Usually it is not possible to identify the species of a bryophyte without a microscope. Characteristics like the form of the leaves, the structure of its lamina and costa, the organization of its stem or of a cross section of a leaf are impossible to ascertain without one. The nature of a transmitted light microscope makes some kind of preparation necessary in almost all cases. If one is only interested in determining a species, preparation should be quick and effortless, ruling out embedding techniques requiring several days or complicated staining protocols. This section gives a quick overview of methods useful in identifying bryophytes. For this usually only preparation of the specimen and sectioning of leaves and stems is necessary. Sometimes staining is needed.
Different methods require the specimen to be either wet or dry. Regardless, if it is very dirty the first step should be cleaning, to get clean preparations. Examples:
If wet material is needed, one can simply put desiccated material in a small bowl or cup with a little water. Most bryophytes will swell in little time. A little detergent can speed the process up [1, S. 5]. One should take note that only individual plants are needed [1, S. 5]. It is unnecessary to rehydrate entire turfs or cushions.
Foliose Marchantiophyta do not need further preparation, instead they are put under the microscope as a whole [1, S. 5].
To pluck leaves from a moss one needs two good tweezers. The gametophyte is held with one, the other is used to pluck individual leaves [1, S. 5]. One should try to pull the leaves of like a sticker so that the sometimes important cells at the base of the leaves are recovered. A stereo microscope is a great aid.
An alternative technique pulls the gametophyte through a half open tweezer, thus scraping the leaves of the stem [1, S. 5]. Personally, i found this technique to be lacking.
Classical methods used to obtain cross sections are of limited use for the purposes of identifying a specimen. Embedding in paraffin requires several days, hand cuts using Styrofoam, carrots or elder marrow are unreliable for leaf cross sections and cryomicrotomes are expensive and also aren’t that fast [1, S. 5].
Instead, a range of other techniques are used. The simplest one arranges some leaves on a hard surface like a slide and fixes them down with a finger. Using a sharp razor blade a first cut is made to establish a perpendicular edge, then the tilt of the razor is changed or it is pushed against the finger to make the cut for the cross section [1, S. 5]. Smith recommends a similar approach. The leaves are held down using a slide, the razor is rested against the upper edge of it. The thickness of the cross section is controlled by the angle of the razor. By progressively reducing the angle one can cut multiple cross sections one after the other [3, S. 5–7]. Both techniques require a stereo microscope.
A disadvantage of above methods is their high demand on motor skills. A small bump against the table, and one must start anew arranging the leaves. A better technique “embeds” the leaves in polyethylene glycol. PEG with a molecular weight around 1500 g/mol melts at similar temperatures to paraffin, but dissolves in water. A small amount of PEG is melted (e.g. using a light bulb) and dry leaves incorporated into it. After the PEG has solidified again, cross sections are cut using a razor and if possible a stereo microscope in the way described above [1, S. 6]. The sections are transferred to a slide using a scalpel, covered with a cover slip, then water is added. This reduces the chance that sections “fall over” [1, S. 6]. In my opinion this is the best technique of the ones described here, but one still needs quite some experience for it to work consistently.
If one wants cross sections of stems, classical hand sectioning techniques can be used successfully. It is described here using elder marrow, but materials like soap, paraffin, stearin [2, S. §74], styrofoam or carrots can be used as well. Using a sharp razor, a piece of marrow approximately 1,5 cm in length is cut axially. On both halves a bit of the marrow is removed to make space for the specimen. The assembly of specimen and marrow is then cut by hand or using a microtome [2, S. §74]. For further notes on embedding and sectioning see the appropriate chapters of the general treatment.
For identification staining is not routinely carried out. Peat mosses sometimes have taxonomically important pores in the cells of leaves and the hyalodermis, which without staining are very hard to see. In this case staining using methylene blue, brilliant cresyl blue, crystal violet, eosin is carried out. Concentration of the dye solution is more important than the type of dye used [1, S. 7]. To stain cross sections of leaves the solution is sucked under the cover slip using filter paper [1, S. 7]. A cheap alternative to buying pure dyes or ready-made staining solution is extracting dye from a ball pen with ethanol [1, S. 7]. A copying pencil dunked into the water drop with the cross sections works as well [1, S. 7].
In some taxons treatment of leaves with 2% KOH solution leads to different colours in cells of the lamina and costa. The stain is high in contrast, the produced colours range from brown over yellowish to reddish. The procedure is very easy, the gametophyte is simply soaked in a 2% KOH solution with a little detergent [1, S. 7].
[1] Frahm, J.-P. u. Müller, R.-D.: Moose unter dem Mikroskop. Archive of Bryology Special Volume (2013) 13
[2] Schömmer, F.: Kryptogamen-Praktikum. Anleitung zur mikroskopischen Untersuchung und Präparation der blütenlosen Pflanzen für Studierende und Liebhaber. Stuttgart: Kosmos Gesellschaft der Naturfreunde Franckh'sche Verlagshandlung Stuttgart 1949
[3] Smith, A. J. E.: The moss flora of Britain and Ireland. Cambridge, UK, New York: Cambridge University Press 2004