1. Injection is the filling of the arteries, veins, or other vessels of animals with some coloured substance, in order that their natural arrangement may be made visible. This is, of course, a delicate operation, and needs special apparatus, which I will now attempt to describe.

2. Syringe.—This is usually made to contain about two ounces. On each side of the part next to the handle is a ring, so that the finger may be thrust through it, and the thumb may work the piston as in an ordinary syringe. The plug of the piston must be “packed” with wash-leather, and fit so closely as to be perfectly air-tight; and if, when it has been used awhile, it is found that some of the liquid escapes past the plug into the back part of the body, it must be repacked, which operation will be best understood by examining the part. These syringes are made of various sizes, but in ordinary operations the above will be all that is needed. The nozzle is about an inch long, and polished so accurately that there is no escape when the pipes are tightly placed upon it dry.

3. The pipes are usually about an inch long, to the end of which are affixed thicker tubes so as to fit the nozzle, as before mentioned, whilst a short arm projects from each side of these, so that the silk or thread which is used to tie this artery, &c., upon the thin pipe may be carried round these arms, and all danger of slipping off prevented. The pipes are made of different sizes, from that which will admit only a very fine needle (and this will need now and then to be cleaned, or to be freed from any chance obstruction), to that which will take a large pin. These sizes must always be at hand, as the vessels of some subjects are exceedingly minute.

4. Stopcock.—This is a short pipe like a small straight tap, which fits accurately upon the end of the syringe like the pipes, and also takes the pipes in the same manner. The use of this is absolutely necessary when the object is so large that one syringe full of liquid will not fill it. If no preventive were used, some part of the liquid would return whilst the syringe was being replenished, but the stopcock is then turned as in an ordinary tap, and all danger of this effectually removed.

5. Curved needles.—These are easily made by heating common needles at the end where the eye is situated, and bending them with a small pair of “pliers” into a segment of a circle half an inch in diameter. They are, perhaps, more convenient when the bent part is thrown slightly back where it commences. The pointed end is then thrust into a common penholder, and the needle needs no re-tempering, as the work for which it is wanted is simply to convey the thread or silk under any artery or vessel where it would be impossible to reach with the unassisted fingers.

6. A kind of forceps, commonly know by the name of “bullnose forceps,” will be constantly required during the process of injecting. These are short, usually very strong, but not heavy, and close very tightly by their own spring, which may be easily overcome and so released by the pressure of the fingers. When any vessel has not been tied by the operator, and he finds the injected fluid escaping, one of these “bulldogs” may be taken up and closed upon the opening. This will cause very little interruption, and the stoppage will be almost as effectual as if it were tied.

7. When the ordinary mode of injection is employed, it is necessary that the preparations be kept warm during the time they are used, otherwise the gelatine or size which they contain becomes stiff, and will not allow of being worked by the syringe. For this purpose we must procure small earthenware or tin pots of the size required, which will differ according to the kind of work to be done; and to each of these a loose lid should be adapted to protect it from dust, &c. These pots must be allowed to stand in a tin bath of water, under which a lamp or gas flame may be placed to keep the temperature sufficiently high to insure the perfect fluidity of the mixture. The tin bath is, perhaps, most convenient when made like a small shallow cistern; but some close it on the top to place the pots upon it, and alter the shape to their own convenience.

8. We will now inquire into some of the materials which are needed in this operation; the first of which is size. This substance is often used in the form of glue, but it must be of the very best and most transparent kind. To make the liquid which is to receive the colours for the usual mode of injecting, take of this glue seven ounces, and pour upon it one quart of clean water; allow this to stand a few hours, and then boil gently until it is thoroughly dissolved, stirring with a wooden or glass rod during the process. Take all impurities from the surface, and strain through flannel or other fine medium. The weather affects this a little as to its stiffness when cold, but this must be counteracted by adding a little more glue if found too liquid.

9. Instead of glue, gelatine is generally used, especially when the work to be accomplished is of the finer kind. The proportions are very different in this case, one ounce of gelatine to about fourteen ounces of water being sufficient. This, like glue, must be soaked a few hours in a small part of the cold water, the remainder being boiled and added, when it must be stirred until dissolved. A good size may be made by boiling clean strips of parchment for awhile, and then straining the liquid whilst hot through flannel; but when the injections are to be transparent, it is of the greatest importance that the size be as colourless as possible. For this purpose good gelatine must be employed, as Nelson’s or Cox’s: some persons of experience prefer the latter.

10. Colours.—The size-solution above mentioned will need some colouring matter to render it visible when injected into the vessels of any animal, and different colours are used when two or more kinds of vessels are so treated, in order that each “set” may be easily distinguished by sight. The proportion in which these colours are added to the size-solution may be given as follows:—

11. For—

Whichever of these colours is made use of must be levigated in a mortar with the addition of a very small quantity of water until every lump of colour or foreign matter is reduced to the finest state possible, otherwise in the process of injecting it will most likely be found that some of the small channels have been closed and the progress of the liquid stopped. When this fineness of particles is attained, warmth sufficient to render the size quite fluid must be used, and the colour added gradually, stirring all the time with a rod. It may be here mentioned that where one colour only is required, vermilion is, perhaps, the best; and blue is seldom used for opaque objects, as it reflects very little more light than black.

12. When it is wished to fill the capillaries (the minute vessels connecting the arteries with the veins), the “Micrographic Dictionary” recommends the colouring matter to be made by double decomposition. As a professed handbook would be, perhaps, deemed incomplete without some directions as to the mode of getting these colours, I will here make use of those given in that work. For red, however, vermilion, as above stated, may be used; but it must be carefully examined by reflected light to see whether it be free from all colourless crystals or not. It must first be worked in a mortar, and then the whole thrown into a quantity of water and stirred about; after leaving it not longer than a quarter of a minute, the larger portions will settle to the bottom, and the liquid being poured off will contain the finer powder. This may then be dried slowly, or added to the size whilst wet in the manner before advised.

13. Yellow injection.—To prepare this, take—

Dissolve the lead salt in the warm size, then add the bichromate of potash finely powdered.

Some of the chromic acid remains free, and is wasted in this solution, so the following is given:—

The first of these has the deepest colour, and is the most generally used.

14. White injection.—This is a carbonate of lead:—

Dissolve the acetate of lead in the warm size, and filter through flannel; dissolve the carbonate of potash in the smallest quantity of water, and add to the size: 143 grains of carbonate of soda may be substituted for the carbonate of potash. 15. For blue injection, which is not, however, much used with reflected light, as before stated, take—

The oxalic acid is first finely powdered in a mortar, the Prussian blue and a little water added, and the whole then thoroughly mixed with the size.

16. It may here be repeated, that it is only when the capillaries are to be filled that there is any need to be at the trouble to prepare the colours by this double decomposition; and, indeed, colours ground so finely may be procured that the above instructions would have been omitted, had it not been supposed that some students might find a double pleasure in performing as much of the work as possible by their own unaided labours.

17. The process of injection may now be considered; but it is impossible for written instructions to supply the place of experience. I will do my best, however, to set the novice at least in the right way. There are two kinds of injection—one where the object and colours are opaque, and consequently fit for examination by reflected light only; the other, where the vessels are filled with transparent colours, and must be viewed by transmitted light. The first of these is most frequently employed, so we will begin with it. In the object which is to be injected a vessel of the kind which we wish to be filled must be found; an opening must then be made in it to allow one of the small pipes before mentioned to be thrust some distance within it. When this is accomplished, thread the curved needle with a piece of silk thread, or very fine string, which some operators rub well with beeswax. This thread must not be too thin, else there is danger of cutting the vessel. The cord is then carried under the inserted pipe, and the vessel bound tightly upon it, the ends being brought up round the transverse arms, and there tied; so that all danger of accidentally withdrawing the pipe is obviated. Care must now be used in closing all the vessels which communicate with that where the pipe is placed lest the injecting fluid escape; and this must be done by tieing them with silk. Should, however, any of these be left open by accident, the bullnose forceps must be made use of, as before recommended.

18. The part to be injected must now be immersed in warm water, not, however, above 100° Fahrenheit, and be left until the whole is thoroughly warmed. Whilst this is being done, the coloured size must be made ready by the pot being placed in the tin bath of warm water, which must be of sufficient temperature (about 110° Fahrenheit) to keep it perfectly liquid. For the same purpose, the syringe is often tightly covered with two or three folds of flannel; and, indeed, there is no part of the process which requires more attention. If the substance to be injected is too hot, it is injured; whilst, if any of the articles are too cold, the gelatine, or size, loses a part of its fluidity, and consequently cannot enter the minute parts. When all is prepared, the syringe, with the stopcock attached, should be warmed, and then filled and emptied with the injecting fluid two or three times, care being taken that the end of the syringe be kept beneath any bubbles which form upon the surface. The syringe may then be filled, and closely attached to the pipe which is tied in the vessel. With a firm and steady pressure the piston must be forced downwards, when the substance will be perceived to swell, and the colour show itself in places where the covering is thin. When the syringe is almost emptied of its contents, the stopcock must be turned to prevent any escape of the injection from the subject. It must then be refilled, as in the first instance, and the process repeated. I say almost emptied, because it is well not to force the piston of the syringe quite to the bottom, lest the small quantity of air which frequently remains be driven into some of the vessels, and the object be injured or quite ruined. As the injection is proceeded with, it will be found that the force required grows greater, yet care must be taken not to use too much, or the vessels will burst, and render all the labour fruitless. The movement of the piston must be occasionally so slow as to be almost imperceptible, and for this reason it is sometimes marked with lines about one-eighth of an inch apart.

19. Of course, during the whole process the injecting fluid and subject must be kept at a temperature high enough to allow the liquid to flow freely; and the escape of a little of it need cause no fears to the student, as it is almost impossible to fill any subject without some loss. When the injected object has received sufficient fluid, it should have a plump appearance, owing to all the vessels being well filled. The vessel must then be tied up where the pipe was inserted, and the whole left in cold water two or three hours, after which time it may be mounted; but it may be well to notice a few things which the beginner ought to know before entering into that part of the process; and he may be here informed that it is not necessary to mount the objects immediately, otherwise it would be impossible for one person to make use of half of any large subject, as it would be in a state of decay long before each part could have been examined and separated. Large pieces should be therefore immersed in equal parts of spirits of wine and water, or glycerine, which some think better still, and thus preserved in bottles until time can be given to a closer examination.

20. In operating upon large subjects, entire animals, &c., the constant pressure required by the piston of the syringe grows wearisome, besides occupying both hands, which is sometimes inconvenient when working without assistance. To obviate this, another way of driving the syringe was published in the “Micrographic Dictionary” which I will quote here:—“We have therefore contrived a very simple piece of apparatus, which any one can prepare for himself, and which effects the object by mechanical means. It consists of a rectangular piece of board, two feet long and ten inches wide, to one end of which is fastened an inclined piece of wood (equal in width to the long board, and one foot high). The inclined portion is pierced with three holes, one above the other, into either of which the syringe may be placed—the uppermost being used for the larger, the lowermost for the smaller syringe; and these holes are of such size as freely to admit the syringe covered with flannel, but not to allow the rings to pass through them. The lower part of the syringe is supported upon a semiannular piece of wood, fastened to the upper end of an upright rod, which slides in a hollow cylinder fixed at its base to a small rectangular piece of wood; and by means of a horizontal wooden screw, the rod may be made to support the syringe at any height required. The handle of the syringe is let into a groove in a stout wooden rod connected by means of two catgut strings with a smaller rod, to the middle of which is fastened a string playing over a pulley, and at the end of which is a hook for supporting weights, the catgut strings passing through a longitudinal slit in the inclined piece of wood.” When in use the syringe is filled with injecting fluid, and passed through one of the three holes which is most suitable. The object being placed so that the pipe and syringe can be best joined, the rod and strings are set in order, and a weight placed on the hook. The stopcock must then be opened gradually, when the operator will be able to judge whether the weight is a proper one or not: if the piston is driven with any speed, there is danger of injuring the subject, and less weight may be used; if, however, the piston does not move, more must be added.

21. Such is the method recommended by the “Micrographic Dictionary,” and perhaps it is as good as any mechanical plan could be; but where the operator is willing to undergo the labour of performing all this with the hand, he has a much better chance of succeeding, because the pressure can be regulated so accurately, and changed so quickly when requisite, that no mere machine can compete with it, however well contrived. 22. When the beginner attempts to inject a subject, one of his difficulties is finding the vessel from which to commence. Another consists in distinguishing the arteries from the veins; but this is partly removed by making a longitudinal incision in the vessel, and with a blunt thick needle probing a little distance into the tube. The artery will be found thicker in the coating than the vein, and the difference is easily perceived by this mode of testing: the vein is also of a bluer colour than the artery. I say above, a “longitudinal incision” must be made: the reason for this is, the artery when cut across contracts considerably, and is lost in the adjoining substance; but where the opening is made longitudinally all danger of this contraction is obviated.

23. The different systems of vessels are often injected with various colours, so that their relative positions, &c., may be shown most clearly. In some specimens, the veins are injected with white, and the arteries with red; in the kidney, the urinary tubes are often filled with white, and the arteries with red. Then, again, the liver affords tubes for three or four colours. But no written instructions on this point can benefit the young student, and he must be content for a while to employ himself with single colours until he has gained the mechanical skill and the primary knowledge which are necessary before he can make any advance.

24. We will now consider the best methods of mounting injected objects. They must always be well washed in water after they have been kept in any preservative liquid, using a camel-hair pencil to clean the surface if necessary. Many parts when injected are in masses, such as the lungs, liver, &c., of animals, and consequently sections of these must be cut. For this purpose Valentine’s knife is very convenient, as the thickness can be regulated so easily; but where the injections are opaque, there is no need to have the sections very thin. Some few of this kind undergo comparatively little change in drying, so that the section may be well washed and floated upon the glass slide in the place desired, where it will dry perfectly and adhere to it. It must be then moistened with turpentine and mounted in Canada balsam like other objects. No great heat should be used with these preparations, as it is very liable to injure them; and some of the colours seem to suffer a slight contraction when any great degree of warmth is applied. There are many objects, however, which must be seen in the mass to be understood, and, indeed, lose all their form and beauty in drying, such as certain parts of the intestines, &c. These must be mounted in fluid, with the precautions noticed at length in Chapter IV., and for this purpose either Goadby’s fluid, the chloride of zinc solution, or spirit diluted with ten parts of distilled water, may be employed. It is a good thing, when practicable, to mount similar objects on two separate slides, using different preservative liquids, and taking the precaution of marking each with the kind of liquid employed. This not only serves as a guide to what is best for certain subjects, but if one is injured, there will probably be a good specimen in the other.

25. It may be here mentioned that many are now mounting sections of injected substances with the balsam and chloroform before mentioned, instead of using balsam alone, and consider that the labour is much lessened thereby.

26. A description of that mode of injection which is most generally employed has now been given, but this is not the only method of effecting our object. A most ingenious process was invented by M. Doyers, requiring no artificial warmth, by which many beautiful objects have been prepared. Make a solution of bichromate of potash, 524 grains to a pint of water, and throw this into the vessels to be injected; then take 1,000 grains of acetate of lead dissolved in half a pint of water, and force this into the same vessels. A decomposition now takes place in the vessels, and the yellow chromate of lead is formed. In this decomposition, however, the acetate of potash also is formed and as this salt has an injurious action upon the cells, Dr. Goadby recommends nitrate of lead to be used, which preserves rather than destroys them. He also advises the addition of two ounces of gelatine dissolved in eight ounces of water, to eight ounces of the saturated solution of each salt; but with this addition the hot-water bath would be required to keep the injecting fluid liquid.

27. Many of these are best mounted in balsam, in the same manner as those made in the ordinary way; whilst others are best shown when preserved in liquids, for which purpose Goadby’s fluid may be employed.

28. This mode of making injections with chromate of lead is deemed by many the best, especially where one colour only is employed. But it must be allowed that there is a little more danger of failure where two separate fluids are used for the same vessels.

29. We will now consider the best manner of making transparent injections, which, for many purposes, possess an undoubted advantage over the opaque ones. But it must be remembered that there are certain subjects to which no transparent injection could be applied, as they are too thick when in their natural state, and cutting would destroy all that beauty which is shown by the different parts in their relative adaptation. For those objects, however, which must be cut into sections to display their wonders, or are naturally thin—such as some of the finer tissues, livers, kidneys, &c.—transparency is a great acquisition, and enables us to understand the arrangement of the vessels more perfectly. Again, another advantage is the simplicity of the process; no hot water is needed with some preparations, either for the subject or the injecting fluid, which runs into the minute vessels thoroughly and easily, whilst the cost is small.

30. For this kind of injection no colour is so commonly made use of as Prussian blue. It is not a good one, as was before stated, for any opaque object, as the light reflected from it appears almost black; yet by transmitted light no colour is more useful, because its distinctness is equally great by artificial light and ordinary daylight. The method of preparing this, as given by Dr. Beale, is as follows:—

Dissolve the ferrocyanide of potash in one ounce of the water; add the tincture of sesquichloride of iron to another ounce. Mix these solutions gradually together, shaking the bottle well which contains them—it is best to add the iron to the potash solution. When thoroughly mixed, these solutions should produce a dark-blue mixture, perfectly free from any perceptible masses or flocculi. Next mix the naphtha and spirits of wine, and add the glycerine and the remaining two ounces of water. This must now be slowly mixed with the blue liquid, shaking the whole well in a large bottle whilst the two come together. The tincture of sesquichloride of iron is recommended, because it can always be obtained of a uniform strength.

31. Dr. Turnbull used a mixture slightly different from the above, which is made with the sulphate of iron:—

Dissolve the sulphate of iron in one ounce of the water, gradually add the ferrocyanide of potassium dissolved in another ounce, and proceed as above. 32. Dr. Beale also gives us the following carmine injection to be employed in the same way as the blue.G Take—

Mix the carmine with a few drops of water, and when well incorporated add about five drops of liquor ammoniÆ. To this dark-red solution about half an ounce of the glycerine is to be added, and the whole well shaken in a bottle. Next, very gradually pour in the acid glycerine, frequently shaking the bottle during admixture. Test the mixture with blue litmus-paper, and if not of a very decidedly acid reaction, a few more drops of acid may be added to the remainder of the glycerine and mixed as before. Lastly, mix the alcohol and water very gradually, shaking the bottle thoroughly after adding each successive portion till the whole is mixed. This fluid may be kept ready prepared, and injections made very rapidly with it.

33. The method of making injections with these colours is the same as with the gelatine mixtures before described, except that no heat is required, and consequently most of the trouble removed. The bottle of the fluid must be well shaken immediately before use; and when the object is injected, we must allow it to remain in a cool place for a few hours before cutting it. Thin sections of the subject may be cut with Valentine’s knife, as before described, and are very beautiful transparent objects. Some of the finer tissues, also, are shown much better by this mode of injection than by the opaque, and are easily mounted by washing in clean water when first separated, and floating upon a slide, where they must be allowed to dry thoroughly. They may then be immediately mounted in balsam, or kept in the dry state until it is convenient to finish them; but in many cases this keeping, if too much prolonged, will injure the object. If it is desired to transfer the section to another slide, it will be necessary to wet it thoroughly with water by the aid of a camel-hair pencil, and then gently strip it off with the forceps. When it is wished to preserve injected subjects in “masses,” it must be done by immersion in spirit, and the sections may be cut at leisure. Most of these transparent objects may be mounted in Canada balsam; but some recommend glycerine or glycerine jelly, as allowing the use of a higher power in their examination, and preserving them in a more natural form.

34. A few subjects may be noticed which are very beautiful when injected, and amongst these are the eyes of many animals. They must be injected by the artery in the back part, and when the blue transparent liquid is employed, nothing can exceed the delicate beauty which some of the membrane bears. It must, however, be dissected with care, but well repays us for the trouble. Water-newts and frogs are not difficult subjects, and in their skin and other parts are many interesting objects. Amongst the commoner animals—rats, rabbits, cats, &c. &c.—almost endless employment may be found, making use either of portions or the whole animal at once. The intestines of many of these are very beautiful. We must divide them with a pair of scissors along the tube, and cleanse them from all the matter; the coating may then be laid upon a slide, and any remaining impurity removed with a camel-hair pencil and water. When dried it may be mounted in balsam, and having been injected with the transparent blue, its minute beauty is shown most perfectly. In injecting a sheep’s foot, which is a good object, the liquid should be forced into it until a slight paring of the hoof shows the colour in the fine channels there.

35. When the lungs of small animals are injected, the finest fluid must be used, as some of the capillaries are so small that it is not an easy matter to fill them properly. And before entering upon these subjects, a certain proficiency in the mode of using the syringe, &c., should be obtained by practising upon simpler parts.

36. No subjects are more difficult to inject than fish, owing to the extreme softness of their tissues. Dr. Hogg recommends the tail of the fish to be cut off, and the pipe to be put into the divided vessel which lies just beneath the spinal column; by which method beautiful injections may be made. The gills, however, are the most interesting part as microscopic objects.

37. These instructions may seem very imperfect to those who have had much experience in this branch; but they will remember that their own knowledge was not gained from any written descriptions, but was forced upon them by frequent failures, some of which probably were very disheartening. As I before stated, it is very difficult (if not impossible) to accomplish much without some knowledge of anatomy.

38. I may here mention that the transparent injections sent over from the Continent are beautifully executed by Hyrtl of Vienna (who states that the injected fluid is composed of gelatine and carmine), Dr. Oschatz of Berlin, the Microscopic Institute of Wabern, Schaffer and Co. of Magdeburg, and others. Some of these will bear examining with a high power. A friend informs me that he measured a vessel in a rat’s tongue by Hyrtl, which was 1-7200th of an inch in diameter, and had a clear outline with quarter-inch objective. He has also made many experiments with the same materials, but has as yet failed in producing perfectly distinct outlines, there being a tendency of the colouring matter (magenta, carmine. &c.) to diffuse itself through the coats of the vessels into the surrounding tissues, although he has varied the pressure from one half a pound to sixty pounds. He believes the vessels are first washed out (injected with warm water and pressure applied), then some fluid introduced which renders the arteries impervious to the coloured fluid afterwards injected.

39. He finds that after washing out the vessels as above, the injecting fluid is much more easily introduced. He has used a strong solution of gallic acid previously to injecting with the colouring matter (in one experiment only), and the result was satisfactory. He puts the query,—Might not carbolic acid have a similar effect? He has often used it with injections to preserve the specimens, but not in sufficient quantity to act in the way indicated above.

Since writing the above, Mr. J.G. Dale, F.C.S., and I have made numerous experiments with carmine injection, and have at length been favoured with what we deem success. Some of the vessels in a kitten lately injected do not exceed 1-2000th of an inch in diameter, and present a clear outline with one-fifth objective. There is no extra-vasation, neither does the colouring matter show any grain except when a very high power is employed. The following is our process:—

Put these into a small flask, and allow them to digest without heat from twenty-four to thirty-six hours, or until the carmine is dissolved. Then take a Winchester quart bottle, and with a diamond mark the spot to which sixteen ounces of water extend. The coloured solution must be filtered into the bottle, and to this pure water should be added until the whole is equal to sixteen ounces.

Dissolve 600 grains potash alum in ten fluid ounces of water, and add to this, under constant boiling, a solution of carbonate of soda until a slight permanent precipitate is produced. Filter and add water up to sixteen ounces. Boil and add the solution to the cold ammoniacal solution of carmine in the Winchester quart, and shake vigorously for a few minutes. A drop of this placed upon white filtering-paper should show no coloured ring. If much colour is in solution the whole must be rejected, because, although it is possible to precipitate all the colouring matter by the addition of ammonia or alum, it is not well to do so, as the physical condition of the precipitate is thereby altered.

Supposing the precipitation to be complete, or very nearly so, shake vigorously for at least half an hour, and allow it to stand until quite cold. The shaking must then be renewed for some time, and the bottle filled up with pure water.

After allowing the precipitate to settle a day, draw off the clear supernatant fluid with a syphon. Repeat the washing until the clear liquid gives little or no precipitate with chloride of barium. So much water must be left with the colour at last that it shall measure forty fluid ounces.

For the injecting fluid take twenty-four ounces of the above coloured liquid, and three ounces of good gelatine. Allow these to remain together twelve hours, and then dissolve by the heat of a water bath; after which it should be strained through fine muslin.

As this injecting fluid contains gelatine, the hot water, and other contrivances mentioned in a former part of the chapter, will be necessary here also, but no peculiar treatment will be required.