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,
Eds. Zoosporic
fungi in teaching and research. Southeastern
Publishing Corporation.
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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