[13]. Should the student, however, determine to prepare this solution for himself, he will find a good formula for the purpose in Schäfer’s “Practical Histology,” p 176. [Note I.]
20. Absolute Alcohol.—As we purpose mounting the sections which have just been stained, in Canada balsam, we will briefly consider the preliminary treatment to which they must be submitted before this can be effected. The object of this is to abstract from the tissue all its water, for if any moisture be permitted to remain in the section it will, when mounted in balsam, become obscured and surrounded by a kind of opalescent halo, due to the imperfect penetration of the balsam into the only partially dehydrated tissue. The old-fashioned plan of dehydration was simple exposure to the air. The method now generally adopted is to bring about the same result by means of absolute alcohol. This fluid has such a strong affinity for water that tissues submitted to its influence are rapidly and effectually deprived of any water they may contain. Absolute alcohol in small quantity may be obtained from the druggist at about sixpence per ounce. It will be necessary for the student to provide himself with a little of this agent, say about two ounces, the method of using which will very shortly be explained. Absolute alcohol must be kept in a bottle with a very accurately-fitting stopper, in order to prevent its absorbing moisture from the air. For our purpose such a bottle, having a neck as wide as possible, is to be selected.
21. Clove Oil.—After being thoroughly dehydrated the sections may, in special instances (§ 48), be at once mounted in balsam; but, as a general rule, it will be found necessary (particularly in the case of animal sections) to treat them with some clarifying agent, in order to remove the cloudiness and opacity which is (in part) due to their previous immersion in alcohol. For this purpose turpentine, or any of the essential oils, may be used: of these, oil of cloves is to be specially recommended. It is rather expensive, ranging from sixpence to one shilling per ounce: but, as a drop or two will be sufficient for preparing each slide, only a small quantity—say half an ounce or an ounce—need be procured. The most convenient vessel in which to keep the oil is one of the small test bottles used by watchmakers. These bottles are provided with a glass cap to exclude dust, and the stopper is prolonged into a glass rod, which dips into the bottle. The use of this rod and the method of employing the oil will be explained shortly.
22. Canada Balsam, as ordinarily met with, is a thick resinous balm of great viscidity, but readily rendered perfectly fluid by the application of heat. Formerly, sections were mounted in this medium in its pure state, but owing to the annoyance which was so constantly being experienced from the tenacity with which intruding air-bubbles were held by the viscous medium, this plan of mounting is rapidly falling out of use.[[14]] It is now usual to employ the balsam in a diluted condition, the two chief diluents being chloroform and benzole. As balsam, however, often contains more or less moisture, it is desirable to drive this off before adding the diluent. A very convenient way of doing so is to expose some pure balsam to the heat of a cool oven for several hours, when the balsam will be found to have assumed a hard, vitreous character. It should now be broken into small pieces, these put into a bottle, and some methylated chloroform added, which in a little while will completely dissolve the hardened balsam. More chloroform is then to be added, until a solution is obtained sufficiently thin to run through filtering-paper. A glass spirit lamp must now be procured, having a capacity of about two ounces, and provided with a cap. Into the wick-holder of this (which must be made of porcelain) a hollow glass tube is to be so fitted that its end dips into the lamp to within about a third of the bottom. (See Fig.) The thin chloroform-balsam is now to be filtered into this lamp, very fine filtering-paper—through which a little chloroform has first been passed—being used for the purpose. When the lamp is full it must (deprived of its cap) be put in a warm place until sufficient of the chloroform has evaporated to leave behind it a fluid of the consistence of thin syrup.
[14]. Although we cannot too strongly insist upon the use of chloroform-balsam wherever practicable, yet it sometimes happens in the mounting of substances of considerable thickness, that after all the chloroform has evaporated an insufficient amount of balsam is left behind to fill up the cavity between slide and cover. In such cases, therefore, it is advisable to use pure balsam, which may be done in the following manner. The object having been previously thoroughly dehydrated by immersion in absolute alcohol, is to be thence transferred to a little good turpentine, or benzole, where it should remain until perfectly transparent. It is now to be placed in the centre of a slide which has been gently warmed, and a drop or two of fresh fluid balsam added, the greatest care being taken to prevent the formation of air-bubbles. Should such arise they must be touched with the point of a heated needle, which will cause them to burst and disappear. The chief difficulty of the process has yet to be encountered in the application of the cover; for it is during this procedure that the development of air-bubbles is most likely to take place. This annoyance may, however, be entirely avoided by taking the simple precaution of dipping the cover into turpentine before it is applied (§ 16), when it will be found that “you can’t get air-bubbles, even if you try.” The courtesy of Mr. J. A. Kay, of Chatham, enables us to give our readers the benefit of this practical “wrinkle.”
SPIRIT LAMP ADAPTED TO CONTAIN BALSAM.
23. Mounting in Balsam.—Let us now return to our sections which, it will be remembered, were left in methylated spirit (§ 19). These we will now mount in balsam, and although, of course, any number may be proceeded with at the same time, yet to avoid confusion, in the following directions one section only will be spoken of. This section then is, with the perforated spoon, to be transferred to the bottle of absolute alcohol, where it may remain for about an hour—considerably less time is actually required, but as from constant use the spirit becomes weakened, it is as well to be on the safe side. It must now be removed to the centre of a clean glass slip, and here the plain end of the spoon comes into use. If this be employed for effecting the transfer, it will be found that when the section is being removed from the alcohol it will bring along with it a small pool of the spirit. A slight touch of the needle applied to the edge of the section will cause it to float from the spoon on to the slide, at the same time carrying the pool of alcohol with it, in which it will gently spread itself out upon the slide without the faintest risk of injury. The superfluous spirit is now to be drained off, and just as the section is becoming glazed and sodden-looking (not dry) we must, by means of the long glass stopper (§ 21), apply to it a large drop of clove oil. The oil, however, should not be placed on the section, but be allowed to drop on to the slide near to its margin. By gently tilting the slide the oil will gradually insinuate itself beneath the section and slowly ascend through it to the surface. The slide should now be covered with a bell-glass (or wine-glass), and about two minutes allowed for the oil thoroughly to saturate the section. As much as possible of the superfluous oil must then be drained off, and the remainder removed with blotting paper. By means of the glass rod a small quantity of chloroform-balsam is now taken from the spirit lamp which contains it, and allowed gently to fall upon the section, which must then be covered with a thin glass circle in the manner previously described (§ 16). When the object is very fragile, it is a good plan, after draining off the clove oil, to apply the cover directly upon the section, and then to place a drop of the balsam near to the edge of the cover. This, by capillary attraction, will speedily diffuse itself beneath the cover, flowing over and surrounding the object, without in the slightest degree disturbing its position. If, during the process of mounting, any air-bubbles arise, we may view their development with equanimity, being well assured that as the chloroform evaporates they too will quickly disappear. When the mounting is completed, the slide should be roughly labelled and placed on a warm mantel-piece for a few days to dry.
24. Finishing the Slide.—In the course of two or three days it will be advisable to take an old penknife, and after heating the blade in the flame of a spirit lamp, gently to run the point of it round the margin of the cover, so as to remove any excess of balsam which may have oozed from beneath it. In a few days more, any remaining balsam may be carefully scraped away with a cold knife. All remaining traces of balsam are then to be removed from around the cover by means of a rag just moistened with methylated spirit, or, what is better, with a mixture of equal parts of spirit and æther, after which the slide is to be thoroughly washed in cold water. The slide is now in reality finished, but, in order to give it a smart appearance, it is usual, with the assistance of the turn-table, to run a ring of colored varnish round the covering glass. A very useful varnish for the purpose is the white zinc cement. To prepare this, dissolve an ounce of gum dammar in an ounce of turpentine by the aid of heat. Take one dram of oxide of zinc and an equal quantity of turpentine; rub them up together in a mortar, adding the turpentine drop by drop, so as to form a creamy mixture perfectly free from lumps or grit. One fluid ounce of the dammar solution previously made must now gradually be added, the mixture being kept constantly stirred (Frey). The cement, when made, should be strained through a piece of fine muslin, previously wetted with turpentine, into a small wide-necked bottle, which, instead of having a cork or stopper, should be covered with a loose metal cap. Instead of a bottle, the varnish may be kept in one of the collapsible tubes used by artists; but though this plan is highly recommended by many, it is not without its disadvantages. If the varnish becomes thick by keeping, a few drops of turpentine or benzole well stirred in will soon reduce it to a suitable consistence for use.