In recent years such progress has been made in culture methods for fungi, especially for those which cause disease, that only the trained pathologist could be expected to know just what is the best medium for each particular fungus. But a student, not trained in pathology, may secure material for preparations. Professor Kleb's methods make it possible to secure material of many forms in various phases of their life-histories.
In general, filamentous fungi are treated like the filamentous algae, while the fleshy forms are cut in paraffin.
Rhizopus (Mucor).-This familiar mold appears with great regularity on bread. The following is a sure and rapid method for obtaining Mucor: Place a glass tumbler in a plate of water, put on the tumbler a slice of bread which has been exposed to the air for a day, and cover with a glass jar. The bread must not become too wet. To obtain a series of stages in the development of the sporangium it is better to use living material. For class work, time the cultures so as to have a plenty of sporangia which have not yet begun to turn brown.
For permanent preparations, fix for at least 24 hours in 10 per cent formalin; wash 1 hour in water, and then follow the Venetian turpentine method. Eosin gives good results. Stain over night or 24 hours, treat with 2 per cent acetic acid, changing several times, and then put into glycerin, merely pouring off the 2 per cent acetic acid and not rinsing the acid out in water. When washing out the glycerin, do it with alcohol which has about 0.5 c.c. of acetic to the 100 c.c. of alcohol, and leave 1 or 2 c.c. of this slightly acid absolute alcohol on the material when you add the 10 per cent Venetian turpentine.
For permanent preparations, fix for at least 24 hours in the special chromo-acetic-osmic solution; wash 24 hours in water and follow the Venetian turpentine method. Eosin gives good results for general topography. Stain over night or 24 hours, treat with 2 per cent acetic acid in water for 5 or 10 minutes, changing several times, and then put the material into 10 per cent glycerin, merely pouring off the acid but not washing it out with water. Wash out the glycerin with alcohol containing about 0.5 c.c. acetic acid to 100 c.c. of the 95 and 100 per cent alcohol. Leave about 1 c.c. of the slightly acid alcohol on the material as you add the 10 per cent Venetian turpentine.
To bring out the nuclei, use iron-alum haematoxylin. Rhizopus stains very rapidly, so that an hour in iron-alum and an hour in the haematoxylin may be long enough. The sporangia stain more readily than the mycelium; consequently to show the coenocytic character of the mycelium, the action of the second iron-alum must be stopped earlier than when staining for the sporangium. Extract the stain until the nuclei of the mycelium show clearly, and then remove part of the material to wash in water. For the rest of the material, continue to extract the stain until the sporangia are satisfactory. Mount some of each lot on each slide.
The finer details of the sporangium can be seen only in thin sections. Rhizopus is the most easily obtained material for showing the progressive cleavage of protoplasm by vacuoles.
The zygosporic stage in the life-history is rarely met in nature or in cultures, but when once secured it may be propagated indefinitely. We have a culture which has been furnishing illustrative material for nearly twenty years. Once in a while, when a particularly good culture appears, lay aside some of it to start the next culture. The best series of stages generally appears between the fourth and seventh days.
While Rhizopus may be grown on bread, it is better to use culture media in Petri dishes. While it grows well on agar media, it is hard to pick it off; and on liquid media, the growth is abnormal. Mrs. Alice A. Bailey has devised a method which is ideal for securing material. She puts about the usual amount of a potato dextrose agar in a Petri dish and pours over it a potato decoction about 2 mm. deep. The + and - strains are then added. The potato decoction is made as follows: Use 300 g. of Irish potatoes and 1000 c.c. distilled water. Peel and slice the potatoes and boil for 1 hour in the distilled water. Strain off the liquid through cheese-cloth and make up to the original quantity by adding distilled water. Flask and sterilize.
The potato dextrose agar is made as follows: to 1,000 c.c. of potato decoction, add 20 g. of dextrose and 30 g. of agar. Boil 1/2 hour in a double boiler. Strain, flask, and sterilize.
The potato dextrose agar is an excellent medium and the thin layer of potato decoction keeps the material from sticking to the agar so that it can be lifted off intact. Rinse it under the tap and fix in the chromo-acetic-osmic solution.
Zygospores may begin to form within 3 days, and mature zygospore may appear within 4 or 5 days, usually at a lower level than the sporangia. Watch the cultures and fix so as to secure a series of stages.
Paraffin sections should not be thicker than 5(, and 3( is better for nuclear detail. Iron-alum haematoxylin is best for nuclei, but safranin, gentian-violet, orange will give beautiful preparations.
In the related genus, Sporodinia, which is rather common in summer on fleshy fungi, especially upon Boletus and its allies, the zygosporic condition is not infrequent, because Sporodinia does not have + and - strains. Rhizopus behaves like a dioecious plant, while Sporodinia behaves like a monoecious one. The very damp atmosphere and the nutrition necessary for the formation of zygospores may be provided in the laboratory in the following way: Put a little water in a glass battery jar and place filter paper around the inside of the jar so that it will take up water and thus keep the sides of the jar moist. Place a small beaker or dish, without any water in it, in the bottom of the jar, and in the beaker place a small piece of bread dampened with the juice of prunes. Infect the bread with spores, or use a piece of bread upon which mycelium is already growing. Sections of the root of Daucus carota may be used instead of the bread. Put a piece of wet filter paper on a pane of glass and cover the jar. Begin to examine after 24 hours. The zygospores may appear in 4 or 5 days.
Zygorhynchus is another interesting relative of Rhizopus, readily distinguished by having suspensors of very unequal size.
Saprolegnia - This is an aquatic mold, very common upon insects and algae. Cultures are easily and quickly made. Bring in a quart of water from any stagnant pond or ditch, and into the water throw a few flies. After 12 to 24 hours throw the water away, rinse the flies in clean water, and put then into tap water. The water must be changed every day to keep bacteria from ruining the culture. The larvae of ants or small pieces of boiled white of egg are sometimes better than flies. Sporangia may appear within 24 hours but may be a day later. Sporangia may be produced in the greatest abundance by cultivating the mycelium for several days and then transferring it to pure water or to distilled water. As long as the nutrient solution is sufficiently strong and fresh, only sterile mycelium will be produced.
To secure oosporic material, mycelium which has been highly nourished for several days in a nutrient solution is brought into a 0.1 per cent solution of leucin, or into a 0.05 to 0.1 per cent solution of haemoglobin. Begin to examine after 24 hours.
Oogonia have been produced in great numbers by the following method: cut ordinary corn (Zea Mais) into small pieces and boil for 20 minutes. When cool, put pieces into a Petri dish and add enough pond water to nearly cover the pieces of corn. Oogonia may appear within 3 or 4 days.
For fixing, the following formula is excellent for material which is to be mounted whole:
Formalin..................10 c.c. Glacial acetic acid........5 c.c. Water.....................85 c.c.
Fix at least 24 hours, but material may be left for months in this fixing agent.
Stain some in Magdala red and anilin blue, and some in iron-alum haematoxylin. Mount some of each lot on each slide (Fig. 54).
For sections, it is better to fix in the special chromo-acetic-osmic-acid solution.
Satisfactory material for general laboratory, purposes can be secured as just described. Absolutely pure cultures can be secured only by observing all the precautions necessary in bacteriological work.
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