employed, but the injection permeated the delicate basement membrane of the tubes and the adjacent textures were equally coloured, so that the nature of the arrangement was still undecided. Freshlyprepared Prussian blue was tried, and with much better success. Now, however, a new difficulty presented itself. The thinnest sections which could be obtained, necessarily consisted of several planes of the cell-containing network and capillaries, and, in consequence of the distension of the former by the injection, the latter could not be distinguished. It then occurred to me to try to inject both the portal vein and the duct; the former with plain size, and the latter with Prussian blue. In a liver prepared in this manner I was fortunate enough to see in several different sections, with a quarter of an inch, the blue transparent injection in one tube in close juxtaposition to a colourless capillary vessel injected with plain size. By varying the kind of injection, and resorting to many experiments which it would be useless to recount, I have gradually arrived at a plan of procedure by which specimens can almost always be obtained which will bear examination with a quarter, or even with an eighth of an inch object glass. Extravasation into the Lymphatics.-Often when too great force is employed, rupture of the walls of a small duct occurs, when the injection not unfrequently passes into a lymphatic vessel, and in this way, as was shown by Kiernan, the abundant plexus of lymphatics in the large portal canals can be injected. In one instance the injection passed into the thoracic duct. A similar result likewise occurred to me in a rabbit. This accidental injection of the lymphatics has been noticed by many. Mascagni long ago noticed that when injection was thrown into the ducts it returned colourless by the absorbents. Although the lymphatics are so easily injected, I have not been successful in my attempts to ascertain how these vessels commence in the liver, and have not yet seen them distinctly in portal canals less than the quarter of an inch in diameter. DIRECTIONS FOR INJECTING A LIVER FOR EXAMINATION WITH HIGH POWERS OF THE MICROSCOPE. The portal vein is to be injected with tepid water until the blood is washed out and the whole organ becomes nearly colour less. After a time water only slightly tinged with blood will escape from the hepatic vein, while at the same time almost pure water will pass out from the gall duct. The force used must be as gentle as possible, for otherwise some of the minute vessels will be ruptured, and the preparation will not succeed. After the vessels have been fully distended in this manner, the water is allowed to escape from them, and the liver wrapped up in two or three soft cloths, which will absorb much more of the water. The cloths are to be changed when soddened with water. In the course of from twelve to twenty-four hours, the liver will appear shrivelled, and of a soft clayey consistence. It is now ready to be injected from the duct. A mixture of about two-thirds water, and one-third spirit, in which recently precipitated Prussian blue is suspended, is strained through two or three layers of the finest muslin, and carefully injected into the duct, applying slight, but gradually increased, pressure. The injection should be performed with a small half-ounce or ounce syringe, or by pressure of the fluid placed in a glass tube about four feet high. As soon as the blue injection is seen at several points at the circumference of the lobules, and more fluid cannot easily be forced into the duct, it is better to wait a few hours, and then inject a little more; but the injection of the duct must not be pushed too far, for although in some places no colour whatever is to be seen, in other parts a good injection has, perhaps, been effected. After the duct has been injected, the liver is again set aside for a few hours in damp cloths, so that the superfluous fluid may be absorbed. Preparation of the Injection.-The Prussian blue is prepared in the usual way, by adding gradually a solution of Ferrocyanide of Potassium to a very dilute solution of Perchloride of Iron. The mixture should be of a dark colour, but should contain no flocculi. It should not deposit a precipitate even after it has been allowed to stand for some time. Without care an abundant deposit of Prussian blue is obtained, which does not flow well. The mixture should have the appearance of a dark-blue solution, rather than of a deposit held in suspension. The addition of a little spirit has the advantage of hardening the delicate walls of the smaller ducts as the injection passes into them, and thus preventing their rupture. The great advantage of the Prussian blue arises from the circumstance that the precipitate is so fine as to appear like a solution; but still the minute particles cannot be made to pass through the basement membrane of the ducts. The addition of glycerine to this fluid causes it to flow better. The following is the composition of the injecting fluid which I have been lately in the habit of using. It is well adapted for all ordinary purposes of injection. It is used cold, and it does not run out from the openings of the divided vessels when a thin section is made a disadvantage from which few injecting fluids which can be used cold are free. The Ferrocyanide of Potassium is to be dissolved in an ounce of the water, and the Tincture of Sesquichloride of Iron added to another ounce. These solutions are to be mixed very gradually, and well shaken in a bottle, the iron being added to the Ferrocyanide of Potassium. When thoroughly mixed these solutions should produce a dark-blue mixture, in which no precipitate or flocculi are observable. The naphtha is to be mixed with the spirit; and the glycerine, and the remaining two ounces of the water added. It is important to mix the solutions in the above order, for otherwise a dense precipitate and free flocculi which will not run well are formed. Lastly, the colourless fluid is to be mixed gradually with the Prussian blue, the whole being well agitated in a large bottle during the admixture. Injection of the Portal Vein and Hepatic Artery with plain Size. -After the duct has been injected in the manner above referred to, the liver is placed in warm water, and when it has become warm through the portal vein and hepatic artery are to be injected with fine size. If the injection of the duct has been well conducted, * The Tinct. Ferri Sesquichlor. of the London Pharmacopoeia, commonly known as Muriated Tincture of Iron, is recommended because it is a solution of a persalt of Iron, which can always be readily obtained of uniform strength, and rupture of its coats has not taken place, the fluid which escapes from the hepatic vein will not have the slightest blue tinge; but if this should be the case, we may still proceed, as it often happens that, although extravasation has occurred from the duct into the vessels, the escape has only taken place into one of the smaller branches; and as the vessels do not anastomose, it only affects the lobules supplied by the branch of vein in which the opening has been made. When size runs out at the hepatic vein, the open end of the latter may be tied, and a little more carefully injected into the portal vessels, in order to distend the capillary plexus. Lastly, a little size is injected into the duct, in order to distend the larger ducts, and to prevent the injection from returning after it has been forced into the smaller branches. When the liver appears filled, it may be placed in cold water until the size sets. By varying the plan above described somewhat, the different vessels may be injected with different colours. I have succeeded in injecting a human liver with four colours. The portal vein is injected with Flake White, the artery with Vermilion, the hepatic vein with Lake, and the duct with Prussian Blue. From this liver I have obtained specimens which show very clearly many of the points which I shall describe, and many of these preparations have been preserved. In this liver, as would be supposed, it is difficult to find a part in which the vessels and ducts are well injected, but such specimens have been obtained. PREPARATION OF SPECIMENS PREVIOUS TO EXAMINATION IN THE MICROSCOPE. When the ducts and portal vein have been successfully injected in the manner described, the greatest care is required in preparing the section for examination. A very sharp knife, or Valentin's knife, in good order, may be employed. The thinnest section, after very careful washing, must be placed in a drop of syrup, or glycerine, and the glass cover so applied as to press very slightly upon the specimen. If the washing be not carefully conducted, the superficial cells will be removed, and the vessels will collapse. On the other hand, if all the cells and debris adhering to the surface of the section are not washed off, the preparation will appear confused and indefinite. The sections should bear examination with a quarter or eighth of an inch object-glass. It is, of course, very difficult, and, in great measure, a matter of chance, to obtain a section including the course of the smallest duct, and it will often be necessary to examine very many sections before a demonstrative specimen can be obtained. When we consider how difficult it is to obtain a section of the mammalian kidney, showing, even for a short distance, the convolutions of a single uriniferous tube, it need not excite surprise that it is rare to obtain a section of the liver, which shows clearly the termination of the smallest branches of the duct, for these tubes in passing only a very short distance, often occupy many very different planes. The circumstances which render the demonstration of the course of the uriniferous tube very difficult, operate still more forcibly in the case of the liver, as the tubes are very much smaller, the tissue of which they are composed infinitely more delicate, and the entire organ so soft that very slight manipulation is sufficient entirely to destroy the relative positions of the anatomical elements of which it is composed. |