What are ascomycetes?

This group, popularly known as the "sac fungi," contains an immense number of saprophytic and parasitic forms. The green mold on cheese and leather, the leaf curl of peach, the black knot of cherry and plum, and the powdery mildews are familiar to everyone. The few objects selected will enable the student to experiment, but he must not be discouraged if success does not crown the first attempt, for some members of the group present real difficulties.

Peziza - The Pezizas and related forms are fleshy, and present but little difficulty in fixing, cutting, or staining. They are abundant in moist places, on decaying wood, or on the ground. The apothecia have the form of little cups, which are sometimes black and sometimes fleshcolored, but often orange, red, or green.

For general morphological work it is better to tease out fresh or preserved material. Sections showing the entire ascus should be 10 to 15( in thickness.

For the free nuclear division in the ascus, and also for the development of the ascospores, Flemming's weaker solution, followed by the safranin, gentian-violet, orange combination has given excellent results. The centrosomes, especially at the first division in the ascus, are sharply defined and the radiations are conspicuous. With iron-alum haematoxylin and orange, the nuclear detail and the centrosomes are better, but the spindle and radiations are not so sharply defined. For such details, sections should not be thicker than 5(, and 3( will give a clearer view.

Morchella esculenta is very good for the development of the ascus because the nuclei are very large.

For showing the ascogonium, ascogenous hyphae, and the origin of the asci, nothing is better than Pyronema. Fix in formalin, acetic acid (10 c.c. formalin, 5 c.c. acetic acid, and 85 c.c. water) for 24 hours or more; wash in water and stain in eosin. Or, fix in the special chromo-acetic-osmic solution and stain in iron-alum haematoxylin. In either case, use the Venetian turpentine method and tease the material so as to obtain instructive views.

Eurotium - Eurotium with its conidial stage, Aspergillus, is a very common mold found on bread, cheese, decayed and preserved fruit, etc. In the conidial stage it is green and in the ascosporic stage yellow, reddish yellow, or reddish brown. Aspergillus is almost sure to appear upon bread which is kept moderately moist, because the conidia are usually abundant in the atmosphere. If the bread be wet with a 10 per cent solution of cane-sugar or with grape juice, this stage appears sooner and in greater abundance. A temperature of 22 to 30 C. is also a favorable condition.

The perithecial stage is not found so frequently, but can sometimes be secured by examining moldy preserves. The sexual stage has been induced. Soak a piece of bread in a 20 per cent solution of grape-sugar in grape juice; upon this sow the spores and keep at a temperature of about 28 C. After 4 or 5 days, begin to examine. A 40 per cent solution of cane-sugar in the juice of prunes is also a good nutrient solution.

For class use or for permanent preparations it is best to select rather young material which shows various stages in development, from the swollen end of the hypha to the ripe spore.

Fix in 1 per cent chromo-acetic acid (1 g. chromic acid and 1 c.c. acetic acid and 100 c.c. water) for 24 hours; wash in water 24 hours, stain sharply in eosin, transfer to 10 per cent glycerin, and follow the Venetian turpentine method.

Material may be fixed in corrosive sublimate-acetic acid (corrosive sublimate 2 g., glacial acetic acid 2 c.c., and water 100). Use it hot (85 C.). One minute is long enough. Wash in water and add, a few drops at a time, the iodine solution used in testing for starch. At first, the brownish color caused by the iodine will disappear, but after a certain amount has been added the brownish color will remain. Stain in eosin or iron-haematoxylin and follow the Venetian turpentine method.

A very rapid method for this and for similar small filamentous forms may be added. Forms as large as Thamnidium elegans can be mounted successfully by this method.

1. 100 per cent alcohol, 2 minutes. 2. Eosin (aqueous), 2 minutes. 3. 1 per cent acetic acid, 2 to 10 seconds. 4. Mount directly in 50 per cent glycerin and seal.

If the material gets through the first three stages without shrinking but collapses at the fourth, put it into 10 per cent glycerin and allow it to thicken, following the Venetian turpentine method.

The earlier perithecial stages are more instructive when mounted whole; but later stages, even before the formation of the asci, are very unsatisfactory by this method, and should be cut in paraffin.

Penicillium - This green mold is found everywhere upon decaying fruit, upon bread, and upon almost any decaying organic substance. Material is even more easily secured than in case of Aspergillus, and Penicillium is an easier type for laboratory study. Such a satisfactory study can be made from the living material that it is hardly worth while to fix and stain. The very rapid method described for Aspergillus will furnish good mounts if permanent preparations are desired.

The Erysipheae - The mildews are found throughout the summer and autumn on the leaves of various plants. Some of the most abundant forms are Microsphaera aim on the common lilac; Sphaerotheca castagnei on Bidens frondosa and other species, on Erechtites hieracifolia, and on Taraxacum officinale; Uncinula necator on Ampelopsis quinquefolia, and U. salicis on Salix and Populus;

Erysiphe commune on Polygonum aviculare; and Erysiphe cichoriacearum on numerous Compositae and Verbenaceae. Podosphaera may be found on the leaves of young cherry trees and apple trees, and on young shoots of older trees. The infected leaves are likely to be more or less deformed. Phyllactinia is sometimes abundant on leaves of Alnus incana. It is also found on Celastrus, Desmodium, Typha, and on various members of the Amentiferae. For herbarium purposes they may be preserved by simply drying the leaves under light pressure. When needed for examination the leaf should be soaked in water for a few minutes, after which the perithecia may be scraped off and mounted in water. In mounting great care must be taken not to break off the appendages. The asci may be forced out by tapping smartly on the cover.

For permanent mounts of entire perithecia with appendages, fix in 5 per cent formalin 24 hours, wash in water 1 hour, stain in aqueous eosin 24 hours, remembering to keep all solutions slightly acid. Use the Venetian turpentine method. If chromic acid, corrosive sublimate, or alcohol be used for fixing, the appendages become brittle and very easily break off. However, the chromo-acetic mixtures are better if it is desired to make paraffin sections showing the developing of the perithecium with its asci and spores. For this purpose the omnipresent Erysiphe commune on Polygonum aviculare is exceptionally favorable, because, after the material has been fixed and has been brought into alcohol, the whole mycelium, with the developing perithecia, may be stripped from the leaf without the slightest difficulty, thus avoiding the necessity of cutting the leaf in order to get the fungus. Material in which the perithecia are still white or yellowish contain stages up to the formation of the uninucleate ascus; brownish perithecia show the development of ascospores, and darkbrown or black perithecia contain the mature asci with fully developed ascospores. In early stages while the perithecia are still yellow or very slightly brownish, the material can be stripped off from the leaves before fixing. An air-pump will remove any air. Use iron-alum haematoxylin and orange, or the safranin, gentian-violet, orange combination. Sections thicker than 5( will be hard to stain effectively.

The Xylariaceae.-Most of these forms, in their mature condition, are black. In younger stages the color is lighter, often showing gray, brick-red, or brownish tints. Nummularia is common on dead branches of beech, elm, oak, locust, and other trees. It is generally flat, orbicular, or elliptical in form. Ustilina is a crustaceous form, rather diffuse and irregular in shape. It is most common on the roots of rotten stumps. Hypoxylon is more or less globose in form, and the color is brick-red, brown, or black. It is found on dead twigs and bark of various trees, especially beech, and is more abundant in moist situations. Xylaria is found on decaying stumps and logs, and often apparently on the ground, but really growing on twigs, wood, and bark just under the surface. When mature it is black outside and white or light-colored within. When young, it is easily cut in paraffin; in some forms the ascospores are fully formed before the stroma becomes hard enough to occasion any difficulty in cutting. When the stroma becomes black, many members of the Xylariaceae become very hard and brittle, so that sections are likely to be unsatisfactory. For general morphological study it is better to break the stroma transversely and examine with the naked eye and with a pocket lens. The asci with their spores can be teased out and mounted in water. For permanent preparations, soak the stroma for a month in equal parts of 95 per cent alcohol and glycerin; then cut sections, and, after leaving them in glycerin for a day or two, mount in glycerin jelly. It is better not to stain the old stages. For illustrative purposes, select forms which can be cut in paraffin. The method just given merely shows that such material can be cut.

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