caustic potash and warm.

The colour changes, and with the spectroscope

a faint shading on the left side of the D line is seen.

6. Hæmochromogen.-Add a few drops of ammonium sulphide to a solution of alkaline hæmatin; observe change of colour, and two bands are seen, one between D and E, and the other nearly coinciding with E. The spectrum of

alkaline hæmatin reappears for a short time after vigorous shaking with air.

7. Hæmatoporphyrin.-To some strong sulphuric acid in a test-tube add a few drops of undiluted blood, and observe the spectrum of acid hæmatoporphyrin (iron-free hæmatin).

8. Map out all the spectra you see on a chart.

The various absorption spectra are depicted in fig. 59.

(See Chemical Physiology and Pathology,' p. 274 et ɛe7.)

K

LESSON XXI

SERUM

1. The following methods of precipitating serum-globulin (paraglobulin) should be performed :

(a) Panum's Method.-Dilute serum with fifteen times its bulk of water. It becomes cloudy owing to partial precipitation of the serum-globulin. Add a few drops of 2-per-cent. acetic acid; the precipitate becomes more abundant, and it dissolves in excess of the acid. It was formerly called 'serumcasein.'

(b) Alexander Schmidt's Method.-Dilute serum with twenty times its bulk of water and pass a stream of carbonic acid through it. A fairly abundant precipitate of serum-globulin falls. Let it settle, and an additional precipitate can be obtained from the decanted liquid by treating it with a trace of acetic acid (the serum-casein' mentioned above). Repeat the carbonic acid method without dilution; no precipitate forms.

6

(c) By Dialysis. Put some serum in a dialyser with distilled water in the outer vessel. The water is frequently changed. In order to prevent decomposition a few crystals of thymol are added. In a few days the salts have passed out; the proteids remain behind: of these the serum-albumin is still in solution; the serum-globulin is precipitated, as it requires a small quantity of salt to hold it in solution.

(d) By Addition of Salts :

(i.) Schmidt's method. Saturate some serum with sodium chloride. A precipitate of serum-globulin is produced.

(ii.) Hammarsten's method. Use magnesium sulphate instead of sodium chloride. A more abundant precipitate is produced, because this salt is a more perfect precipitant of serum-globulin than sodium chloride. In order to obtain complete saturation with these salts it is necessary to shake the mixture of salt and serum for some hours.1

(iii.) Kauder's method. Half saturate serum with ammonium sulphate. This is done by adding to the serum an equal volume of saturated solution of ammonium sulphate. This precipitates the globulin. Complete saturation with the salt precipitates the albumin also.

2. Heat Coagulation.—Saturate serum with magnesium sulphate and filter off the precipitate; preserve the filtrate and label it 'B.' Wash the precipitate on the filter with saturated solution of magnesium sulphate until the washings do not give the tests for albumin,2 then dissolve the precipi

'This may be conveniently done by a shaking machine before the class meets. 2 On account of the prolonged nature of these operations they must necessarily be performed by the demonstrator beforehand.

tate by adding distilled water. It readily dissolves owing to the salt adherent to it. The solution is opalescent. Label it 'A.'

Render A faintly acid with a drop of 2-per-cent. acetic acid, and heat in a test-tube. The temperature of the test-tube may be raised by placing it in a flask of water gradually heated over a flame. A thermometer is placed in the test-tube, and should be kept moving so as to ensure that all parts of the liquid are at the same temperature. The quantity of liquid in the test-tube should be just sufficient to cover the bulb of the thermometer. A flocculent precipitate of coagulated serum-globulin separates out at about 75°.

Now take the filtrate B. This contains the serum-albumin. Dilute it with an equal volume of water; render it faintly acid as before, testing the reaction with litmus paper. Heat. A flocculent precipitate («) falls at about 73° C.; filter this off; note that the filtrate is less acid than that from which the precipitate has separated, or it may even be alkaline. If so, make it acid again, and heat; a precipitate falls at 77-79° C. (B). A third precipi

tate is similarly obtained at 84-86° C. (y). In the serum of the ox, sheep, and horse the ẞ and y precipitates only occur; in cold-blooded animals only the a variety.

3. Take a fresh portion of B, and saturate it with sodium sulphate. The serum-albumin is precipitated (completely after prolonged shaking). This is due to the formation of sodio-magnesium sulphate. B was already saturated with magnesium sulphate (MgSO4 + 7H2O); on adding sodium sulphate this double salt (MgSO4.Na2SO4 + 6H2O) is formed. Shake some serum with sodium sulphate alone. A small precipitate of globulin is produced. Saturate another portion of the serum with sodio-magnesium sulphate; both globulin and albumin are precipitated.

Of the methods used for precipitating serum-globulin practically only two are used now. These are Hammarsten's and Kauder's. The other methods only precipitate the globulin incompletely. Kauder's method is rapid and efficacious: if the globulin is filtered off, the albumin may be precipitated in the filtrate by complete saturation with the same salt, ammonium sulphate. This method avoids the trouble of using two salts as described under 3. This last method is instructive, but not nearly so quick as Kauder's.

With regard to the separation of serum-albumin into a, B, and y varieties by the use of the method of fractional heat coagulation, it must be mentioned that at present no further difference has been shown to exist between them, and the opinion has been very freely expressed that the results obtained are not trustworthy. I am convinced that the method is a good one, especially as in other cases (see MUSCLE) the proteids so separated can be shown to possess other differences. In the case of serum, however-and the same is true for egg-albumin-the matter must still be considered sub judice. (See 'Chemical Physiology and Pathology,' p. 230 et seq.)

LESSON XXII

COAGULATION OF BLOOD

1. Sodium sulphate plasma has been prepared by receiving blood into an equal volume of saturated solution of sodium sulphate. After the corpuscles have been separated by the centrifugal machine (one form of this is represented in the next figure), the supernatant plasma is pipetted off.

Dilute some of this with ten times its bulk of water and divide it into five parts-A, B, C, D, and E.

FIG. 60.-Centrifugal machine as made by Runne, of Basel. Glass vessels containing the substances to be centrifugalised are placed within the six metallic tubes which hang vertically while the disc is at rest; when the machinery is set going they fly out into the horizontal position. A water motor or gas engine may be used to work these instruments. A small but effective hand-centrifuge is made by Watson, Laidlaw & Co., Glasgow.

Leave A at the temperature of the air. It clots slowly or not at all.

Put B into the warm bath at 40° C. It clots more quickly. This shows the influence of temperature in accelerating coagulation. It coagulates because the dilution with water has removed the inhibitory influence of the

strong saline solution on the fibrin ferment which is present as the result of corpuscular disintegration.

To C add a few drops of serum.

To D add a piece of buffy coat.

To E add solution of fibrin ferment dissolved in a weak solution of calcium chloride. Put C, D, and E into the warm bath at 40° C. On account of the presence of abundance of fibrin ferment they clot more quickly than B. The presence of calcium is also essential (see OXALATE PLASMA, Lesson IX.).

3. Heat a portion of the undiluted plasma to 60° C. The fibrinogen is precipitated (coagulated by heat) at 56° C. Filter. Treat the filtrate as in E, diluting it first. No coagulation occurs.

4. Hydrocele Fluid. This does not clot spontaneously, or only very slowly. Divide it into four parts—A, B, C, and D.

To A add an equal volume of serum.

To B add a few drops of fibrin-ferment solution.

To C add a piece of buffy coat.

Put them into the warm bath, and coagulation takes place in each. The serum or the buffy coat supplies the missing fibrin ferment. The serum does not produce its effect in virtue of the serum-globulin it contains; hydrocele fluid contains both fibrinogen and serum-globulin, as the following experiment shows:

Take the portion D and half saturate it with sodium chloride by adding to it an equal bulk of saturated solution of sodium chloride. Fibrinogen is precipitated. The precipitate is a small one, and on standing aggregates together, and so becomes more apparent. Filter, and saturate the filtrate with sodium chloride, or, better, magnesium sulphate; serum-globulin is precipitated.

5. Demonstration.—A solution of nucleo-albumin from the thymus has been prepared beforehand by the demonstrator. It may be prepared in one of two ways.

(a) Wooldridge's Method. The gland is cut up small and extracted with water for 24 hours. Weak acetic acid (0.5 c.c. of the acetic acid of the 'Pharmacopoeia' diluted with twice its volume of water for every 100 c.c. of extract) is then added to the decanted liquid. After some hours the precipitated nucleo-albumin (called tissue-fibrinogen by Wooldridge) falls to the bottom of the vessel. This is collected and dissolved in 1-per-cent. sodium carbonate solution.

(b) The Sodium Chloride Method. The finely-divided gland is ground up in a mortar with about an equal volume of sodium chloride in a mortar. The resulting viscous mass is poured into excess of distilled water. The nucleo-albumin rises to the surface of the water, where it may be collected and dissolved as before.

A rabbit is anesthetised, and a cannula inserted into the external jugular vein. The solution is injected into the circulation through this. The animal soon dies from cessation of respiration; the eyeballs