If one should master the technic for all features of this group, he would be prepared to deal with all the rest; for embryo sacs are so delicate that they are as difficult as the free nuclear stage in the female gametophyte of a gymnosperm, while the peach stone needs a petrotome rather than a microtome. Between these extremes there is everything imaginable in structure, chemical composition, and consistency.
Some hints will be given, but the student will gradually learn what should be cut freehand and what should be imbedded; what stages in floral development, what stages in the development of the embryo sac, or what stages in spermatogenesis are likely to be correlated with easily recognized field characters, and what fixing agents are likely to give the best results with various kinds of material.
The Vegetative Structures - In stems, roots, and leaves the more delicate structures should be imbedded in paraffin and the more rigid structures should be cut without imbedding at all; but it should be remembered that the range of structures which can be imbedded in paraffin can be increased by the use of hydrofluoric acid; and also that hydrofluoric acid or cellulose acetate will extend almost indefinitely the range of structures which can be cut without imbedding.
The stem - The vascular cylinder of the angiosperms is either an endarch siphonostele, or a polystele derived from it. For a study of the development of the stem, the common geranium (Pelargonium) may be recommended. Near the base of afresh stem, about 1 cm. in diameter, cut freehand sections and fix them in 95 per cent alcohol for 10 to 20 minutes; transfer to 70 per cent alcohol to extract the chlorophyll, and then stain in safranin and light green. Such sections will show both primary and secondary structures in .the stele and in the cortex. Higher up, there will be secondary structures only in the stele; and still higher up will be found the origin of interfascicular cambium. All these can be cut without imbedding, but the earlier stages showing the differentiation of protoxylem, metaxylem, and the origin of secondary xylem are too soft for successful freehand sections. Fix in alcohol-formalin-acetic acid (10 c.c. formalin, 5 c.c. acetic acid, and 100 c.c. of 50 per cent alcohol) and imbed in paraffin.
For a study of woody stems, Tilia americana (basswood) is good, and shoots from 5 to 10 mm. in diameter are easy to cut. Very hard stems like Hicoria (hickory) and Quercus (oak) must be boiled and treated with hydrofluoric acid, if you expect to cut shoots more than 5 to 7 mm. in diameter. However, with a good sharp knife and a rigid microtome much larger sections can be cut without resorting to hydrofluoric acid. Of course, veneer machines cut very large and fairly thin, smooth sections from the most refractory woods.
While a random selection of stems would furnish material for practice in technic, we suggest that the stem of Clintonia shows a good siphonostele in a monocotyl; the rhizome of A corns calamus is a good type for the amphivasal bundle and, although a monocotyl still shows a differentiation into stele and cortex; Zea Mais, universally used but not characteristic of monocotyls, shows scattered bundles, but not the amphivasal condition; Aloe, Dracaena, or Yucca will illustrate secondary wood in monocotyls. Iris has a highly developed endodermis in the rhizome; and Nymphea or Nuphar will show scattered bundles in a dicotyl.
Lenticels and tyloses are abundant and typical in Menispermum, and very thin sections can be cut without imbedding; but both these structures are well developed while the stem can still be cut in paraffin without previous treatment in hydrofluoric acid.
The sieve tubes of the phloem are easily demonstrated in Cucurbita Pepo, the common pumpkin; other members of the family furnish good material. Take pieces of stem about 1 cm. long and not too hard to cut in paraffin, fix in formalin alcohol, and stain in safranin, gentian-violet, orange. The tropical Tetracera, one of the Dilleniaceae, has sieve plates so large that they are easily seen with a pocket lens. The phloem area is so large in the larger stems that it can be cut out for imbedding in paraffin long after the entire stem has become too hard for paraffin sections. Safranin and gentian-violet is a good stain for sieve tubes. It was once thought that these large sieve tubes afforded an obvious illustration of the continuity of protoplasm; but, as a matter of fact, the actual protoplasmic connections are scanty and hard to demonstrate. Iron-alum haematoxylin and orange will differentiate the strands if you are careful.
Roots.-It has long been known that the root-tip furnishes constantly available material for a study of mitosis. An onion thrown into a pan of water will soon send out numerous roots. Soak beans in water for several hours and then plant them in loose, moist sawdust. In a greenhouse, with "bottom heat," the primary root will be long enough in 2 or 3 days. The large, flat beans, especially Vicia Faba, are very favorable. The root-tips of Trillium grandiflorum, Tradescantia virginica, Podophyllum peltatum, Arisaema triphyllum, and Cypripedium pubescens may be mentioned as known to be favorable; but it is very possible that the best root-tip has not yet been tried.
Cell division does not proceed with equal rapidity at all hours of the day. Kellicott has shown that in the root-tips of Allium there are in each 24 hours two periods at which cell division is at the maximum, and two at which it is at the minimum. The maximum periods are shortly before midnight (11:00 P.M.), and shortly after noon (1:00 P.M.). The minima, when cell division is at the lowest ebb, occur about 7:00 A.M. and 3:00 P.M. When cell division is most vigorous, there is little elongation, and when cell division is at the minimum, cell elongation is at the maximum. Consequently, root-tips of Allium should be collected about, 1:00 P.M. or 11:00 P.M. Lutman, later, made observations upon periodicity of mitosis in the desmid, Closterium; and in 1915, Karsten made a comparatively extended study of periodicity in various stems and roots, together with notes on algae.
It is safe to say that the maximum number of mitoses in root-tips will be found shortly after noon (1:00 P.M.) and shortly before midnight (11:00 P.M.) (Fig. 104). It is certain, however, that abundant mitoses may be found at other times-even at 3:00 P.M.-in sporangia of ferns, in anthers of angiosperms, in endosperm, and in free nuclear stages of the embryo of gymnosperms.
Mitotic figures play such an important part in the development of the plant and in modern theories of heredity that it is worth while to acquire a critical technic in fixing and staining these structures. Use the various fixing agents-Flemming's weaker solution, chromo- acetic acid with or without a little osmic, Benda's fluid, Bouin's fluid, corrosive sublimate with acetic acid, and any others. Make yourself master of Haidenhain's iron-alum haematoxylin; then add the safranin, gentian-violet, orange combination; then safranin and anilin blue; and then experiment for yourself, but remember that the triumphs of modern cytology have been won with iron-haematoxylin and that you cannot read intelligently the literature of the past twenty-five years until you have gained at least an approximate mastery of this stain. Of course, dehydration, clearing, and infiltration must be very gradual. The schedules by Yamanouchi and by Sharp, on pages 45 and 46, will repay careful study.
In staining with safranin, gentian-violet, orange, allow the alcoholic safranin to act for 16 to 24 hours; then extract it with 50 per cent alcohol, slightly acidulated with hydrochloric acid, if necessary, until the stain has almost disappeared from the spindle; then pass through 70, 85, 95, and 100 per cent alcohol; stain in gentian- violet dissolved in clove oil, or in a mixture of clove oil and absolute alcohol, for 5 to 20 minutes; follow with orange dissolved in clove oil, remembering that this will weaken the safranin and sometimes the gentian-violet; finally use pure clove oil to differentiate the gentian-violet. Leave the slide in xylol for 2 to 5 minutes to remove the clove oil and to hasten the hardening of the balsam.
If you use aqueous gentian-violet or crystal-violet, after the safranin is satisfactory, transfer to water and then to the violet. After staining in violet, dip in water to remove the excess of stain and then dehydrate rapidly in 95 per cent and absolute alcohol, differentiate in clove oil, and then transfer to xylol.
The structure and development of the young root will be shown, to some extent, in preparations made for mitotic figures. The origin of dermatogen, periblem, plerome, and also of protoxylem, is well shown in Zea Mays, An ear of sweet corn, as young and tender as you can find on the market, will furnish material. Cut out from the grain a rectangular piece about 2*3 mm. and 4 or 5mm. long; if you want to show also the structure of the entire grain, take a section the entire length of the grain, perpendicular to the flat side of the grain, and about 2mm. wide. Cut the latter longitudinally; the rectangular pieces are sufficient for transverse sections. Fix in chromo-acetic acid. The roots of Hordeum vulgare (barley) might also be suggested.
The roots of Ranunculus repens and Sambucus nigra furnish good illustrations of the radial arrangement of xylem and phloem.
Smilax shows the radial arrangement, with a large number of poles and a very highly differentiated endodermis. The origin of secondary xylem and phloem is well shown in Sambucus nigra. Vicia Faba shows very clearly the origin of secondary roots. Pistia stratiotes, although not so generally available, is splendid for showing the origin of secondary roots. The arrangement of cells in the young roots of aquatic or semi-aquatic plants is often extremely regular.
The leaf - Young and tender leaves should be fixed in formalin alcohol and cut in paraffin. Cut sections freehand whenever there is sufficient rigidity. Resort to pith only when necessary. In cutting sections of a leaf like that of Lilium, lay one leaf on another until you have a bundle of them which will be nearly square in transverse section. Wrap the bundle with string for about 15 mm.; cut the bundle transversely so that about 5 mm. of the bundle will project beyond the tied portion. Dip in melted paraffin, as already suggested for Pinus, fasten the tied portion in the sliding microtome, and cut with the knife placed obliquely. About 15 to 20 cc is a good thickness for general leaf structure. In case of large leaves, cut 1 cm. wide and 3 cm. long and tie them together to make a good bundle for cutting.
Of course, for the finest preparations, imbed in paraffin. The common lilac, Syringa, has a good leaf to illustrate palisade and spongy parenchyma; the privet, Ligustrum, is also excellent.
Buds will furnish beautiful preparations of young leaves and, at the same time, will show the vernation. Cut the bud transversely, a little above the middle; remove the bud scales, if they promise to cause trouble; retain only enough tissue at the base of the bud to hold the parts in place. Fix in formalin alcohol and stain in safranin and light green.
Epidermis stripped from the leaf, fixed in 10 per cent formalin in water for a day or two, and then stained in safranin and anilin blue, will give excellent views of stomata. The development of stomata is particularly well shown in Sedum purpurascens, even in leaves which have reached the adult size. The epidermis is very easily stripped from a leaf of Sedum. If the big Sedum maximum is available, pieces of epidermis 6 or 7 cm. long and 2 or 3 cm. wide are easily stripped off, almost free from any underlying tissue. The epidermis of Lilium and Tradescantia show fine, large stomata, but it is not so easy to strip off. In these two genera the stomata, as in nearly all leaves, show only the adult structure.
Floral Development - For a study of floral development very young buds are necessary, and it is best to select those forms which have rather dense clusters of flowers, in order that a complete series may be obtained with as little trouble as possible.
The usual order of appearance of floral parts is (1) calyx, (2) corolla, (3) stamens, and (4) carpels; but if any of these organs is reduced or metamorphosed, their order of appearance may be affected.
Floral development is easily studied in the common Capsella bursa-pastoris. The best time to collect material is late in March or early in April. Dig up the plant, carefully remove the leaves, and in the center of the rosette a tiny white axis will be found. A series of these axes from 3 to 9 mm. in length, and from 1.5 to 3.5 mm. in diameter will give a very complete series of stages in the development of the floral organs. Preparations from the apex of the shoot taken after the inflorescence appears above ground are not to be compared with those taken early in the season, because the pedicels begin to diverge so early that median longitudinal sections of the flowers are comparatively rare. Fix in chromo-acetic acid and stain in Delafield's haematoxylin. The sections should be longitudinal and about 5( thick. Capsella shows the hypogynous type of development. The order of appearance of floral parts is (1) calyx, (2) stamens, (3) carpels, and (4) petals. The ovary is compound (syncarpous).
Ranunculus, which is also hypogynous, will illustrate the development of the simple (apocarpous) ovary. The ovules appear quite early, so that the archesporial cell, or even the megaspores, may be seen while the carpel is still as open as in any gymnosperm. The whole structure is a simple strobilus.
Rumex crispus (Yellow Dock) is also a good hypogynous type, and the densely clustered flowers afford a fine series of stages. Besides, in transverse sections, the early stages in spermatogenesis are very clear.
In the willows, Salix, the bud scales must be removed and the copious hairs should be trimmed off as much as possible with scissors, after which the catkin should be slabbed a little on opposite sides to facilitate penetration. This is a fine illustration of a compound strobilus.
The cat-tail, Typha, presents a simple type of floral development. The leaves should be dissected away long before the flowers can be seen from the outside. The cylindrical clusters, varying in diameter from 2 or 3 mm. up to the size of one's finger, will afford a complete series of stages. Until the spadix reaches the diameter of a lead pencil, transverse sections are easily cut. For later stages, the outer part of the spadix should be sliced off so that only enough spadix is retained to hold the florets in place.
Prunus and many other members of the Rosaceae furnish examples of the perigynous type of development. In many of them the floral parts do not occur in the usual succession.
The epigynous type is well shown in the Compositae. The order of appearance is (1) corolla, (2) stamens, (3) carpels, and (4) calyx (pappus).
The common dandelion, Taraxacum officinale, affords an excellent series with little labor. Examine vigorous plants which have, as yet, no flowers or buds in sight. Dig up the plant and dissect away the leaves. If there is a white cluster of flower buds, the largest not more than 4 mm. in diameter, cut out the cluster, leaving only enough tissue at the base to hold the buds in place. Larger heads should be cut separately.
Our most common thistle, Cirsium lanceolatum, shows the floral development with unusual clearness, but the preparation of the material is somewhat tedious. The involucre, which is too hard to cut, must be carefully dissected away. Retain only enough of the receptacle to hold the developing florets in place. A series of sizes with disks varying from 3 to 10mm. in diameter will show the development from the undifferentiated papilla up to the appearance of the archesporial cell in the nucellus of the ovule. The Canada thistle, Cirsium arvense, is equally good, but it is more difficult to dissect out the desirable parts. In the common sunflower, Helian- thus annuus, the young floral parts, like the mature head, are so very large that a satisfactory study may be made with a low-power objective. As in the case of the thistle, the involucre must be trimmed away and only enough of the receptacle retained to hold the florets together.
Erigeron (we have cut E. philadelphicus and E. annuus) furnishes a beautiful example of epigynous floral development, and the heads are so densely clustered that, in a single section, one may find various stages from heads with undifferentiated disk up to heads with florets showing pappus, corolla, stamens, and carpels.
Spermatogenesis - The earlier stages in spermatogenesis will be found in the preparations of floral development. The origin of the archesporium, the origin of sporogenous tissue, and the formation of the tapetum are beautifully shown in longitudinal and in transverse sections of the anthers of Taraxacum and many other Compositae. Transverse sections of the head of Taraxacum or any similar head at the time when pollen mother-cells are rounding off in the center of the head, will show various stages from the mother-cells in the center to the tetrads of spores at the periphery. Transverse sections of the anther of Polygala give exceptionally well-defined views of the archesporial cells and sporogenous areas.
Lilium, Trillium, Galtonia, Iris, Tradescantia, Vicia, and Podophyllum can be recommended for demonstrating the nuclear changes
involved in the formation of spores from the mother-cell. Several species of Lilium are common in greenhouses, and these may be used where wild material is not available. In early stages, where the sporogenous cells have not yet begun to round off into spore mother-cells, it is sufficient to remove the perianth, retaining just enough of the receptacle to hold the stamens in place. Transverse sections show the six stamens and also the young ovary. After the spore mother-cells have begun to round off, each stamen should be removed so as to be cut separately. In securing the desirable stages showing the division of the mother-cell into microspores, much time and patience will be saved by determining the stage of development before fixing the material. Mitosis is more or less simultaneous throughout an anther. Long anthers are particularly favorable, since they may show a very closely graded series of the various phases of mitosis. An anther of Lilium may show mother-cells with nuclei in synapsis at the top, while the mother-cells at the bottom have reached the equatorial plate stage of the first division; or, the mother-cells at the top may show the first division, while those at the bottom show the second. Determine the stage by examining a few mother-cells before fixing.
From what has been said, it is evident that longitudinal sections should be cut to show mitosis. Transverse sections should be cut to show the general structure of the anther. It is not necessary to cut the stamens into pieces before fixing, since they are easily penetrated and infiltrated; in later stages the stamens must not be cut into pieces, since the pollen grains and even the pollen mother-cells are easily washed out.
Return to Methods in Plant Histology