THE ORDER MONOBLEPHARIDALES
MONOBLEPHARIS MACRANDRA (ISOLATE M# 42) IN THE CLASSROOM
Isolate M# 42 of Monoblepharis macrandra reproduces asexually and sexually in nutrient growth medium and can be used to demonstrate the following featreleaseures of Monoblepharidales: highly vacuolate, “foamy” appearance of the hyphal contents, development of asexual zoosporangia, and swimming of posteriorly uniflagellate zoospores, germination of encysted zoospores, antheridia and oogonia, release of antherozoids, fertilization of oospheres, release of oospore from oogonium, and mature oospores. In Monoblepharis macrandra, the antheridia develop separately from the oogonia. In young thalli the antheridia are often on separate branches, but on older thalli they develop on the same branch.
CAUTION - Use sterile technique at all times. Sterilize all glassware and media by autoclaving before use.
LONG TERM MAINTENANCE - Cultures are maintained on mPmTG agar slants stored in a refrigerator and transfered every three months.
PREPARATION TIME - Start stock plate A 35 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 in ambient light for 10 days at room temperature (~22E C) to increase stock. (Note: If desired for long term storage, 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 in ambient light for 7 days at room temperature (22E C).
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 small pieces and transfer the pieces into a 150 ml Erlenmeyer flask containing 50 ml of T/2 broth. Incubate in ambient light for 8 days at room temperature (22E C); shake gently, occasionally.
DAY 25 - Carefully pour the liquid from the flask and replace with sterile 1/3 PW. Keep in light at room temperature (22E C) overnight.
DAY26 - 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 bottoms of the dishes with a sterile transfer needle. Use a sterile pipette to transfer broth containing zoospores from the flask with 1/3 PW made day 25 to each Petri dish with cover slips. Incubate 1 Petri dish at room temperature and light for 8 days and place the other Petri dish in the dark at room temperature (~22E C) for 8 days.
DAY 34 - Remove the T/2 from the Petri dish kept in the light and replace with sterile 1/3 PW. Do not wash the Petri dish kept in the dark.
IN CLASS - DAY 35 - Use sterile forceps to remove individual cover slips, wipe the under sides dry and invert on clean microscope slides for observation. Thalli from the dish incubated in light should demonstrate all stages of zoospore production, and thalli from the dish incubated in the dark should demonstrate all stages of sexual reproduction.
SCHEDULE ADJUSTMENTS - This schedule can be adjusted by changing the incubation times of the agar plates. Times may vary from a minimum of 7 to a maximum of 10 days. The 8th day after inoculation is the most favorable for observation of the cover slips, but satisfactory observations should be possible from the 7th to the 9th 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
- 1/3 PW - Collect water from a quiet, weedy area of the nearest small pond and dilute, 1 part pond water to 2 parts distilled water. Sterilize in bottles in autoclave. Keep unsterilized water in refrigerator.
MATERIALS NEEDED - In addition to screwcap test tubes for agar slants (for long term maintenance if desired), you will need sterile plastic Petri dishes for agar plates, 150 ml Erlenmeyer flasks (screw caps preferred), glass Petri dishes with 5 cover slips in each, microscope slides, cover slips and bottles for sterile 1/3 PW..
REFERENCES - The following papers may provide useful information.
Karling, J. S. 1977. Chytridiomycetarum iconographia. Lubrecht & Cramer.
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
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 Publishing
Whisler, H. C. And Marek, L. E. 1987. Monoblepharis macrandra. P.54-55. In: Zoosporic
fungi in teaching and research. Eds. M.S. Fuller
and A. Jaworski. Southeastern
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