Part IV

nuclei, which are still connected by fine threads. 4. The subsequent dilatation of the spindle, the threads becoming more curved, while from it new con- necting threads pass to the peripheral protoplasm. 5. Meanwhile microsomata collect at the equator of the dividing cell. 6. Note also the involution and their subsequent division. of the chromatophores 446 PRACTICAL BOTANY 7. The gradual formation of the septum, beginning at the periphery and proceeding towards the centre. III. In summer or autumn the process of conjugation and formation of the zygospores may frequently be observed in Spirogyra : filaments which are about to conjugate assume a position parallel to one another, and on them the following observations are to be made 1. Cells opposite one another put out rounded processes, which meet. 2. The wall at the point of junction is absorbed, and thus the canal of communication is formed. 3. Meanwhile the protoplasm of the two cells has rounded off, one (the male) usually doing so earlier than the other (the female). 4. The protoplasm of the male cell passes through the canal, and coalesces with the female to form the zygote. 5. The zygote surrounds itself with a thick stratified cell-wall, which is smooth or shows various markings of the surface according to species. By fixing specimens in course of conjugation and staining with haematoxylin, Schmitz has been able to observe that the nuclei of the conjugating cells coalesce to form the single nucleus of the zygospore, but according to recent observations this coalescence only takes place after a considerable time, when the zygospore is nearly mature. For comparison with Spirogyra observations should be made on Mesocarpus, a filamentous fresh-water Alga, having in each cell a single straight flattened chromato- phore, in a central position. The conjugation differs from that of Spirogyra in two points: (1) that -the DESMIDIE.E 447 zygospore is formed in the conjugation-canal, and not in the cavity of one of the conjugating cells ; (2) that a part only of the protoplasm of the conjugating cells is used up in forming the zygospore. DESMIDIE.E Members of the closely-allied group of the Desmidiece should alse be examined ; e.g. species of Closterimn, which is not uncommonly to be found in standing pools of fresh water. Mount specimens in fresh water, and examine under a high power : note 1. The more or less semilunar curved form of this unicellular Alga. 2. The smooth cell-wall, often marked by transverse striaB, about the equatorial part of the cell. 3. The colourless protoplasmic basis, which includes a. Two large chromatophores of equal size, disposed symmetrically on either side of a central clear space. A b. single nucleus, which occupies the central space, but is sometimes difficult to observe owing to the fact that the chromatophores often extend across the central space, and may even be connected. Observe further the peculiar plaited form of the chromatophores, which also include several disk-shaped, highly refractive bodies (pyrenoids). Stain with an iodine solution : this will make the nucleus more ap- parent, and the pyrenoids will stain a dusky purple. The plant multiplies by cell-division : try to observe cells in which the process is going on. 448 PRACTICAL BOTANY It has recently been shown that the cell-wall of most of the Desmids is composed of two parts which fit together like a pillbox and its lid : it is even stated that in Closterium it consists of four parts, there being two external shells, and two girdles which fit within them, about the equatorial position : this is the explanation of the transverse striae above noted, but it requires high powers to recognize these striae with certainty as the margins of the shells and girdles above alluded to. In most of the Desmideaa there is a gelatinous sheath, of greater or less extent, covering the cell-wall externally, similar to that in Spirogyra. Other Desmids may also be compared, e.g. Micrasterias, which is to be found in peaty pools : it is of flattened form with a deep median constriction, and stellate outline : the pyrenoids are very well seen in this species. For description of other forms of this family, many of which are of great beauty, reference must be made to books dealing specially with the subject : it should be noted, however, in examining them that in the larger majority of them the cell is divided into two symmetrical halves, and that frequently there is a sharply marked equatorial constriction, as in Micrasterias. Conjugation takes place in the Desmids, and it may frequently be observed in Closterium, the main points being as follows: two cells approach one another, usually placing themselves with their longer axes parallel, but sometimes at right angles. Processes are then formed from the two cells, as in Spirogyra, which meet, and fusion takes place at the point of contact. Meanwhile the protoplasm passes from the ends of the two cells towards their equators, coalesces, and the zygote is formed in central position : it is an oblong cell, which finally becomes rounded, and covered by a thick cell-wall, while the tests of the original cells fall away. DIATOMACE.E 449 DIATOMACE^E Observations should also be made on some of the Diatomacece, a family of somewhat doubtful affinity, and remarkable for the variety and beauty of form of its members. Almost any sediment from a fresh- water pool, mounted on a slide and examined under a high power, will show specimens of Navicula : it is to be recognized as a unicellular organism pointed at both ends, and showing active movement. Note the yellowish brown colour characteristic of Diatoms, and observe carefully that within the cell-wall there is a basis of colourless protoplasm, in which are embedded a nucleus, and a yellowish brown coloured chromatophore, of variable shape and position. Specimens of Pinnularia, which is also a common form, should be observed, since in it the peculiar structure of the cell-wall is to be seen : it is composed of two halves, tests, which fit one inside the other like a pill-box and its lid : this structure is characteristic of the Diatoms, and evidence of it may be found in the various forms examined : but it is only under high powers that the peculiarity can be clearly made out. Compare the structures above noted in the Desmids. A third form, Gomphonema, is to be found attached by a thin transparent stalk to the surface of almost any submerged plant : in this the characters above noted may be again seen. Attention is to be paid to the varieties of surfacemarking in the cell-walls of these and other Diatoms : GG 450 PRACTICAL BOTANY for further details and description of other forms reference is to be made to special treatises. When cell-division takes place in Diatoms, new tests are formed, fitting within the old ones : specimens dividing, or which have recently divided, should be looked for, and examined minutely, under high powers, in order to verify this point. As the tests once formed do not grow, it is obvious that repeated division will lead to a diminution of size : this is met by the formation of auxospores, which may be observed in various Diatoms, especially in summer. There are various details of the process in different Diatoms, but the essential point is that the tests are thrown off, and the contents elongate greatly, and ultimately forming new tests, enter again on a course of division. The tests are silicified, and by treatment with a little potassium chlorate and nitric acid, and subsequent ignition on platinum foil, skeletons of silica may be prepared, which retain in minutest detail the configuration and surface-markings of the tests of the Diatoms so treated. NOSTOC 451 CYANOPHYCE^: NOSTOC This plant is to be found growing on turf, or Moss, but not attached to it. It appears as irregular, more or less flattened, olive-green masses, which are brittle when dry, but soft and gelatinous when wetted. Mount a small piece, or a section of one, in water, and it will be seen under a low power to consist of numerous irregularly coiled filaments, embedded in a gelatinous matrix. Under a high power each filament may be seen to consist of series of small cells, with granular, blue-green contents, which constitute the great proportion of the filaments ; these series are interrupted here and there by larger cells with firm, clearly-marked walls, and trans- parent contents : these are the heterocysts. Observe cases of cells which have recently undergone division. Attempts may be made, by cultivating Nostoc in water, to observe the separation and further development of new masses by means of the hormogonia : these are short pieces of filaments which escape freely into the water ; after a period of movement these settle, form a gelatinous sheath, and grow into a new Nostoc mass. Compare the above observations with those on Oollema (see below) : it is to be noted that Nostoc and GG 2 452 PRACTICAL BOTANY Gollema are frequently to be found closely associated together, so that it is difficult to tell where the Alga ends and the Lichen begins. OSCILLATORIA Organisms belonging to this genus are to be found as dark blue-green, olive-green, or black coverings on damp walls where water is constantly trickling, on wet soil, &c. Mount a small quantity in water, and examine under a high power : they will appear as fine cylindrical, unbranched filaments, with very delicate cellwalls, and cell-contents of various blue, green, and olive tints. Note the septa dividing the whole into diskshaped cells : also the convex ends, and the constant oscillating movements, from which they derive their name. Staining with the usual reagents will disclose no nuclei, though minute granules may be stained. GLCEOCAPSA This organism may be found, usually associated with others, in those slimy masses which frequently grow on the inside of the glass in hot-houses. Examine it under the microscope, and note the numerous cells with pinkish cell-contents, and much swollen cell-walls. The cells will usually be associated in groups, which, from the course of the lines of stratification of the cell-walls, may be seen to have had a common origin. The slimy masses of Glceocapsa will rarely be found to consist of it alone; other organisms being almost always mixed with it. FUNGI I. BASIDIOMYCETES AGARICUS CAMPESTRIS, L. (The Common Mushroom) I. Examine a brick of " mushroom spawn," such as is sold in the shops for the artificial culture of the Mushroom (Agaricus campestris). It will be found to consist of a compost of dried cows'-dung, loam, and clay, in which numerous very fine microscopic filaments are present, or irregularly branched white bands which may be easily recognized with the naked eye : this is the mycelium. The best spawn is that in which the mycelium is generally distributed, so as to give the whole mass a uniformly grey appearance. It is to be remembered that the mycelium of other Fungi may, and most probably will, be present in greater or less amount. Tease out with needles in water some of the mycelium, mount in water, and examine under a low power : note that the white bands recognized with the naked eye are composed of numerous colourless filaments (mycelial hyphae), associated together in a parallel course, while 454 PRACTICAL BOTANY here and there single hyphse diverge from the rest, and ramify through the compost. II. In order to obtain an actively growing mycelium, bearing "mushrooms," the brick is to be broken into pieces, and these must be buried a few inches deep in a compost of similar nature to that of the bricks : the whole is to be kept moist, at a moderately high temperature, and in the dark : the stoke-hole of a con- servatory is well adapted for the cultivation of mush- rooms. After a period of a few weeks, the compost will be found to be permeated by a mycelium, similar to that in the brick of " spawn," while numerous mushrooms of various sizes will be found connected with it : such a culture as this will suffice for the study of Agaricus campestris in the laboratory. III. Eemove a small piece of the mycelium of an actively growing culture, mount it in water, and observe under a low power that it is similar in its main characteristics to that in the dormant state in the brick of spawn. Having teased it out carefully with needles, examine it in detail under a high power, and observe 1. The hyphae, of cylindrical form, and with rounded free ends. 2. The irregular branching of the hyphse. 3. The septa, which are transverse, and situated at irregular intervals. 4. Hypha3 may frequently be seen to be incrusted by numerous rod-like crystals : these are especially numerous in the dormant mycelium : it is to this, in great measure, that the mycelium owes its chalky white appearance. AGARICUS 455 By applying the ordinary tests, it may be shown that the incrustation consists of calcium oxalate. IV. Examine a portion of the mycelium which has begun to produce " mushrooms " : with a little care the compost may be entirely removed from considerable tracts of the branched mycelium, and then the relation of the latter to the young mushrooms may be clearly seen. If a series of specimens illustrating the develop- ment of the mushroom be examined with the naked eye, the following observations may be made 1. That the mushrooms arise from the mycelium itself. 2. That they appear first as irregular rounded, or oval, upward growths, of denser texture than the mycelium itself. 3. That on cutting one of the smaller mushrooms longitudinally it appears to the naked eye to be of homogeneous structure. 4. That older mushrooms acquire an enlarged head (the pileus), which is supported on a cylindrical stalk (the stipe). In this state they are termed " button mushrooms." 5. That as the pileus dilates horizontally, a rupture of a veil of tissue (velum partiale) about its lower margin exposes a complicated laminated structure formed internally (the gills, or hymenial lamellae). 6. Note further the ring or annulus, which remains persistent on the stipe of the mature mushroom, and marks the line of rupture of the velum : the corresponding, irregular fringe at the margin of the pileus is also to be recognized. 7. Removing the mature pileus, examine its lower 456 PRACTICAL BOTANY surface, and note the radiating, more or less darkly coloured lamellae,, some of which extend the whole way from the margin to the insertion of the stipe, others only a part of that distance. Lay the pileus of a mature mushroom with its lamellae downwards on a sheet of white paper for a few hours : on removing it there will be seen on the paper a sort of print of the configuration of the under surface of the pileus, produced by the fall of the minute, dark-coloured spores : the white lines of the print correspond to the spaces between the lamellae. This may be particularly well seen in the small Coprinus, which appears with great constancy on horse-dung after it has been kept at a moderate temperature under a bell-glass for three or four weeks. This fungus shows on a small scale a structure similar to Agaricus, and will serve well as a second type, and it is especially useful since it can be obtained at any time of year. V. In order to study the structure of the mushroom by means of sections, it is a great advantage to harden the material, and the following treatment has been found to produce good results : treat the fresh material for about twenty-four hours with 1 to *5 per cent, chromic acid wash ; with water, and then succes- sively with 50, 70, and 90 per cent, methylated spirit : the tissues will assume a cartilaginous charac- ter, which makes it possible to cut fine sections : in preparing large specimens it is an advantage to cut them up into pieces of moderate size, so that the reagents may gain more ready access to the internal parts. From material thus treated cut longitudinal sections AGAIUCUS 457 of the stipe so as to include both peripheral and central tissues : mount in glycerine, and examine first with a low power : observe 1. The whole is a spurious tissue, composed of elongated septate tubes (hyphae), which are closely interwoven. 2. The diameter of the individual hyphse is less, and they are more closely packed towards the periphery than near the centre of the section. Examining the sections under a high power it will further be observed 1. That the hyphse are branched, while occasionally their endings are to be seen. 2. That they are thin-walled, the transverse or oblique septa being so disposed that the cells are not much longer than broad. 3. Here and there are to be found hyphse with fewer septa, and highly refractive contents. 4. The protoplasmic contents of the hyphse which make up the bulk of the tissue are far from being copious, while no single, well-marked nucleus is to be found in the individual' cells. By careful staining it is possible to demonstrate the presence of numerous very small nuclei in. the protoplasm of the hyphse. If fresh material be used, the sections may be stained with methyl-green in 1 per cent, acetic acid, washed with 1 per cent, acetic acid, and mounted in weak glycerine and acetic acid if ; chromic acid material be used, it should be care- fully washed from the acid, and be stained with hsematoxylin. VI. Cut transverse sections of the stipe, and, treating as before, observe that the hyphse appear circular in section, that they are more loosely packed towards the 458 PRACTICAL BOTANY centre than at the periphery, and that throughout, intercellular spaces are to be recognized. Observe under a high power that where the section includes one of the septa (which will thus be seen in surface view), a central highly refractive spot is to be seen, which may also be recognized in the septa in longitudinal sections as a slight aggregation about the centre of the septum. VII. Passing to the pileus of the mature mushroom, cut tangential vertical sections through it in such a way as to traverse the vertical gills at right angles to their surface : great care must be taken that the surfaces of the gills shall not be injured in the process of preparation, otherwise the basidia and spores which project from their surfaces cannot be observed. Mount in glycerine and examine under a low power : the chief bulk of the section will consist of the massive tissues of the pileus, which show little or no differentiation ; passing downwards to the lower surface where the gills or lamellae have been traversed, the sections of these will be seen as fringe-like projections from the lower surface : occasionally branching of the gill may be recognized. Examine the sections in detail under a high power : the following observations are to be made 1. The mass of tissue of the pileus consists of a complicated plexus of much-branched hypha3, with large intervening spaces : it is composed of short cells, similar in their characteristics to those which compose the stipe : the chief difference lies in their arrangement. This spongy tissue becomes denser about the insertion of the lamellae. AGAIUCUS 459 2. The sections through the lamellae show a differ- entiation into a. The central portion (trama), in which the sep- tate hyphal filaments are easily recognized running longitudinally down the middle of each lamella, and curving outwards at their ends towards the free surface. &. The sub-hymen ial layer, composed of shorter, closely-packed cells, constituting a pseudo-parenchyma : it may, however, be recognized, and especially in sections of young mushrooms, that this pseudoparenchyma consists of the short-celled, terminal parts of the hyphal filaments which compose the trama. c. The hymenial layer, consisting of oblong, closely packed cells, having their longer axes perpendicular to the outer surface : of these cells two types are to be distinguished i. The paraphyses, which are somewhat narrower and have smooth rounded ends, ii. The basidia, which are more bulky, and longer : each bears on its end two fine processes (sterigmata) ; at the extreme tip of each of these there appears a swelling which develops into the mature spore. Note various stages of development of the sterigmata, and spores. VIII. Remove a whole gill carefully from a fresh mushroom, mount it on a slide, without any reagent or cover-slip, and examine its surface with a medium power : it may then be seen that the dark colour is due to the dusky spores, which are thickly distributed over the surface of the gill in pairs, two being pro- 460 PRACTICAL BOTANY duced from each basidium : note further the pale colour of the tissue of the hymenium, and the rounded ends of the paraphyses, and of those basidia which are young, or have already produced mature spores. Attempts should be made to germinate the spores in a decoction of horse- or cow-dung : a very small num- ber of the spores should with a sterilized needle be introduced into a drop of the decoction recently boiled, the drop having been placed in the centre of a cover- slip which had been recently heated. The cover-slip is then to be inverted and placed over a moist chamber constructed as described in Appendix A. The spores can be examined at intervals under the microscope, and the germination followed. A comparison may be made with Coprinus micaceus : this Fungus appears almost with certainty if cow-dung be kept for three or four weeks at a moderate temperature under a bell-glass : if sections be cut from the lamellae, the structure of the hymenium will appear similar to that above described, but the number of sterigmata on each basidium is four. On species of Coprinus large bladder-like cells (cystidia) are found projecting from the hymenial layer. Examine specimens of other Hymen omycetous Fungi, e.g. species of Polyporus, Dcedalea, Boletus, and Hydnum: note in them the difference of conformation of the thallus, and especially of the hymenial surface ; also the difference of texture : thus in Dcedalea quercina, and in some species of Polyporus, the thallus is hard, and of a corky or woody nature, while in others (e.g. Polyporus giganteus) it is soft and succulent. Sections should be cut from the thallus of Dcedalea quercina, or some other woody form : an examination of them will show that, though the thallus is hard, the structure of it is similar to that of the mushroom, the whole being composed of branched septate hyphse. AGARICUS 461 Sections should also be made through the hymenium of some species of Polyporus in such a way as to cut the pores transversely : examination of these will show the hymenial layer lining the pores, and consisting, as in the mushroom, of paraphyses and basidia, the latter producing sterigmata, and spores ; but in Polyporus the hymenial layer is less closely packed and regular. A comparison should also be made of some types of the Gastero- mycetes, e.g. Lycoperdon, Geaster, Crucibulum, and Phallus. In these forms it will be seen that there is considerable variety in detail of arrangement of the hyphse, resulting in a marked difference of external conformation of the mature Fungus. Still, sections of them in relatively young stages will show that they also are composed of branched and septate hyphse, while the mode of formation of the spores on the basidia of more or less regular internal hymenial layers, corresponds essentially to that already described for the mushroom. Dacryomyces may also be examined as an example of the Tremellini. It appears as gelatinous orange masses on rotting wood, especially of the