THE ORDER MONOBLEPHARIDALES
MONOBLEPHARIS POLYMORPHA ISOLATE M#45E IN THE CLASSROOM
Isolate M#45E of Monoblepharis polymorpha reproduces readily both sexually and asexually in nutrient growth medium and can be used to demonstrate the following features of Monoblephariceae: the highly vacuolate, “foamy” appearance of the hyphae, development of asexual sporangia, release and swimming of posteriorly flagellate zoospores, germination of encysted zoospores, antheridia and oogonia, release of antherozoids, fertilization of oospheres, release of oospore from oogonium, and mature oospores. In this species the antheridia typically develop on the oogonia. M#45E reproduces sexually in both light and dark conditions, but it is more copious in the dark.
CAUTION - Use sterile technique at all times. Sterilize all glassware and media by autoclaving before use.
LONG TERM MAINTENANCE - Cultures can be maintained on mPmTG agar slants stored in a refrigerator and transferred every three months.
PREPARATION TIME - Start “stock plate A” 27 days before scheduled class. Consider this Day 0.
DAY 0 - Transfer a piece of thallus from stock culture to an mPmTG agar plate, stock plate A, and incubate 10 days at 22E C in light to increase stock. (Note: It is advisable to make new stock culture slants at the same time.)
DAY 10 - Using stock plate A, with a sterile transfer needle cut the newly grown thallus into 8 or 10 slivers and transfer the slivers to a new mPmTG agar plate, stock plate B, for further increase. Incubate 7 days at room temperature (22E C) and light.
DAY 17 - Using stock plate B, with a sterile transfer needle move the slivers of old agar aside, cut the agar containing each new thallus into several pieces and transfer the pieces into a 250 mL Erlenmeyer flask containing 50 ml of T/2 broth. Incubate 5 days at room temperature (22E C) and light, shaking gently occasionally. Check for the presence of zoospores by holding the flask
toward a light source and examining the thallus with a 10-14 power hand lens, or use a sterile Pasteur pipette to withdraw a drop of broth for microscopic examination. The zoospores produced by M#45E grown on agar plates tend to germinate while still in the sporangium. Swimming zoospores may not be evident. Regular shaking of the flasks during growth may loosen some zoospores.
DAY 22 - Pour 50 ml of T/2 broth into each of 2 sterilized glass Petri dishes containing cover slips. If necessary, redistribute the cover slips evenly over the bottom of the dish with a sterile transfer needle. Use a sterile Pasteur pipette to transfer 4-5 ml of broth containing zoospores from the flask inoculated on Day 17 to each of the 2 Petri dishes. Since the zoospores often germinate in the sporangium swimming zoospores may not be evident; consequently, it is advisable to include a few pieces of cut up agar colony in each Petri dish of cover slips. Incubate 1 Petri dish at room temperature (22E C) and light for 5 days. Place one Petri dish inside an envelope of heavy-duty aluminum foil and incubate at room temperature (22E C) for 5 days.
DAY 26 - To assure that students can see asexual reproduction remove at least 1 Petri plate of cover slips from the dark and leave it in the light over night. A crop of sporangia will form among the sexually reproducing thalli.
IN CLASS - DAY 27 - Use sterile forceps to remove an individual cover slip, wipe the under side of the cover slip dry, and invert the cover slip over a small drop of distilled water on a clean microscope slide for observation. Thalli from the dish incubated in light should demonstrate all stages of zoospore production but may be overwhelmed by the copious sexual reproduction of
this isolate. Thalli from the dish incubated in darkness should demonstrate all stages of sexual reproduction.
SCHEDULE ADJUSTMENTS - This schedule can be adjusted by changing the incubation time of the agar plates. Times may vary from a minimum of 7 to a maximum of 10 days. The 5th day after inoculation is the most favorable for observation of the cover slips, but satisfactory observations should be possible from the 4th to the 7th day if care is taken with sterile technique
and the plates remain uncontaminated.
RECIPES -mPmTG agar for plates and slants.
0.4 g peptonized milk
0.4 g tryptone
2 g glucose
10 g agar for plates, 12 g for slants
1000 ml distilled water
- T/2 broth for flasks and Petri dishes with cover slips
5 g tryptone
1000 ml distilled water
MATERIALS NEEDED - In addition to screw cap test tubes for agar slants (for long term maintenance, if desired), you will need for each 5-8 students: 2 sterile plastic or glass Petri dishes for agar plates, three 150 ml Erlenmeyer flasks (screw cap preferred), 2 glass Petri dishes for cover slip cultures, 16 glass cover slips, thickness 1 ˝, and heavy duty aluminum foil.
REFERENCES - The following papers may provide useful information.
Karling, J. S. 1977. Chytridiomycetarum iconographia. Lubrecht & Cramer. Monticello, N. Y.
Marek, L. E. 1984. Light affects in vitro development of gametangia and sporangia of Monoblepharis macrandra (Chytridiomycetes, Monoblepharidales). Mycologia 76: 420-425.
Perrott, P. E. T. 1955. The genus Monoblepharis. Trans.Brit. Mycol. Soc.38: 247-282.
Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd ed. Univ. Michigan Press, Ann Arbor, Michigan. 1187 pp.
Whisler, H. C. 1978. Monoblepharis polymorpha. P. 57. In : Lower fungi in the laboratory. Ed., M. S. Fuller. Department of Botany, University of Georgia, Athens, Georgia.
Whisler, H.C. 1987. On the isolation and
culture of water molds: the Blastocladiales and Monoblepharidales. P. 121--124 in:
Fuller, M. S. And A. Jaworski,
fungi in teaching and research. Southeastern
Whisler, H. C. and Marek, L. E. 1987. Monoblepharis macrandra. P.54-5. In: Zoosporic Fungi in Teaching and Research. Eds. M. S. Fuller and A. Jaworski. Southeastern Publishing Corporation. Athens, Georgia.
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