are added, and the mixture is kept in a loosely stoppered bottle till the smell of ammonia has become faint.

The fibrin is stained by taking it perfectly fresh and clear. It is chopped fine and placed in the carmine solution for twenty-four hours. The fluid is strained off and the fibrin washed in water till the washings are colourless. It is kept in a stoppered bottle with just enough ether to cover it. (See 'Chemical Physiology,' pp. 645, 654–663.)

ADDITIONAL EXERCISES

Among the important reactions of proteids is Piotrowski's reaction—that is, the coloration produced by copper sulphate and a caustic alkali; the term 'biuret reaction' is applied to the rose-red colour which proteoses and peptones give with these reagents, because biuret (a derivative of urea) gives a similar colour. It does not, however, prove that biuret is contained in the proteid molecule. Biuret and proteid both contain some radicle to which the colour is due. Gnezda1 thought it was cyanogen, and that the cyanogen was differently combined in the peptones and native proteids (albumins and globulins) respectively; hence the rose-red given by one group and the violet by the other. More recent work by Pickering, however, points to a CONH group rather than cyanogen. Gnezda found that if a dilute solution of nickel sulphate is used instead of copper sulphate, the native proteids give different colours from the peptones and proteoses, and Pickering has found the same with cobalt. Their results may be given in the following

table :

2

Another delicate test recently introduced by McWilliam may here be mentioned: Salicyl-sulphonic acid precipitates albumins and globulins; on heating the precipitate is coagulated. The same reagent precipitates proteoses. On heating the precipitate dissolves and reappears on cooling. It does not precipitate peptones.

Tests for Free Hydrochloric Acid

(a) Gunsberg's reagent consists of 2 parts of phloroglucinol, 1 part of vanillin, and 30 parts of rectified spirit. A drop of filtered gastric juice

1 Proc. Roy. Society, vol. xlvii. p. 202.

2 Journal of Physiology, vol. xiv. Most of the other colour reactions of proteids depend on the aromatic radicle they contain (see Chemical Physiology and Pathology, p. 123).

is evaporated with an equal quantity of the reagent. Red crystals form, or, if much peptone is present, there will be a red paste. The reaction takes place with 1 part of hydrochloric acid in 10,000. The organic acids do not give the reaction.

(b) Tropæolin test. Drops of a saturated solution of tropaolin 00 in 94 per cent. methylated spirit are allowed to dry on porcelain slab at 40° C.

A drop of the fluid to be tested is placed on the tropaolin drop, still at 40° C.; and if hydrochloric acid is present, a violet spot is left when the fluid has evaporated. A drop of 0 006 per cent. hydrochloric acid leaves a distinct mark.

LESSON XIX

SECRETION OF SALIVA. ZYMOGEN GRANULES.
BLOOD CRYSTALS

1. Demonstration. In a dog which has been anesthetised the submaxillary gland with its vessels, nerves, and duct has been exposed. A cannula is inserted into the duct. Stimulation of the chorda tympani leads to (a) vaso-dilatation; (b) secretion of saliva. Stimulation of the cervical sympathetic leads to (a) vaso-constriction; (b) a scanty secretion of very viscid saliva. After the administration of atropine, the effects of stimulating the nerves on the vessels is still seen, but there is no secretion of saliva.

2. Examine microscopically, mounting in aqueous humour or serum (or in glycerine after treatment with osmic acid vapour) small pieces of the pancreas, parotid and submaxillary glands in a normal guinea-pig, and also in one in which profuse secretion has been produced by the administration of pilocarpine.

Note that zymogen granules are abundant in the former, and scarce in the latter, being situated chiefly at the free border of the cells.

3. The guinea-pigs in the above experiments should have been killed by bleeding; collect the blood and defibrinate it by whipping. It may then be utilised as follows:

(a) To a drop add a drop of water and cover; in a short time crystals of oxyhæmoglobin are formed.

(b) Mount another drop in Canada balsam. Crystals of oxyhemoglobin soon form. These preparations keep for a long time.

(c) Shake up some of the blood with a sixteenth of its volume of ether; let it stand for 20-30 minutes, and mount a drop of the laky fluid. Crystals again form.

These three methods are those most frequently used for obtaining oxyhæmoglobin crystals; but there is a considerable difference in the readiness with which crystallisation occurs in the blood of different animals. The guinea-pig is one in which crystallisation occurs readily. The crystals are, however, of an exceptional form. In most animals they are rhombic prisms. In the guinea-pig they are rhombic tetrahedra. In the squirrel they are hexagonal (see fig. 30, p. 73).

(d) Instead of ether in experiment c, use amyl nitrite; on shaking vigorously the liquid changes to a mahogany colour, and crystals also form. These consist of methæmoglobin, and resemble in form those of oxyhæmoglobin.

(See Chemical Physiology and Pathology,' on Saliva, pp. 616–629; on Zymogens, pp. 634-636, 655; on Blood Crystals, pp. 268–274.)

1 Directions for this dissection will be found in the Handbook for the Physiological Laboratory, p. 470.

LESSON XX

HEMOGLOBIN AND ITS DERIVATIVES

Defibrinated ox-blood suitably diluted may be used in the following experiments as in those described in Lesson IX.

1. Place some in a hæmatoscope (see fig. 33, p. 77) in front of the large spectroscope. Note the position of the two characteristic bands of oxyhemoglobin, which is replaced by the single band of hæmoglobin after reduction by the addition of Stokes' reagent or ammonium sulphide. By means of a small rectangular prism a comparison spectrum showing the bright sodium line (in the position of the dark line named D in the solar spectrum) may be obtained, and should be focussed with the absorption spectrum.

2. Obtain similar comparison spectra by the use of the microspectroscope.. For this purpose a cell containing a small quantity of oxyhemoglobin solution may be placed on the microscope stage, and a test-tube containing carbonic oxide hæmoglobin in front of the slit in the side of the instrument. Notice that the two bands of carbonic oxide hæmoglobin are very like those of oxyhæmoglobin, but are a little nearer to the violet end of the spectrum.

Carbonic oxide hæmoglobin may be readily prepared by passing a stream of coal gas through the diluted blood. It has a cherry-red colour, and is not reduced by the addition of ammonium sulphide.

3. Methæmoglobin. Add a few drops of ferricyanide of potassium to dilute blood and warm gently. The colour changes. Place the test-tube in front of the small direct vision spectroscope. Note the characteristic band

in the red. On dilution other bands appear. Treat with ammonium sulphide, and the band of hæmoglobin appears.

4. Acid Hæmatin.-Add a few drops of glacial acetic acid to dilute blood, and examine with the spectroscope. Compare the position of the absorption band in the red with that of methæmoglobin; that of acid hæmatin is further from the D line.

Take some undiluted blood and add glacial acetic acid as before. Extract this with ether by gently agitating it with that fluid. The ethereal extract should then be poured off and examined spectroscopically. The band in the red is seen, and on further diluting with ether three additional bands make their appearance.

5. Alkaline Hæmatin.-Add to diluted blood a small quantity of strong

FIG. 59.-1, Solar spectrum. 2, Spectrum of oxyhæmoglobin (0.37 p.c. solution). First band, λ 589564; second band, A 555-517. 3, Spectrum of hæmoglobin. Band, A 597-535. 4, Spectrum of COhæmoglobin. First band, à 583-564; second band, A 547-521. 5, Spectrum of methæmoglobin (concentrated solution). 6, Spectrum of methæmoglobin (dilute solution). First band, X 647622; second band, A 587-571; third band, à 552-532; fourth band, à 514-490. 7, Spectrum of acid hæmatin (ethereal solution). First band, A 656-615; second band, λ 597-577; third band, A 557-529; fourth band, A 517-488. 8, Spectrum of alkaline hæmatin. Band from A 630-581. 9, Spectrum of hæmochromogen (reduced hæmatin). First band, A 569-542; second band, A 535-504. 10, Spectrum of acid hæmatoporphyrin. First band, à 607-593; second band, A 585536. 11, Spectrum of alkaline hæmatoporphyrin. First band, à 633-612; second band, à 589-564; third band, A 549-529; fourth band, à 518-488. The above measurements (after MacMunn) are in millionths of a millimetre. The liquid was examined in a layer 1 centimetre thick. The edges of ill-defined bands vary a good deal with the concentration of the solutions,