Plant histology: The Paraffin method



The paraffin method is still the most important of all histological methods now in use. The results obtained by this method would have been regarded as almost miraculous by the histologists of one hundred years ago. At that time it was customary to observe things dry, and no cover-glasses were used. Section-cutting with sharp knives or razors did not become general until about 1830. The need for an instrument which would cut sections without demanding an extreme degree of manual dexterity was soon felt, but a successful microtome did not appear until much later. The latest microtomes, while rather complicated, give wonderful results. The Spencer microtome, with the cooling attachment devised by Dr. Land, will cut even ribbons, from such material as the antheridial receptacles of Marchantia. This means that a series of sections can be cut from pollen grains or spores too small to be seen by the naked eye. Many of the principles involved in this method are general in their application, and some of the processes are common to other methods. Before attempting the freehand sectioning, the beginner should read the following paragraphs on killing and fixing, washing, hardening and dehydrating, and on clearing.

KILLING AND FIXING

The purpose of a killing agent is to bring the life-processes to a sudden termination, while a fixing agent is used to fix the cells and their contents in as nearly the living condition as possible. The fixing consists in so hardening the material that the various elements may retain their natural condition during all the processes which are to follow. Usually the same reagent is used for both killing and fixing. Zoologists, from humane motives, may use chloroform for killing, while other reagents are used for fixing. In fixing root-tips, anthers, and other material for a study of mitotic figures, it is necessary that killing be very prompt. In a weak solution of chromo-acetic acid, nuclei which have begun to divide may complete the division, although the reagent might hinder nuclei from entering upon division. By treating for 20 minutes to 1 hour with Flemming's weaker solution, or with a chromo-acetic solution containing a much smaller proportion of osmic acid, the killing will be greatly accelerated and the proportion of nuclei in division will be correspondingly greater. If filamentous algae are placed for 10 or 20 minutes in a chromo-acetic solution containing a little osmic acid, all the advantages of immediate killing will be secured. Material is then transferred to chromo-acetic acid containing no osmic acid. The short treatment with an osmic solution is not likely to cause any serious blackening. Take the killing and fixing fluids into the field. If one waits until the material is brought to the laboratory there may be some fixing, but it will, in many cases, be too late to do much killing. Material which has begun to wilt is not worth fixing. Material like Spirogyra, however, may be wrapped in several thicknesses of newspaper, placed in a botany can and brought into the laboratory. Before fixing, it should be placed in water for half an hour. Such forms suffer more from lack of air when placed in a bottle or a can than. from lack of water when wrapped in wet newspaper. Branches with developing buds may be brought in and kept in water. Cones of the cycad, Ceratozamia, sent from Jalapa, Mexico, have arrived in Chicago with cell division still going on at a rapid rate. But such cases are extremes; as a rule, take the killing and fixing fluids into the field.

Always have the material in very small pieces, in order that the reagents may act quickly on all parts of the specimens. Pieces larger than cubes of 1 cm. should be avoided whenever possible. While one sometimes needs sections 2 or even 3 cm. long, it is not likely to be necessary to fix pieces more than 4 or 5 mm. in thickness. For very fine work no part of the specimens should require the reagent to penetrate more than 1 or 2 mm.

For fixing agents of the chromic-acid series, the volume of the reagent should be about 50 times that of the material.

Fixing agents with alcohol as an ingredient will fix a larger proportion of material. It must be remembered that the water, which is always present in living tissues, weakens the fixing agent.

The-time required for fixing varies with the reagent, the character of the tissue, and the size of the piece. About 24 hours is a commonly recommended period for chromic-acid solutions, but 2 or even 3 days will do no harm.

WASHING

Nearly all fixing agents, except the alcohols, must be washed out from the material as completely as possible before any further steps are taken, because some reagents leave annoying precipitates which must be removed, and others interfere with subsequent processes. Aqueous fixing agents with chromic acid as their principal ingredient are washed out with water; aqueous solutions of corrosive sublimate are also washed out with water; but alcoholic solutions should be washed out with alcohol of about the same strength as the fixing agent; picric acid, or fixing agents with picric acid as an ingredient, must not be washed out with water, but with alcohol, whether the picric acid be in aqueous or alcoholic solution. When washing with water, running water is best, and where this is not convenient the water should at least be changed frequently. The washing-out process usually requires about 24 hours.

HARDENING AND DEHYDRATING

After the material has been washed, it is necessary to continue the hardening and also to remove the water. Alcohol is used almost entirely for these purposes. It completes the hardening and at the same time dehydrates, that is, it replaces the water in the material, an extremely important consideration, for the least trace of moisture interferes seriously with the infiltration of the paraffin.

The process of hardening and dehydrating must be gradual; if the material should be transferred directly from water to absolute alcohol, the hardening and dehydrating would be brought about in a very short time, but the violent osmosis would cause a ruinous contraction of the more delicate parts.. In recent years, cytologists have been making the dehydration process more and more gradual. Twenty years ago most workers began the dehydration process with 35 per cent alcohol and used the series 35, 50, 70, 85, 95, and 100 per cent alcohol. Some placed an intermediate grade between water and 35 per cent alcohol. If plasmoylsis-the tearing away of the protoplast from the cell wall-was avoided, the series was thought to be sufficiently gradual; but a series which may avoid plasmolysis may not be adequate if one is to study the finer details of cell structure. The following series is recommended: 2 1/2, 5, 7 1/2, 10, 15, 20, 30, 40, 50, 70, 85, 95, and 100 per cent. There is no particular virtue in the fractions: it is convenient to make 10 per cent alcohol, dilute with an equal volume of water for the 5 percent, and dilute the 5 per cent with an equal volume for the 2 1/2 per cent. It will be noted that the series begins with very close grades and that the intervals are gradually increased. The claim is that by beginning with very weak alcohols in close grades, more perfect dehydration can be secured at the end of the series. Various devices, like constant drip and osmotic apparatus, have been proposed to secure a more gradual transfer, but it is very doubtful whether these possess any real advantages. It is not necessary to use a large amount of alcohol: 2 or 3 times the volume of the material is sufficient.

The grades of alcohol may be used several times, but it must be remembered that pollen grains, fungus spores, starch grains, and various granules are likely to be left in the alcohol, so that it is wise to pour back through a filter each time, thus keeping the alcohols clean.

As the alcohols absorb water from the material, they become weaker and weaker. If the various alcohols be poured in a large "waste alcohol" bottle, when a couple of liters has been accumulated, the strength may be determined by testing with an alcoholometer. Then any grade of less strength can be made from this stock.

The time necessary for each of the stages has not been determined with any certainty. About 2 hours seems to be long enough for each of the grades from 2 1/2 to 70 per cent; for 70, 85, and 95, about 4 hours each; for absolute alcohol, 4 to 12 hours, changing 2 or 3 times. If material is to be kept in alcohol, leave it in 85 per cent, but where labor is no object, it is better to go on and imbed it in paraffin.

CLEARING

Let us suppose that the material has been thoroughly dehydrated, so that not the slightest trace of water remains. If the supposition chances to be contrary to fact, all the work which has preceded, as well as all which is to follow, is only an idle waste of time. The purpose of a clearing agent is to make the tissues transparent, but clearing agents also replace the alcohol. At this stage the latter process is the essential one, the clearing which accompanies it being incidental. The clearing, however, is very convenient, since it shows that the alcohol has been replaced and that the material is ready for the next step.

Various clearing agents are in use. Xylol is the most generally employed, and for most purposes it seems to be the best. Bergamot oil, cedar oil, clove oil, turpentine, and chloroform are used for the same purpose. Cedar oil and chloroform may, in some cases, be as good as xylol.

Only a small quantity of the clearing agent is necessary, enough to cover the material being sufficient.

The transfer from absolute alcohol to the clearing agent should be gradual, like the hardening and dehydrating processes. The most successful workers have been making this transfer more and more gradual. Thirty years ago it was customary to transfer from absolute alcohol directly to xylol; then a mixture of equal parts of absolute alcohol and xylol was interpolated; in the second edition of this book three grades were placed between the absolute alcohol and xylol. It is undoubtedly better to make the transfer still more gradual. The following series seem to be safe, 2 1/2, 5, 10, 15, 25, 50, 75, and 100 per cent xylol. These mixtures of absolute alcohol and xylol can be made with sufficient accuracy without measuring in a graduate. The 50 per cent grade is made by mixing equal parts of absolute alcohol and xylol; the 25 per cent, by adding to the 50 per cent an equal volume of absolute alcohol; make the 10 per cent grade from the 25 per cent by adding a little more than an equal volume of absolute alcohol; in the same way, make the 5 per cent from the 10 per cent, and the 21/2 per cent from the 5 per cent. The different grades may be kept in bottles and may be used repeatedly. A couple of drops of safranin dissolved in absolute alcohol, added to the 50 or 75 per cent xylol, will color the material a little and will often be helpful in orienting after the imbedding in paraffin.

About 2 or 3 hours is enough for each grade. The pure xylol should be changed once or twice. Throughout the dehydrating and clearing it is a good plan to keep the material in Number 4 shells, which are made from glass tubing about 25 mm. in diameter.

Other clearing agents may be used, but the process must be just as gradual.

THE TRANSFER FROM CLEARING AGENT TO PARAFFIN

This should also be a gradual process. The most convenient method is to place a small block of paraffin in the pure clearing agent with the material, but the block of paraffin should not rest directly upon the objects. Dr. Land uses coarse wire gauze, cut into strips about 15 mm. wide and tapered at both ends. The strip is then bent so that the pointed ends rest upon the bottom of the Number 4 shell, while the middle portion forms a flat table upon which the paraffin may rest. Dip the wire gauze table into xylol and then slip it carefully into the Number 4 shell. The table portion should be 10 or 15 mm. above the material, and there should be enough xylol to extend a few millimeters above the table. Place on the table a block of paraffin about equal to the volume of the xylol in the shell. The table not only prevents the paraffin from injuring the material by mechanical pressure, but insures considerable diffusion before the mixture of paraffin and xylol reaches the specimens. After 24 hours (or several days, if time permits) at room temperature, place the shell on a pasteboard box-slide boxes are good-on top of the paraffin bath. Do not place the shell directly upon the metal of the bath, since it is better to minimize heat. As soon as the paraffin is dissolved, add some more, this time leaving the cork out, in order that the xylol may evaporate. About 24 hours on the top of the bath should be sufficient.

THE PARAFFIN BATH

This step is usually called infiltration, but when the transfer from the clearing fluid to paraffin is made gradually, as has just been indicated, the process of infiltration is already begun. It is now necessary to get rid of the xylol or other clearing agent. This is accomplished, to a considerable extent, by pouring off the mixture of xylol and paraffin and replacing it with pure melted paraffin. Pour off the pure paraffin immediately. This is important. You will notice that often, when the pure paraffin is poured on, a froth or scum will appear on the surface. Much of the xylol will be in this scum, and, if allowed to remain, it would diffuse into the mass and greatly prolong the time needed for infiltration. So, pour it off and add more pure paraffin, for some xylol remains in the tissues and must be removed. Do not put the shell into the bath, but use a flat dish of some sort. The main object is to have a fairly large surface exposed, so that the remaining xylol may evaporate as rapidly as possible. Change the paraffin 2 or 3 times. Soft paraffin (about 45 C.) may be used at first, but the second should be the paraffin of the grade in which the material is to be imbedded. If there are two baths, one should be kept at 46 C. and the other at 52 C., if the material is to be imbedded in 52 C. paraffin. While using the soft paraffin, keep the material in the 46 C. bath; for the harder paraffin, use the 52 C. bath. If there is only one bath, there is no object in using the 45 C. paraffin. Do not throw away the paraffin which you pour off, but put it in a waste jar or beaker, or, still better, in a small tin lard pail, in which you have made a lip to facilitate pouring. This can be placed in the bath, or, in winter, on the radiator, and the xylol will gradually evaporate. After long heating, the paraffin not only becomes as good as new, but even better, since it becomes more homogeneous and tenacious. If it contains dust or debris of any kind, it may be filtered with a hot filter. The time required varies with the character of the material and the thoroughness of the dehydrating and clearing. If this schedule has been followed up to this point, the time will be much shorter than most investigators now deem necessary. Fern prothallia infiltrate perfectly in 15 to 20 minutes; onion root-tips in 20 to 30 minutes; ovaries of Lilium at the fertilization stage, 30 minutes to 1 hour; 5 or 6 mm. cubes of endosperm of cycads, containing archegonia, 2 to 21 hours; median longitudinal sections, 4 or 5 mm. thick, through ovulate cones of Pinus Banksiana may require 6 or 8 hours; if serial sections through the entire cone are wanted, Miss Aase found that the time must be prolonged to 24 or even 48 hours. When one is dealing with many lots of the same kind of material, as in research work, the time required for infiltration is easily determined. As a rule, minimize heat. It is, probably, never necessary to use paraffin with a melting-point higher than 52 C. With Land's cooling device sections 1( in thickness can be cut from 52 C. paraffin.

IMBEDDING

Material may be imbedded in paper trays, watch crystals, or any apparatus made for the purpose. The most satisfactory imbeddingdish we have used is a thin rectangular porcelain dish glazed inside. This dish, called a Verbrennungsschale, is made by the Konigliche Porzellan-Manufactur, Berlin, Germany. The most convenient sizes are 40*50*10 mm., 68*45*10 mm., and 91*58*15 mm. As listed, these dishes are not glazed; care should be taken to indicate that the dishes must be glazed inside {innen glasiert). Where these dishes are not available, any dishes of convenient size and shape can be used. The paper tray, if well made, is as good as anything. Thick ledger linen or thin, smooth cardboard make good trays. Smear the dish or tray with glycerin or soapy water to prevent sticking. Another way to prevent sticking is to put a piece of tissue paper in the dish, pour on water and make the tissue paper fit the inside of the dish, and then pour on the paraffin with the material to be imbedded. The paraffin will not stick to the paper. If several objects are to be imbedded in one dish, it is best to have the dish as near the temperature of melted paraffin as possible; otherwise the objects may stick to the bottom, and it will be impossible to arrange them properly. Hot needles are good for arranging material. Great care should be taken not to have the dish too hot, since too high a temperature not only injures the material, but also prevents a thorough imbedding. Pour the paraffin with the objects into the imbedding-dish and arrange them so as to facilitate the future cutting-out from the paraffin cake.

After the objects have been arranged, cool the cake rapidly by allowing the bottom of the dish to rest upon cold water. As soon as a sufficiently firm film forms on the surface of the cake, let water flow gently over the top. After the cake has been under water for a few minutes, the paraffin will either come out and float on the water or, at least, it will be easily removed from the dish. If paraffin cools slowly it crystallizes and does not cut well. The layer of paraffin should be just thick enough to cover the objects, not only as a matter of economy, but because a thick layer retards the cooling. Very small objects, like the megaspores of Marsilea, ovules of Silphium, etc., may simply be poured out upon a cool piece of glass, which has been smeared with glycerin or soapy water. In this way, thin cakes are made which harden very rapidly. If one is doing much imbedding, it is worth while to have the paraffin cakes uniform in size and to have a convenient method of filing. In our own collection, there are more than 6,000 paraffin cakes. They are filed in pasteboard boxes 28 cm. long, 10 cm. wide, and 2 cm. deep.

With the generic name written on the box, it is easy to find anything in the large collection.

CUTTING

As soon as the paraffin is thoroughly cooled, it is ready for cutting. Trim the paraffin containing the object into a convenient shape, and fasten it upon a block of wood. Blocks of pine 3/4 inch long and 3/8 inch square are good for general purposes. Put paraffin on the end of the block so as to form a firm cap about 1 inch thick. Warm the cap and the bottom of the piece containing the object, and press them lightly together; then touch the joint with a hot needle, put the whole thing into cold water for a minute, and it is ready for cutting. Cutting can be learned only by experience, but a few hints may not come amiss:

a) Keep the knife sharp. There should be two hones, one for use when the knife is rather dull and the other for finishing. For the first hone, nothing equals a fine carborundum hone. About 5.5 * 22.5 cm. is a good size. A hard Belgian hone, of the same size, may be a little better for finishing. Flood the stone with water, and rub it with the small slip which accompanies all high-grade hones; this not only makes a lather which facilitates the sharpening, but it also keeps the surface of the hone flat. As soon as the edge of the knife appears smooth and even under a magnification of 30 or 40 diameters, the sharpening is completed with a good strop. It is better to sharpen the knife every time you use it. A first-class microtome knife, in perfect condition, is unsurpassed for cutting paraffin sections, but it requires both time and skill to keep the edge perfect. More than 20 years ago we began to experiment with the Gillette safety-razor blade and devised several holders for it, some of them more or less successful. Mr. Strickler finally perfected a holder which has already been mentioned. In using this holder the blade should not project more than 1 mm. The Gillette blade is harder than the ordinary microtome knife, and is so sharp that the edge appears smooth, even under a high-power dry lens. The bevel is about the same as that of a microtome knife which has been " backed for sharpening. With this blade in the holder as made by Mr. Strickler or Mr. Larsen, we have cut smooth ribbons of Selaginella strobili, sections through the sporangium region of the whole plant of Isoetes, sections of stems of Cucurbita, in fact, we have not used an ordinary microtome knife for cutting paraffin ribbons for more than 15 years. Many fail at the first attempt and go back to the continual drudgery of sharpening microtome knives. If the holder is placed in the microtome at the angle used for a microtome knife, failure is certain, because the blade will scrape rather than cut. The angle should be considerably nearer vertical than in the case of a microtome knife.

b) Keep the microtome well oiled and clean. c) Trim the block so that each section shall be a perfect rectangle.

A ribbon of sections is much better, because sections will usually come off in neater ribbons if the knife strikes the longer edge of the rectangle, so that the sections are united by the longer sides rather than by the shorter. Crooked ribbons are caused by wedge-shaped sections, and are always to be avoided, because they make it difficult to economize space, and also because they present such a disorderly appearance. The knife, which should be placed at a right angle to the block and not obliquely, should strike the whole edge of the block at once, and should leave in the same manner.

If sections stick to the knife, it may be that the knife is too nearly parallel with the surface of the block. By inclining the knife this difficulty is often obviated. A split or scratch in the paraffin ribbon may be caused by a nick in the knife.

Use some more favorable position of the edge, or sharpen the whole knife. A split or a scratch in the ribbon is often caused by some hard granule which becomes fastened to the inner side of the edge of the knife. This is the most common cause of the difficulty. Simply wipe the knife by an upward stroke of the finger, slightly moistened with xylol. Do not use a cloth.

Sometimes good sections can be cut with a rather slow stroke when a rapid stroke fails. When paraffin is rather hard, sections may sometimes cut nicely at 5(, when, at 10(, ribbons cannot be secured. If very thin sections are desired and the paraffin seems too soft, cool the paraffin and the edge of the knife with ice, by Land's cooling device, or by pressing a piece of ice against the paraffin block and the knife. Sometimes hard paraffin does not ribbon well. This difficulty may be remedied by dipping a hot needle in soft paraffin and applying it to the opposite edges of the block to be cut. Often the mere warming of the opposite edges of the block with a hot needle is sufficient.

Another method, suggested by Dr. Land to facilitate the cutting of difficult material, has been tested in this laboratory and has been found to be very effective. Paraffin absorbs a small amount of water, or water penetrates between the crystals of paraffin. At any rate, water reaches cell walls and, perhaps, other structures which have not been completely infiltrated, and thus softens them. The paraffin cakes may be left for weeks in water. Cakes of class material may be put in water in a fruit can and kept until ready for use. After such treatment, smooth ribbons may be cut from material which would hardly cut at all without it.

A ribbon carrier is very convenient. A good carrier can be made by mounting a couple of spools 15 or 20 inches apart, with a strong piece of cloth for a band. More elaborate carriers may be made if one has tools.





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