2 I. GLOBULIN. ORDINARY ALBUMIN AND ALKALI ALBUMIN. Take some blood serum and dilute to ten times its volume with distilled water. Pass a current of CO, through it, globulin (§ 12) will be deposited. Filter. Heat filtrate to 70° C., ordinary soluble albumin (§ 11) will be coagulated and precipitated. Filter; add to filtrate a drop of acetic acid or a pinch of magnesium sulphate in bulk, a precipitate of alkali albumin, casein (§ 16). 2. FIBRIN. Beat up some freshly drawn blood, or better still, blood flowing from an animal, with a bundle of fine twigs; when all the fibrin is withdrawn, separate the fibrin from the twigs, place it in a muslin bag, tie the bag securely to the water tap, and allow the water to run through for some hours, from time to time removing the bag and rinsing the fibrin; continue this till the fibrin becomes free from colouring matter. (Test according to § 14). * ESTIMATION OF FIBRIN. Carefully clean and dry a bottle capable of holding 8 ounces, provided with a stopper, and introduce into it several strips of lead; then weigh the whole, and after recording the weight, place in it 5 ounces of fresh uncoagulated blood, and agitate briskly for twenty minutes; at the end of that time the fibrin will have separated and attached itself to the fragments of lead. The bottle is again weighed, to ascertain the exact quantity of blood employed, and the blood removed; the bottle, with the fragments of lead and the adhering fibrin, are carefully washed in cold water till perfectly free from colouring matter, and then dried over a water-bath, and when thoroughly dry again weighed, the increase in weight of the bottle and lead corresponds to the amount of fibrin removed from the blood. 3. HÆMOGLOBIN. This name was given by Hoppe Seyler in 1864 to the red colouring matter of the blood contained in the corpuscles, and which separates from them in crystalline forms at a temperature of o° C. * Preparation.-Freshly drawn blood (that from the rat, guinea-pig, or dog yields the best defined crystals) is received into a saucer surrounded with ice, de-fibrinated, and a 10 per cent. solution of sodium chloride added. This mixture is allowed to stand in a beaker surrounded with ice some time till the corpuscles are deposited. The supernatant liquid is then decanted off and the mass washed on a filter surrounded with ice repeatedly with ice-cold sodium chloride solution. When the mass is free from serum, it is to be agitated with a mixture of 1 vol. of water and 4 vols. of ether; the water dissolves the hæmoglobin, the ether, the cholesterin, and phosphorised fats. The red aqueous solution is then filtered, received into a beaker or watch-glass surrounded by ice, and alcohol added till a precipitate begins to appear. The mixture is then set aside for some hours. If the blood used in this process be obtained from the dog, rat, squirrel, or guineapig, the crystals will be abundant and well defined, but in the case of the blood of man, ox, sheep, or horse the hæmoglobin is generally deposited in an amorphous state, the crystalline form being rare. Chemical and physical properties. The crystals of hæmoglobin are formed upon the rhombic system, the forms varying in different animals; thus, in man, though obtained with difficulty, the crystals consist of four-sided prisms with dihedral summits; in the guinea-pig the crystals are tetrahedral; in the rat tetrahedral and octohedral; in the dog and cat the crystals are needle-shaped terminated by one plane surface; in the squirrel the crystals are hexagonal. The crystals are soluble in water and in alkaline solutions, but insoluble in alcohol, chloroform, ether, fatty oils, benzole, turpentine, and carbon bisulphide. Hæmoglobin, though a crystalloid, does not diffuse through parchment paper, thus forming an exception to Graham's theory. * ESTIMATION OF THE RED CORPUSCLES.-Stir a definite quantity of freshly drawn blood with the plume of a feather to remove the fibrin; add a 10 per cent. solution of sodium chloride, and set aside the mixture in a cool place till the corpuscles are deposited. The mass is then to be collected on a filter, and washed with the solution of sodium chloride till it is perfectly freed from serum; the mass is then placed in a weighed capsule and dried, the weight after drying representing the amount of corpuscles present in a certain quantity of blood. Corpuscles are formed of a delicate membrane or stroma, which contains the colouring matter, hæmoglobulin, cholesterin, phosphorised fat, paraglobulin and inorganic salts, chiefly potassium Decomposition of hæmoglobin.—Solutions of hæmoglobin readily decompose at temperatures above o° C., and on the addition of acids, and caustic alkalis, it breaks up into hæmatin and globulin yielding about 4 per cent. of the former to 96 of the latter. With glacial acetic acid and any metallic chloride, it is decomposed into hæmin and globulin. Hæmatin (Hoppe Seyler) is best prepared by mixing fresh defibrinated blood with a strong solution of potassium carbonate, till the liquid adhering to the separated coagulum becomes colourless. The coagulum is then dried at 50° C. and digested for some days in absolute alcohol; the alcoholic solution after concentration will deposit rhombic crystals. Hæmatin crystals are of a bluish black colour with a metallic lustre, becoming brown on trituration. They are insoluble in water, alcohol, ether, and chloroform; but soluble in acids and alkalis. Hamin, or hæmatin hydrochloride; if a small quantity of blood is rubbed up with sodium chloride and boiled for a few minutes with glacial acetic acid, and the mixture evaporated to dryness and placed in a glass slide, and then a drop of acetic acid added, on warming carefully for some little time over the flame of a spirit-lamp, in the residue mixed with colourless crystals of sodium chloride, and sodium acetate, will be found rhombic tablets of hæmin; which are of a bluish red colour when viewed by reflected, and brownish red by transmitted light. The crystals are insoluble in hot and cold water, in alcohol and ether. Soluble in alkaline solutions. All acids with the exception of hydrochloric and acetic acid decompose them. chloride and sodium phosphate. The stroma is the colourless portion of the living blood corpuscle, it is insoluble in water, and in sodium chloride solutions, but freely soluble in ether, chloroform, caustic soda, ammonia, and in solutions of the bile acids and urea. The stroma appears to combine with the hæmoglobin and, so to speak, fixes it, but the union is very feeble, and very slight disturbing influences set free the colouring matter. The hæmoglobin in the living blood is combined with an alkali, probably potash, to keep it in solution, as otherwise it is very insoluble and would crystallize out. Optical Properties of Hæmoglobin and its Compounds.It has already been stated that hæmoglobin has the power of forming a loose combination with oxygen, and in this state presenting two absorption bands in the spectrum characteristic of arterial blood. This and other changes produced by different reagents will now be more fully described. If we take a concentrated solution of oxyhæmoglobin or undiluted blood, and place it in a tube in the slit of the spectroscope, we find the whole spectrum is obscured with the exception of the extreme red rays; on gradual dilution of the solution the spectrum clears up, allowing light to pass in the green beyond E to F, and in the blue towards G in the violet end of the spectrum; at the same time in the yellow and beginning of green between D and E, the spectrum is still obscure; as the dilution is continued the dark space disappears, leaving two absorption bands a and B with a clear space intervening; the band a, which is near D, is smaller and darker than b, which is near E (see Fig. 9, No. 1). Now if we deprive the blood or oxy-hæmoglobin of its oxygen by the action of reducing agents, as ammonium sulphide, or ammoniacal ferrous sulphate, to which enough tartaric acid has been added to prevent precipitation, being careful at the same time to exclude the air, we find the two absorption bands fade away and a single broad shadow y take the place of the previously clear space; this band represents the spectrum of reduced hæmoglobin (see Fig. 9, No. 3). In the spectrum of venous blood this single band is always present, but as venous blood is never quite free from oxygen there is always some trace of the spectrum of oxy-hæmoglobin as well. Carbon Monoxide Compound.-On passing a stream of carbonic oxide through a solution of oxy-hæmoglobin, the oxygen is displaced; at the same time, the solution acquires a dark bluish red tint, but the spectrum is only a little altered; the line a being shifted slightly towards E (see Fig. 9, No. 2). The absorption bands, however, do not disappear on the addition of reducing agents, and it is by this that the presence of carbon monoxide may be detected in the blood of animals poisoned by it. Spectrum of Hæmatin.-If a little acetic acid be added to a solution of hæmoglobin the two absorption bands a and b vanish, and another absorption band appears G which covers C and extends slightly towards D (see Fig. 9, No. 4); this is the spectrum of hæmatin in an acid solution. If an alkali, such as ammonia, be added, a broad stria appears which touches C and extends nearly to D; this is the spectrum of hæmatin in an alkaline solution (see Fig. 9, No. 5). If a solution of ferrous Absorption Spectra of the Blood and of its Colouring Matter. 10 "I FIG. 9. 14 Solar spectrum with the lines of Fraun hofer. sulphate with tartaric acid and ammonia be added, the 4. FATTY MATTERS.-Evaporate to dryness over a |