simply of a graduated glass tube, in which the suspected milk is allowed to remain for 24 hours, at a temperature of 15°. After the lapse of this time, the cream present completely separates as a supernatant layer, the thickness of which indicates the quality of the sample taken.

_M. Lacomte_ recommends the addition of glacial acetic acid, in order to cause the more rapid separation of the cream.

The estimation of the butter being accomplished, it is frequently needful to determine the amount of lactose present. For this purpose, recourse is had to Barreswil's method, based upon the reduction of cupro-potassic tartrate by milk-sugar in the presence of alkalies. A solution is prepared containing 40 grammes of pure crystallized sulphate of copper, 600 or 700 grammes of caustic soda lye of sp. gr. 1.12, and 160 grammes of neutral tartrate of potassa. The sulphate of copper and tartrate of potassa are previously dissolved separately in a little water, the three solutions united, and water added until the fluid acquires a volume of 1154.4 cubic centimetres. In order to standardize this test solution, a known weight of pure lactose is dissolved in water and the fluid added, drop by drop, from a graduated burette, to a small flask containing 10 cubic centimetres of the copper solution, diluted with 40 cubic centimetres of distilled water, and heated to boiling. At first a yellow precipitate forms, which gradually turns red, and is deposited on the bottom of the flask, leaving the solution colorless. As soon as the test solution is completely decolorized, the addition of the lactose solution is discontinued, and the weight of lactose corresponding to 10 cubic centimetres of the test fluid calculated from the quantity used. The standard of the test solution having been determined, the above operation is repeated, the milk under examination being substituted for the solution of pure lactose. The quantity of milk necessary to decolorize 10 cubic centimetres of the copper solution will evidently contain the same amount of lactose as the quantity of solution used in the preliminary test, and the actual amount of lactose present is very easily calculated. When an estimation of the solid matter contained in the milk is required, a known weight is evaporated to dryness over a water-bath, and the residue weighed. In performing this evaporation, the addition of a known amount of sand, or ground glass, is advisable. The amount of ash present is determined by incinerating the residue left by the evaporation.

Foreign substances are sometimes added to milk, for the purpose of disguising the presence of an abnormal quantity of water, the principal of which are: chalk, bicarbonate of soda, emulsion of almonds, gum tragacanth, gum arabic, starch, flour, decoction of barley or rice, sugar, and cerebral substances. These bodies are detected as follows:

_Chalk._--If chalk is contained in the milk, it readily subsides upon allowing the sample to remain at rest for some time in a flask, forming a deposit which effervesces when heated with hydrochloric acid, and dissolves to a solution, in which the characteristic properties of a lime salt can be recognized.

_Bicarbonate of soda._--In presence of this compound the milk possesses a strongly alkaline reaction, furnishes a serum having a sharp and bitter taste, and leaves a residue of the salt upon evaporation.

_Emulsion of almonds._--The milk has a specific gravity of at least, 1.033. If it is passed through a gauze, small opaque lumps are separated. When examined under the microscope, numerous minute globules, having a diameter of 1/400 of a millimetre, are observed, and, upon adding a few centigrammes of amygdaline to one or two grammes of the milk, the characteristic odor of bitter almonds is produced.

_Gum tragacanth._--When shaken in a glass flask and allowed to rest, the milk deposits on the sides small transparent lumps, which usually present a slightly elongated or angular form.

_Gum arabic._--The addition of alcohol produces an abundant white opaque precipitate.

_Starch, flour, decoction of barley, rice, etc._--Upon boiling the suspected milk, and adding tincture of iodine, the amylaceous substances present produce a blue coloration in the fluid.

_Sugar._--If yeast is added, and the mixture allowed to stand for some time at a temperature of 30°, alcoholic fermentation ensues; under these circumstances, lactose does not undergo fermentation.

_Cerebral substances._--Adulteration by these substances is probably of much less frequent occurrence than was formerly supposed. The admixture is detected by evaporating the milk to dryness, dissolving the residue in ether, evaporating the etherial solution, and fusing the second residue, which consists of fatty matters, with nitrate of potassa in a platinum crucible. The mass is then taken up with water, and chloride of barium added to the solution. If cerebral substances were contained in the milk, ether will dissolve the fatty matters present, the phosphorus of which is converted into a soluble phosphate by the calcination with nitrate of potassa and is thrown down as a white precipitate, upon the addition of a solution of chloride of barium. This test may be confirmed by a microscopic examination of the milk, when the peculiar appearance of cerebral matter will be detected.[U]

[U] Fragments of nerves, and other organic structures, are frequently
observed in this examination.--_Trans._

WINE.

The most common adulteration to which wines are subjected is the addition of water: wines having a rich color are frequently mixed by the dealer with lighter wines, and the fraud consummated by adding water. The detection of this adulteration is somewhat difficult, as water is a normal constituent of wine. In Paris the following method is usually employed: As soon as the wine is confiscated, it is ascertained what kinds of wine are manufactured by the inculpated dealer, and a statement obtained from him, giving the proportions of alcohol, etc., contained in the various brands. A wine is then prepared, according to the information received, an estimation of the alcohol contained in the prepared sample made, and the results compared with those furnished by a similar examination of the suspected wine. In case the proportion of alcohol is less in the suspected wine than in the prepared sample, it is evident that a fraudulent adulteration has been committed. If, however, the quantity of alcohol is the same in both wines, it does not necessarily follow that the wine has escaped admixture, since this body may have been added after the adulteration with water. In addition to the estimation of alcohol, it is also necessary to determine the amount of cream of tartar (bitartrate of potassa) present, as the proportion of this salt would be sensibly decreased by the addition of alcohol and water to the wine. This fraud could, however, be disguised by subsequently adding the proper amount of cream of tartar.

It is also well to ascertain if two equal quantities of the prepared sample and the wine under examination require the same amount of solution of hypochlorite of lime for decolorization. In case the suspected wine has been adulterated, the quantity of hypochlorite solution used will be less than the amount necessary to decolorize the prepared wine. Foreign coloring matter may be added by the adulterator, but this fraud is easily detected by adding potassa to the sample: if its coloration is natural, a green tint is produced; whereas, if foreign matter has been introduced, the wine assumes various other colors upon the addition of the alkali.[V]

[V] _Cotlini_ (_Ann. du genie civil_, No. 3, 1873) states that the
following reactions occur when artificially colored wines are heated
with potassa:

Pure wine no precipitate greenish hue
Elderberry violet "
Beet-sugar red "
Logwood red violet-red "
Privet violet-blue "
Turmeric light-blue "

According to _M. de Cherville_ (_Quar. Jour. Sc._), a bright violet
coloration is produced in the above test, if litmus be present.

Fuchsin is separated by treatment with subacetate of lead and
addition of amylic alcohol (_Jour. de Ph. et de Ch. Mar.
1873_).--_Trans._

The indications furnished by the above test are rendered valueless, if the wine has been artificially colored by the addition of the coloring matter of grape-skins; but the execution of this fraud would require some knowledge of chemistry, and fortunately adulterators, as a class, are deficient in this branch of science.

Another method for detecting the addition of water is based upon the fact that fermented liquors do not contain air in solution, but only carbonic acid; whereas, water dissolves oxygen and nitrogen. It is executed as follows:

The wine to be tested is placed in a flask, the delivery-tube of which is also filled, and heated; the evolved gas being collected in a tube filled with mercury. In case the wine is pure, the disengaged gas will be completely absorbed by potassa; if, on the other hand, water has been added, an unabsorbed residue, consisting of oxygen and nitrogen, will remain.

This test is useless in case water, through which a current of carbonic acid gas has been passed for a considerable time, has been employed. Under these circumstances, however, the presence of the gas would probably be detected by the taste of the wine, as well as by the estimation just mentioned, since the sample would invariably contain a larger proportion of the gas than the standard with which it is compared; indeed, it would be almost impossible to prepare a solution which contained exactly the proportion of carbonic acid ordinarily present in wine.

It remains to mention the methods employed in determining the amount of alcohol and cream of tartar contained in wine.

The alcometrical method usually employed is based upon the difference in density possessed by pure alcohol and by mixtures of alcohol and water. _Gay-Lussac_ has proposed an areometer (_alcoholmeter_), provided with a scale which directly indicates the proportion of alcohol contained in a mixture. As the indications furnished by this instrument vary with the temperature, and the scale is constructed on the basis of a temperature of 15°, a correction of the results obtained is necessary if the determination is made at other temperatures. Gay-Lussac has compiled a table which indicates at once the required correction; the following formula can also be used: _x = c ± 0.4 t_, where _x_ is the quantity of alcohol present in the sample; _c_ the degree indicated by the alcoholmeter, and _t_ the number of degrees differing from the temperature of 15°: the second member of the formula is subtracted from, or added to the first, as the temperature at which the estimation is made is greater or less than 15°.[W]

[W] Tralles alcoholmeter is almost exclusively employed in this
country.--_Trans._

In case the wine to be examined contains substances other than water and alcohol, which would affect its density, it is necessary, before making use of the alcoholmeter, to distil the sample and subsequently examine the distillate, which will consist of a simple mixture of water and alcohol. Usually the distillation is discontinued as soon as one-third of the sample has passed over, and a quantity of distilled water, sufficient to render the volume of the mixture equal to the original volume of the wine, added to the distillate: the fluid remaining in the flask will be entirely free from alcohol. The addition of water to the distillate is not indispensable, but otherwise it is necessary to divide the degrees indicated by the alcoholmeter by 3, in order to reduce the result to the original volume of the wine taken.

_M. Salleron_ offers for sale a small apparatus (Fig. 20) used in examinations of this character, consisting of a flask, closed with a gutta-percha cork, containing a tube which connects with a worm passing through a cooler. The flask is supported by an iron stand, and heated with a gas or spirit lamp.

In order to estimate the cream of tartar, the wine is evaporated to the consistency of an extract, alcohol of 82° B. added, and the residue obtained calcined in a crucible. The amount of salt present in the fused mass is then determined by the alkalimetric method, as directed in all works on quantitative analysis. The carbonate obtained from 1 gr. of cream of tartar exactly saturates 9.75 cubic centimetres of a solution containing 100 grammes of sulphuric acid of 66° B., and 1800 grammes of distilled water.

The detection of toxical substances, often contained in wine, is accomplished by the methods described under the head of detection of poisons.

VINEGAR.

Vinegar is frequently adulterated with water, and occasionally sulphuric acid is added to artificially increase its acidity.

The ordinary reagents--such as chloride of barium, or nitrate of silver--are not adapted to the direct detection of sulphuric acid, or of other mineral acids, as sulphates and chlorides, which are as readily precipitated as the free acids, may also be present.

The following method, proposed by _M. Payen_, is usually employed:

Five centigrammes of starch (fecula) are added to a decilitre of table vinegar, the mixture boiled for 12 or 15 minutes, and, after the fluid has become _completely cooled_, a few drops of iodine solution added: dilute acetic acid does not affect starch, and, in case the vinegar is pure, a blue coloration is produced; if, on the other hand, even a minute quantity of a mineral acid be present, the starch is converted into dextrine, and the addition of iodine fails to cause a blue coloration.

The water present is indirectly estimated by determining the amount of acetic acid contained in the vinegar. This can be accomplished in different ways: either the quantity of a standard solution of an alkali, necessary to exactly neutralize a measured quantity of the vinegar, is ascertained, or the vinegar is supersaturated with solution of baryta, the excess of the salt eliminated by conducting carbonic acid through the fluid, the precipitate removed by filtration, and the baryta salt in the filtrate precipitated by the addition of sulphuric acid. The second precipitate is then collected on a filter, washed, weighed, and the amount of acetic acid present calculated: this is done by multiplying its weight by 0.515.

SULPHATE OF QUININE.

Owing to the high price of this salt, it is frequently adulterated. The substances used for this purpose are: crystalline sulphate of lime, boric acid, mannite, sugar, starch, salicine, stearic acid, and the sulphates of cinchonine and quinidine. These bodies are detected as follows:

_a._ Upon slightly warming 2 grammes of sulphate of quinine with 120 grammes of alcohol of 21° B., the pure salt completely dissolves; if, however, starch, magnesia, mineral salts, or various other foreign substances are present, they are left as insoluble residues.

_b._ Those mineral substances that are soluble in alcohol are detected by calcining the suspected sample: pure sulphate of quinine is completely consumed; whereas, the mineral substances present remain behind as a residue.

_c._ In presence of salicine, the salt acquires a deep red color, when treated with concentrated sulphuric acid.

_d._ Stearic acid remains undissolved upon treating sulphate of quinine with acidulated water.

_e._ To detect sugar and mannite, the sample is dissolved in acidulated water, and an excess of hydrate of baryta added: a precipitate, consisting of quinine and sulphate of baryta, is produced. Carbonic acid is then passed through the fluid, in order to precipitate the excess of baryta as insoluble carbonate, the fluid saturated with ammonia, to throw down the quinine which may have been re-dissolved by the carbonic acid, and the mixture filtered. If the salt be pure, no residue will be obtained upon evaporating the filtrate; a residue of sugar or mannite is formed, if these substances are present.

_f._ Sulphate of quinine invariably contains 2 or 3 per cent. of cinchonine, originating, not from a fraudulent admixture, but from an incomplete purification of the salt. One of the best methods for detecting the respective quantities of quinine and cinchonine, present in a sample of the sulphate, is the following: Several grammes of ammonia and ether (which has previously been washed with water) are added to one or two grammes of the salt under examination, the mixture thoroughly agitated, and then allowed to remain at rest. The supernatant etherial solution contains all of the quinine; the cinchonine, which is almost completely insoluble, both in water and ether, remaining suspended between the layers of the two fluids. The ether is next removed by means of a stop-cock funnel, evaporated to dryness, and the weight of the residue obtained determined. The operation is then repeated, the ether being replaced by chloroform in which both quinine and cinchonine are soluble. The residue, formed by the evaporation of the second solution, will be heavier than the first residue: the difference between the two weighings gives the weight of the cinchonine present.

_g._ The detection of the presence of sulphate of quinidine is based upon the difference in the solubilities of the oxalates of quinine and quinidine. Oxalate of quinidine is sufficiently soluble in cold water not to be precipitated by double decomposition when solutions of oxalate of ammonia and sulphate of quinidine are mixed. Under the same circumstances, quinine is almost completely thrown down. The test is applied as follows:

The suspected salt is dissolved in water, a slight excess of oxalate of ammonia added, and the precipitate formed separated by filtration. If the salt be pure, the filtrate is scarcely rendered turbid by the addition of ammonia; when, however, sulphate of quinidine is present, it will be entirely contained in the filtrate, in which ammonia will produce an abundant precipitate.

EXAMINATION OF BLOOD STAINS.

This branch of legal chemistry formerly gave but very unreliable results. It is scarcely ten years since the reactions that are now regarded as only secondary and confirmative in their character, and far from conclusive, were the only ones in use: these are the tests based upon the presence of iron and albumen in the blood. Since then, great progress has been made in the methods employed. It must not be understood, however, that the question under consideration always admits of an easy and decisive solution: the stains are sometimes too greatly altered to be identified; but in cases where the distinctive reactions of blood can be produced, the real nature of the stains under examination can, at present, be determined with certainty.

The tests more recently introduced consist in the production of small characteristic crystals, termed _haemin_ crystals, and in the use of the spectroscope. Crystals of haemin (first discovered by _Teichman_) are formed when dry blood is dissolved in concentrated acetic acid, and the solution evaporated to dryness: they are of a brownish-red color. _Brücke_ first suggested an analytical method, based upon this property of blood, which is equally characteristic and sensitive: It is only necessary to dissolve a minute portion of the matter to be examined (dried blood, or the residue left by the evaporation of the fluid obtained by treating the stain, or the dried blood, with cold water) in glacial acetic acid and evaporate the solution to dryness in order to obtain crystals of haemin, which can be readily recognized by means of a microscope having a magnifying power of 300 diameters. If the crystals originate from fresh blood, they appear as represented in Fig. 21; crystals from old blood are represented in Fig. 22.

The former possess a reddish-brown, the latter a lighter color.

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).

If upon adding some of the substance under examination to the above mixture a clear blue coloration ensues, and the insoluble matter thrown down possesses a deep blue color, the presence of coloring matter of the blood is indicated. The mixture also imparts a blue color to moistened spots from which the blood stains have been as completely extracted as possible. Unfortunately sulphate of iron gives the same reaction.[X]

[X] Fresh gluten, gum arabic, and caseine also cause the blue
coloration.--_Trans._

_b._ Upon heating the fluid obtained by treating the stains with cold water in a test-tube, its brown or reddish color disappears, and greyish-white flakes of coagulated albumen are thrown down. The precipitate acquires a brick-red color, when treated with an acid solution of nitrate of mercury containing nitrous acid. The albumen is also coagulated by the addition of nitric acid: it assumes a more or less yellow color, if heated with a slight excess of the acid. Chlorine-water, especially upon heating, likewise precipitates albumen in the form of white flakes.

_c._ If the fluid is acidulated with a few drops of acetic acid, and a drop of ferrocyanide of potassium added, a white precipitate, or, at least, turbidity is produced.

_d._ The flakes of albumen, separated by heating, dissolve in caustic alkalies to a solution, from which they are re-precipitated by nitric acid, or chlorine water.

_e._ Upon treating blood stains with chlorine-water, a solution which contains chloride of iron, and acquires a red coloration by the addition of sulphocyanide of potassium, is formed.

_f._ Should the stains have failed to be affected by cold water (which, as has already been remarked, is the case when they have been previously washed with hot water), they are treated with weak soda lye. Nitric acid, hydrochloric acid, and chlorine water will produce in the solution so obtained a white precipitate, which exhibits the general properties of albumen previously described. In case the stains are deposited upon linen, it is necessary to replace the soda by ammonia, in order to avoid dissolving the fabric.

_g._ Solutions of the alkalies, which dissolve the albumen, leave the coloring matters intact, and consequently do not decolorize the fabric. If the latter is afterwards subjected to the action of hydrochloric acid, the coloring matter is dissolved, forming a solution that leaves upon evaporation to dryness a residue containing iron, which gives a blue coloration with ferrocyanide of potassium, and a red coloration with sulphocyanide of potassium.

_h._ The coloring matter of blood dissolves in boiling alcohol, to which sulphuric acid has been added, to a brown dichroic fluid (appearing green by transmitted light, and red by reflected light). A mixture of rust and blood exhibits the same phenomenon.

_i._ If substances containing blood are heated in a dry tube, an odor resembling that of burnt horn is emitted. In case the stained fabric is a substance that would produce this odor, (such as wool, silk, or hair), the test naturally loses all value.

_j._ If the fluid obtained by treating the stains either with water or alkali is evaporated with a little carbonate of potassa, and the residue heated, at first at 100°, then to redness, in a glass tube to which a fresh quantity of carbonate of potassa has been added, cyanide of potassium is formed. When cold, the tube is cut above the part containing the fused mixture, the mass heated with iron-filings and water, the fluid filtered, and the filtrate then acidulated with hydrochloric acid: ferrocyanide of potassium will be present in the fluid, and upon adding a drop of solution of perchloride of iron a green, or blue, color will be produced, and a precipitate of Prussian blue gradually thrown down.

If the stained cloth is non-nitrogenous (_per ex._: hemp, linen, or cotton), instead of treating it with water, it may be heated until pulverulent, mixed with carbonate of potassa, the mixture calcined, and the operation then completed as just described. This test having given affirmative results, the operations should be repeated with an unstained portion of the cloth, to remove all doubt that the indications obtained do not really originate from the fabric.

In the present state of science, it is impossible to discriminate chemically between human and animal blood. _M. Barruel_, it is true, is able, not only to accomplish this, but also to distinguish the blood of the various species of animals by its odor! But this test has a somewhat hypothetical value for scientific purposes. In regard to the crystals of haemin, they do not present sufficient difference to allow the blood of different animals to be distinguished. We have not yet treated of the globules. It often occurs that these minute organs are so altered as to be no longer recognized in the microscopic examination; when, however, the stains are tolerably recent, they may be detected by examining the moistened stained cloth, directly under the microscope: a discrimination between animal and human blood is then possible: corpuscules of human blood possess the greater size: those of the sheep, for instance, have only one-half the diameter of the former. It is, however, but seldom that this distinction can be made use of.[Y]

[Y] _Menstrual blood_ is recognized by the presence of epithelial
cells.--_Trans._

EXAMINATION OF SPERMATIC STAINS.

In cases where attempt at violence, rape or pederasty is suspected, the expert may be required to determine the nature of stains found on clothing, sheets, etc. The fact that the stains were produced by semen, may often be regarded, _per se_, as criminating evidence. This class of investigation possesses, therefore, considerable importance.

_External appearance of the stains._--Dry spermatic stains are thin, and exhibit a greyish or, occasionally, a citron-yellow color, if present on white cloth. In case the fabric is colored, they appear whitish and, if on linen, present a glossy aspect. They are translucid, when observed by transmitted light. If the fabric, upon which the stains are deposited, is of a heavy texture, they are visible only on one side: under all circumstances, their circumference is irregular and undulated. These indications, however, are not conclusive, but vary according to whether the stains were produced by the thick semen of a vigorous man, or the aqueous seminal fluid of an aged and diseased person, or by semen more or less mixed with the prostatic fluid. Upon moistening spermatic stains, the distinctive stale odor of fresh semen is sometimes emitted, but this characteristic is usually obscured by the presence of foreign substances.

Semen stains are soluble in water, forming a gummy fluid, in which chlorine, alcohol, bichloride of mercury, acetate and subacetate of lead produce a white precipitate, but which fails to be coagulated by heating. Plumbate of potassa does not impart a fawn-color to these stains, at a temperature above 20°, as is the case with those produced by albuminous substances.

Persulphate of iron imparts to spermatic stains a pale yellow color,

Sulphate of copper, a bluish grey color,

Cupro-potassic tartrate, a bluish grey color,

Nitrate of silver, a pale grey color,

Nitric acid, a pale yellow color.

The above reactions, separate or united, are insufficient; they are not very delicate, and are likewise produced by stains originating from the other varieties of mucus: the indications furnished by a microscopic examination of the stains are alone conclusive.

_Microscopic examination._--Semen contains as its principal and fecundating constituent, peculiar vibratory filaments, (_spermatozoa_), held suspended in a viscous fluid. These filaments, when preserved in a warm and moist place, retain their activity for a considerable time: it is even possible that they may exhibit vitality in the organs, into which they have been voluntarily or forcibly ejaculated, for ten, or even twenty-four hours. When exposed to cold air, the spermatozoa quickly expire; still, they preserve their form for some time, and, as this is very characteristic, it is then easy to identify them; moreover, since they originate exclusively in the testicles, their detection may be considered as certain evidence of the presence of semen. In stains produced by aged persons, and by persons enfeebled by excesses, the spermatozoa fail to be presented; in case they are discovered, this fact evidently does not affect the certainty of the spermatic origin of the stains. The contrary conclusion is never absolutely certain: still, if the use of the microscope fails to establish the presence of spermatozoa, it is almost certain that the stains were not produced by semen.

Of the various methods for obtaining from the stains a preparation adapted to the microscopic examination, the one proposed by M. Charles Robin is the most simple and reliable.

A strip, 1 c. c. in size (comprising the entire stain, if this be small, containing its inner portion, if it be large), is cut from the fabric under examination, care being taken that the two extremities of the sample extend beyond the stained portion.

One end of the cloth is then immersed in a capsule, or watch-glass, containing pure water: the stains become moistened by capillary attraction, and, in a space of time varying from twenty minutes to two hours, acquire the appearance of fresh semen. As soon as the stained portion becomes swollen and softened, the surface of the cloth is gently scraped with a spatula, and the substance removed placed on the slide of the microscope. The particles are next slightly detached, a drop of water added, if necessary, and the whole covered with a small plate of very thin glass. The preparation is then examined by a microscope, having a magnifying power of from 500 to 600 diameters. In this way, the presence of either entire or broken spermatozoa is readily detected. Their existence is rendered still more apparent, if the mucus present is dissolved by adding a drop of acetic acid to the preparation.

Entire spermatozoa consist of long slender filaments, having a length of 0.04041 to 0.04512 millimetre; the anterior extremity presents an oval enlargement, either round or pyriform, exhibiting a double outline, when magnified to 500 diameters. This enlarged end is termed the "head;" the entire remaining portion being regarded as the "tail." In case the spermatozoa are broken, they are severed either near the head or in the middle of the tail, and a mass of detached fragments will be observed in the microscopic examination. The spermatozoa are not the only corpuscules revealed by the microscope; other substances, entirely different in character, are often observed. Although the detection of these bodies is, in itself, of no value, it will be well to enumerate and characterize them; they are:

_a._ Oily globules.

_b._ Leucocytes, or spherical and finely granulous globules of mucus.

_c._ Corpuscules, originating from the seminal vesicles, termed sympexions. These are rounded or ovoid, possess an irregular outline, and are usually mixed with the spermatozoa and globules of mucus.

_d._ Crystals of phosphate of magnesia, varying greatly in size; the largest are from 0.mm. 001 to 0.mm. 002 in length. The crystals formed upon cooling the semen, present the form of an oblique prism, with a rhomboidal base. Occasionally they are elongated and flattened; they then assume the form of a rhomboid.

_e._ Epithelial cells; originating from the mucous follicles of the urethra.

_f._ Irregular grains of dust; soluble in acetic and hydrochloric acids, with gaseous evolution.

_g._ Brownish-red grains of rust; only slightly soluble in acetic acid, but easily soluble in hydrochloric acid.

_h._ Filaments of the strained fabric; detected by their texture, and general appearance.

_i._ Grains of starch, in case the cloth has been stiffened. These are almost invariably swollen, and are frequently broken and deformed.

If the examination is to be secretly executed, and the cloth cannot well be cut, it is rolled in a cone, in such a way that the external side contains the stained portion. The lower extremity of the cone (which should be free from stains) is dipped in a watch-glass containing water, so as to avoid directly wetting the stains. The cone soon becomes moistened by absorption, and the operation is then completed in the same manner as when the fabric has been cut; which is always preferable, when possible.

The examination of spermatic stains consists, then, in moistening the stains with water, separating them as completely as possible from the stained cloth, and determining the presence of the spermatozoa by means of the microscope.

All other tests are valueless; even their execution for confirmatory purposes is not advisable; inasmuch as they fail to possess a distinctive character, and the reagents employed in their production may destroy the fabric, and thus prevent the formation of the only conclusive reaction--the detection of the spermatozoa.

In case the stains are deposited upon a woman's chemise, they are usually present on both the front and back portions, and are sometimes to be found on the sleeves. When a man's shirt is under examination, especial attention should be given to the anterior portions. The pantaloons are also often stained; usually in the interior, but sometimes also on the exterior, just above the thighs. In reporting the decision to the court, as to the nature of the stains, their precise position should invariably be stated, as, by this means, the circumstances attending the commission of the crime may be, at least partially, elucidated.

THE END.