In general, the mosses are more conspicuous than the liverworts and easier to collect. Many of the most desirable forms fruit only in the spring, but something can be found throughout the summer and autumn and some, like species of Sphagnum, pass the winter with the antheridia and archegonia in advanced stages of development.
Material is more troublesome to fix than in the liverworts because small bubbles of air hinder penetration of the fixing fluid. Use an air-pump. Older archegonia and capsules which have turned brownish add to the difficulties of the technic.
The special chromo-acetic-osmic-acid solution, or this solution without the osmic acid, fixes well. If an air-pump is not available at the time of fixing, Land's formalin alcohol solution (6 c.c. commercial formalin to 100 c.c. of 70 per cent alcohol) will be more satisfactory.
Protonema - Protonema of some moss can always be found at any season. Look for greenish patches resembling Vaucheria. Such mats show the developing protonema and young leafy plants. Very young mats of moss will also show good protonema, but are not likely to show young buds. The brownish bulbils, which are quite common in mosses, can be seen with a good pocket lens. The little Webera, almost always found on the pots in the fernery or on the benches in greenhouses, quite frequently shows this mode of reproduction. Protonema is easily grown from spores.
Permanent mounts are very easily made. Simply wash away the dirt with water and put the material into 50 per cent glycerin, and let the glycerin concentrate. Mount in glycerin or glycerin jelly for permanent mounts. Seal thoroughly. Such mounts, with no fixing or staining, may retain the green color for many years.
If you do not insist upon keeping the green color, much clearer mounts can be made by fixing in formalin acetic acid, about 10 c.c. of formalin and 5 c.c. of acetic acid to 100 c.c. of water, and staining in eosin, or in Magdala red and anilin blue. Mount in Venetian turpentine.
Antheridia - It is easy to find material for a study of antheridia, because, in so many cases, the antheridial plants can be detected at once without even a pocket lens. Funaria, with its bunch of antheridia as large as a pinhead, is extremely common everywhere. Spring is the best time to collect it, but it is found fruiting in the autumn and sometimes in summer; besides, it is easily kept in the greenhouse, where it may fruit at any time. Bryum roseum has a large cluster of antheridia surrounded by radiating leaves, making it easy to recognize. Other species of Bryum and species of Mnium, like M. cuspidatum, make good sections. Polytrichum has a large cluster of antheridia surrounded by reddish leaves, so that the whole is sometimes called the moss "flower." In fixing this or the closely related Atrichum (Catharinea), cut a small slab from two sides, so as to leave a flat piece to cut for longitudinal sections. This trimming will greatly facilitate fixing and infiltration. A single antheridial plant of Polytrichum often furnishes a fairly complete series of stages in the development of antheridia. Transverse sections show not only the antheridia, but also good views of the peculiar leaf of this genus. In all cases the stem should be cut off close up to the antheridia, for many of the moss stems, after they have begun to change color, cut like wire.
Sections to show the development of the antheridium should be 5 to 10( in thickness. The safranin, gentian-violet, orange is a good combination. For details of spermatogenesis, sections should not be thicker than 3(. Iron-haematoxylin is a better stain for the chromatin and blepharoplasts.
Although sections 20 to 50 cc in thickness can be cut to show topography, it is far better to study such stages in the fresh material. When a particularly fine view is secured in this way, a permanent preparation may be made by putting the piece into 10 per cent glycerin without any fixing or staining, and allowing the glycerin to concentrate. Then mount in glycerin jelly.
Archegonia - Since the necks of the archegonia are usually long and more or less curved, it is necessary, for habit work, to cut sections as thick as 20 or 30( in order to get a view of an archegonium in a single section (Fig. 73B). Mayor's albumen fixative is not likely to hold such sections to the slide. Use Land's fixative. Here, as in case of antheridia, it is better to use fresh material, putting particularly good pieces into 10 per cent glycerin for glycerin jelly mounts.
For the development of the archegonium, trim away the leaves which usually cover the cluster. Fix in chromo-acetic acid with a little osmic acid and cut 5 to 10( thick. For a study of the ventralcanal cell and fertilization, sections should not be thicker than 5(. There is a general impression that the antheridia and archegonia of Sphagnum are rare and hard to find. Dr. George Bryan, who made an extensive study of Spagnum subsecundum, found that antheridia appear in August and archegonia in September. In examining acres of this species, he did not find a sterile plant.1
Sporophyte - It is often difficult to get good mounts of sporophytes. In the younger stages the calyptras are likely to interfere with cutting, while in the older stages the peristome, or hard wall of the capsule, occasions the trouble. If an attempt is made to remove the calyptra in young stages, the apex of the sporophyte usually comes with it. While picro-acetic acid material cuts more easily, chromo-acetic acid followed by Delafield's haematoxylin gives so much sharper differentiation in stages.
Later stages, after the peristome has begun to differentiate, are likely to occasion difficulty in cutting. Bryum cuts as easily as any (Fig. 76). For the development of the peristome, fix in formalin alcohol and stain in safranin and anilin blue, or in safranin and light green. Safranin and Delafield's haematoxylin is also an excellent stain for the older stages in the differentiation of the capsule.
Beautiful mounts of the peristome are easily and quickly made. Take a capsule at the stage when the operculum is just ready to fall off, or has just fallen off; with a sharp razor cut off the end of the capsule just below the line of the annulus; put it into absolute alcohol for 10 minutes, clear in clove oil, transfer to xylol, and mount in balsam. If several are placed on a slide, some one side up and some the other, with some complete and some teased a little, there will be good views of the entire peristome and also good views of teeth and cilia.
The mature sporophytes of Sphagnum are exceptionally hard to cut. It will be worth while to prick the capsule with a needle when the material is collected. This will allow the fixing agent to penetrate readily, and will also facilitate the infiltration of paraffin or celloidin. The puncture causes only a slight damage, and need not reach the really valuable portion which is to furnish the median longitudinal sections.
The younger stages in the sporophyte of Sphagnum, and also the antheridia, archegonia, and the peculiar development of the leaves cut easily in paraffin.
Return to Methods in Plant Histology