error arises from the fact that, in washing out the blood vessels, much solid matter besides that belonging to the blood is taken from the tissues, and thus an excess is found.

Valentine's Method.-A small measured quantity of blood is drawn from a vein and its percentage of solids accurately estimated; a known quantity of water is then injected into the vessels. When some time has been allowed for proper distribution of the water, a sample of the diluted blood is taken and its solids estimated. The difference in solid contents of the two samples shows the degree of dilution caused by a known quantity of water introduced into blood of ascertained strength, and thus the amount of the diluted fluid (the blood) may be calculated and added to the amount of the first sample to make the absolute quantity.

This method cannot give accurate results, because in the time necessary for the distribution and mixture of the water with the circulating blood much of the former is excreted by the kidneys and skin, and the second sample of blood is more concentrated than should result from such dilution.

Welcker's Method depends upon the estimation of the coloring matter of the blood. He connected the carotid with a small T piece, and allowed the animal to bleed into a bottle in which the blood could be defibrinated by shaking with pieces of glass. One cubic centimetre of this defibrinated blood was carefully measured off and saturated with carbon monoxide (CO), which gives a permanent and equally bright red color. It was diluted to 500 cc. with distilled water and kept as a standard color solution. The blood vessels of the animal were then washed out with .6 per cent. solution of sodium chloride until the solution flowing from the jugular vein was colorless. The tissues of the animals were chopped up, steeped in water and pressed. The washings of the vessels and the infusion from the tissues were added together and diluted until they had the same color intensity as a layer of the standard solution of the same thickness. Every 500 cc. of these diluted washings corresponds to 1 cc. of blood.

By this method the following estimates have been made of the relation of the blood to the body weight:-.

Only approximate estimates of the distribution of blood in the body during life can be made, since there can be no accurate method of investigation, and the amount varies considerably, according as the organ or part is in a state of rest or activity. It is supposed that a quarter of the entire amount is habitually flowing through each of the following regions:

1. The heart, great vessels and lungs.

2. The skeletal muscles.

3. The liver.

4. Skin and other tissues.

PHYSICAL CONSTRUCTION OF THE BLOOD.

As already stated, the blood is not really a red fluid. It is seen with the microscope to be made up of a clear fluid called

Human Blood after death of the elements. The red corpuscles are seen in different positions showing their shape, some also are seen in rolls. Only one white cell (w) is seen, misshapen and entangled in fibrin threads.

plasma or liquor sanguinis, which contains an immense number of little disc-shaped bodies-red corpuscles-and a few colorless protoplasmic cells-white corpuscles-so that the living blood may be physically tabulated, giving approximately an estimation of the relative amounts, thus::

PLASMA.

The fluid part of the blood, plasma or liquor sanguinis, is of a pale straw color, when pure and free from the coloring matter of the corpuscles, and of slightly less density (p. 215).

Unless special precautions have been taken, the plasma is altered when removed from the blood vessels and coagulation of the blood takes place, so that plasma does not come under observation, except when suitable methods are employed to separate it from the corpuscles. It was first separated from the corpuscles

Reticulum of Fibrin Threads after staining has made them visible. The network (b) appears to start from granular centres (a). (Ranvier.)

by the filtration of frog's blood to which strong syrup had been added to delay coagulation and destroy the flexibility of the corpuscles, so that they were rapidly caught in the meshes of the filter and the clear plasma passed through.

To obtain mammalian plasma free from corpuscles it is necessary to use some other method, as the small elastic corpuscles easily run through the meshes of the thickest filter paper.

The blood of the horse is chosen because it coagulates more slowly than that of most mammals, and delay in the coagula

tion or postponement of the change in the plasma is the chief object to be obtained. To encourage this delay the blood is drawn from a vein into a cylinder surrounded with a freezing mixture. The cold, however, must not be so intense as to absolutely freeze the blood, for the wished for subsidence of corpuscles could not go on if the blood become solid. It is then left quite motionless for twenty-four hours, after which time it will be found that the heavy corpuscles have fallen and left a clear supernatant fluid, which is plasma containing some white cells. This can be removed with a cool pipette and passed through an ice-cold filter to remove the cells, then tolerably pure plasma is obtained which soon coagulates at the ordinary temperature.

Another method of checking coagulation consists in letting the blood flow into a 25 per cent. solution of magnesium sulphate (about three volumes of blood to one of the solution). This, if left in a cool place, will not coagulate, and the corpuscles will separate by subsidence from the plasma and salt solution, which form an upper layer of clear fluid. If the salt be removed by dialysis or weakened by dilution with water, coagulation com

mences.

The coagulation of plasma can be seen with the microscope to depend upon the appearance of a close feltwork of exquisitely delicate, finely granular, elastic fibrils, which pervade the entire fluid and cause it to set into a soft jelly. The substance forming the meshes is called fibrin.

Some time after the plasma has gelatinized, the threads of fibrin break away from their attachment to the vessel in which the coagulum is contained, and owing to their elasticity the general mass of fibrin contracts, squeezing out of its meshes clear drops of fluid termed serum.

The fibrin clot gradually shrinks to small size and floats in the abundant fluid serum.

The separation of the serum is accelerated by agitation of the soft clot; and if brisk agitation, such as whipping, be kept up for a few minutes in recently drawn blood, the plasma does not form a jelly, but the fibrin firmly adheres to the stirring rods and at once contracts around them.

CHEMICAL COMPOSITION OF PLASMA.

On account of the rapid spontaneous formation of fibrin and serum when the plasma is removed from the body and allowed to die, the exact chemical condition of the liquor sanguinis during life cannot be investigated, the separation occurring before the simplest chemical method can be carried out.

We have no reason to suppose that fibrin exists normally in the blood, but it would appear that this substance is only formed at the moment of coagulation, its appearance being one of the most obvious of many changes which take place at the time of the death of blood plasma.

The chemical changes comprehended under the term coagulation occurring when plasma is deprived of its means of vitality, and ending in the production of fibrin and serum, are naturally of the first importance in studying the chemical relationships of living plasma. They can best be followed out in the coagulation of plasma when separated from the corpuscles, for (although the stages in the coagulation of blood are the same, the appearance of an insoluble albumin-fibrin-being the one essential in either case), the corpuscles complicate the process and modify the appearance of the clot.

Not only is the fibrin not present as such in the living plasma, but it requires for its production the presence of other substances which either do not exist in the living plasma, or are there so chemically associated as not to bring about the change which occurs when the plasma dies.

The reasons for believing this are the following: Fluids which sometimes collect by a slow process in the serous cavities of the body, e. g., hydrocele fluid, pleural effusion, etc., if kept quite clean do not generally undergo spontaneous coagulation. If to one of these some serum or recently washed blood clot be added, coagulation takes place just as in plasma (Buchanan). That is to say, we have here two fluids, neither of which coagulates when left to itself, but which do if mixed together. From each of these fluids a substance can be precipitated by passing a stream of carbon dioxide (CO2) through the fluids. Both precipitates readily redissolve in weak saline solutions.