On the use of phenol (carbolic acid)in microscopic technic*
It is now about forty years since Lister introduced phenol, or carbolic acid, as the chief agent in antiseptic treatment. This substance, which until then was little known, attained such popularity as to be considered the antiseptic par excellence. The toxic properties, which correspond to its microbicid action, cannot however be ignored. In fact the common solutions of 2 to 5% may become very dangerous when freely used in the spraying or in the irrigation of large cavities. On the other hand, the thorough study of this and other antiseptics shows that their microbicid action is greatly inferior to what has been generally supposed, and this fact has led to their substitution by other disinfectants and to replacing antiseptics by asepsis. Phenol, notwithstanding, is widely used as a domestic remedy, and not always with the necessary precaution. Being easily and universally obtainable, it has become deplorably common as a means of suicide.
It is strange that the great advantages offered by phenol in microscopic technic and principally in the study of minute organisms should have been mostly overlooked. This fact may be due to its being considered very destructive to tissues, owing to its caustic action and to its special effects on the horny layer of the epidermis.
By degrees, however, I have come to the conclusion that its properties are of great value and have used it extensively in my work on Diptera, as well as in helminthologic researches.
In Ehrlich's Encyclopedia of Microscopic Technic, phenol is cited as mordant and also [adequate] for clearing sections by being mixed with other liquids, less tolerant of water.
Pyrolignic acid and creosote have been for a long time used in microscopic work, the first for fixing and the second for making objects transparent. Pyrolignic acid, which is of no special advantage, has been quite abandoned. As to creosote, care must be taken to distinguish the creosote of vegetable origin obtained from beech-tree and composed of a mixture of guaiacol, cresol and other substances and the creosote of mineral origin extracted from coal, which contains an approximate proportion of 20% phenol mixed with many other substances. Both are certainly inferior to pure phenol for dehydrating and clarifying purposes. The use of mineral creosote gave me the idea of substituting it by pure phenol and I soon perceived the enormous advantage gained by this substitution.
Before discussing the different uses to which phenic acid is put, I will just recall some of its properties. Pure phenol is solid at room temperature, but has a low degree of fusion (about 40%); it forms needle-like crystals which may become red when exposed for some time to the action of light, apparently under the influence of the glass of the [lab] jars. Liquid phenic acid is obtained by the addition of alcohol or glycerine; this is the phenic acid of druggists, commonly used in our research-work.
Liquid phenic acid as a preserving fluid
Small zoological and botanical specimens may be kept indefinitely in phenic acid without showing any change in their structure, after being transferred to other fluids such as alcohol, glycerine, solutions of formaline, etc. No turbidity arises from transferring them to phenol (in excess), when they have been in watery, alcoholic or glycerine solutions, or even in pure glycerine. These objects may be inversely transferred from pure phenol to the greater number of other reactives employed in microscopic technic. These same specimens may be transferred quite as easily from the above mentioned liquids (in excess) to phenic acid because phenol can be mixed with them without turbidity, unless it has previously absorbed a greater quantity of water.
In this case, essential oils and rosins may become turbid, which may be avoided by passing the specimens trough anhydrous phenic acid or other dehydrants.
Penetrating power of liquid phenol
Liquid phenol in excess has great penetrating power. I have found that small insects (adults and larvae), arachnids, worms and other organisms are penetrated and dehydrated in a short time, as it may be seen by the transparency of their tissues.
The chitinous skeletons do not form as obstacle, as the action of phenic acid on chitin is analogous to its action or keratin. The substitution by phenol is still more rapid when the objects have been preserved in alcohol.
Phenol as a clarifier
Liquid phenol is an ideal clarifier. Its refractory index is superior to that of glycerine and perhaps to that of balsam. I have used it extensively for larvae of files and for worms, which become completely transparent as if they were made of glass.
Among clearing fluids, phenol has the rare, if not unique, property of not making the specimens shrink; on the contrary, they become more turgid, so that the segments of the abdomen of insects, for instance, become much more distinct. This I have found of great advantage in studying ovipositors and genital appendices that are retracted when treated which other liquids. Many internal organs, as for instance spermatocysts, are clearly seen on account of their more intense pigment. The same may be said of maxillary hooklets and stigmata of file larvae; it is really wonderful to note how clearly hairs, spines, nails and other more pigmented parts are seen. Specimens that have been dirtied in alcohol and are seemingly of no further use for examination reassume their natural intense turgidity, which is easy to diminish by adding a little alcohol to the solution, as its action is just the opposite of that of phenol.
The use of phenol with the freezing microtome
Liquid phenol easily freezes, and it is then of a favorable consistency for cutting the cleared objects with a freezing microtome.
Specimens colored all through by carmine or hematoxyline can be used, but aniline dyes are extracted by phenol.
It is evident that phenol offers advantages for serial cuts, but it allows the dividing of small specimens, and makes the study of the same easier; at the same time, it preserves the stereoscopic image of the thicker sections. To make preparations of small diptera, adults, larvae and pupae, they should be placed on the table of a freezing microtome using CO2, so that sagittal section is perpendicular to the table; when treating arthropodes, their legs must be turned upwards; they are then frozen and a cut made which divides the body into two halves, each of which includes the appendices of the sides of the body. These halves are useful for microscopic study, and much easier to observe than the organism as a whole. Sometimes it is advisable to separate the head and place it in the position found most appropriate. The target of the sagital section is just the rest of the body. When larvae, pupae and worms are used, it is better to separate the head and the tail, which may be placed in the most favorable position. The body may then be divided into two or three longitudinal sections which allow the studying of cutaneous and internal structure. These cuts may be made by a scalpel; I however prefer the use of Gillette razor-blades.
For clearing opaque objects
Opaque bodies like fleas, ticks and spiders, which are not sufficiently cleared by glycerine or glycerine-gelatine, become transparent when treated with phenol. I have found that the larvae of Blepharoceridae, the tegument of which is quite dark and opaque, are made so transparent that the new skin may be perfectly seen through the old one when they are about to moult; this fact I have found of the greatest importance in identifying the different phases of evolution. Little mollusks may also be cleared so as to show their internal anatomy. Even their shells become transparent. Phenol may also be used for studying eggs with thick shells, and organisms which either contain fat of milklike appearance or are covered by silky hairs, like some species of the genus Trombidium.
Objects cleared by phenol may be easily included in balsam. They must first be transferred to another solution to extract the remaining water, thence passing them into essence of turpentine or cloves, before including in balsam. Phenol is of not great utility for sealed microscopic preparations (unless they are very thin, on account of its fluidity). There are however methods for using it even so, which will be explained in another paper.
Objections
A few objections may be made to the use of phenic acid. The chief disadvantage is that it changes its colour after being a long time exposed to active light. As it becomes red it gets less transparent. The experiments that I made in order to do away with this disadvantage have not given much result. I tried several other substances added to the phenol; a mixture with thymol seemed to delay the change of colour, without wholly preventing it; it is better to change the phenol and put the specimen to be studied into a fresh and hylaine solution when the other is already coloured. Another way is to change the specimens for study into another clearing medium, but these may reduce the turgescence, or they may also change colour with time. I experimented with some substances, but as it was during the war I found it impossible to obtain others. Up till now guayacol gave the best result. Phenol cannot be entirely substituted by it, as its penetrating and dehydrant powers are weak, but bodies cleared by phenol may be preserved in guayacol.
It is strange that, before forming their barrel-shaped cocoon, file larvae darken after being some time in phenol. This shows that the blackening process, which takes place in their skin when they form their pupae in life, also takes place in the dead skin preserved in alcohol. The second disadvantage of phenol is its action on human skin; this however can be avoided by taking the precautious procedures generally used when handling colouring substances.
Human skin, even when thick, as in the palm of the hand, is permeable to phenol, which used for some time, even is small quantities, produces general phenomena such as nausea, uneasiness and elevated temperature as I have twice experienced myself. The resorption of phenol is easily demonstrated by the characteristic colour of the urine some time after emission. Sulphate of sodium is considered as an antidote to phenol, but it is doubtful if it would prove so to the phenol absorbed by the skin. The parts of the skin brought in contact with phenol should be washed with alcohol or alkaline solutions.
It must be remembered that liquid phenol runs on the slides more like alcohol than like water, and that a drop splashed or conveyed by the fingers to the face leaves a spot which takes some days to disappear.
Conclusions
It is not difficult to avoid the inconveniences arising from the use of phenol, once they are known. They are not serious enough to prevent its use in microscopic technic.
I can recommend it specially for examining small bodies which are naturally opaque, and which become transparent after imbibing the liquid; also for objects become dry either voluntarily or by accident. For this I think it has no equal, unless it is a modification such as chlorophenol, which cannot be recommended on account of its pungent smell.