THE ORDER MONOBLEPHARIDALES
GONAPODYA PROLIFERA IN THE CLASSROOM
Isolate
M#6 of Gonapodya prolifera grows
on mPmTg agar and demonstrates the following features
of the genus: subumbellate branching of the pseudoseptate mycelium, scalariform
hyphal contents, proliferation of sporangia and gametangia, oogonia containing one
to many oospheres (female gametes) that leave the oogonium propelled by the antheridial
flagella and form oospores in the medium. Hyphae of G. prolifera
are narrower than those of G. polymorpha and may be 200—500 µm
long when grown on seeds. They are more
regularly pseudoseptate than G. polymorpha. Older hyphae are
often misshapen and distorted with uneven swellings.
The
available isolates of G. prolifera seldom form reproductive structures but, if
they do, look for the following features of the species: siliquiform
zoosporangia with long drawn out tips that proliferate freely, antheridia and oogonia that occur together in subumbellate
clusters and both proliferate freely. Clusters of gametangia and zoosporangia are subtended by cantenulate swellings separated by pseudoseptations. The narrow siliquiform
antheridia are smaller than zoosporangia and contain numerous small antherozoids. The oogonia are wide at the basal end and contain several large
oospheres. Zygotes and mature oospores
may form inside the oogonium, but usually the female
gametes emerge and are fertilized outside the oogonium. The antheridial flagella propells the
zygotes away and mature oospores form in the medium
some distance from the point of origin. A
hyaline membrane surrounds the round oospores.
CAUTION - Use sterile technique at all times. Sterilize all glassware and media by
autoclaving before use.
LONGTERM
MAINTENANCE – If desired, cultures can be maintained on mPmTG
agar slants stored in a refrigerator and transferred every three months.
PREPARATION TIME - Start “stock plate A” 23 days before scheduled class. Consider this
Day 0.
DAY
0 - Transfer a piece of the stock
culture to an mPmTG agar plate, stock plate A, and
incubate 7 days at room temperature (22E C) to increase stock.
(Note: If wishing to maintain this isolate in culture, it is advisable
to make new stock culture slants at the same time.)
DAY
7 - Using stock plate A, with a
sterile transfer needle cut the newly grown thallus
into small slivers and transfer the slivers to 2 new mPmTG
agar plates, stock plates B, for further increase. Incubate
7 days at room temperature (22E C), one in light and one in dark.
Day
14 - Using stock plates B, with a
sterile transfer needle move the pieces of agar aside, cut the agar containing
each new thallus into small pieces and transfer the
pieces into 150 mL Erlenmeyer flasks containing 50 mL of T/2 broth. Incubate
5 days, one in light, one in dark, checking daily for the presence of
spores. If spores are
found, immediately inoculate Petri plates with cover slips in T/2 broth
and incubate both light and dark for 5 to 7 days for class.
DAY
19 - If spores were not found
earlier, wash the thalli by pouring the contents of
the flasks into sterile wire baskets and place the baskets in Petri dishes of
sterile 1/3 PW for 10 minutes. With
sterile forceps lift the wire baskets, empty the Petri dishes, refill with sterile
1/3 PW and replace the wire baskets. Let
stand in both light and dark.
DAY
20 - Check for any response. It will probably happen quickly if at all.
DAY
22 - If no response is found, remove
the sterile 1/3 PW and replace with unsterile 1/3 PW.
DAY
23 - Check the thalli
daily for production of zoosporangia or gametangia.
SCHEDULE
ADJUSTMENTS - Incubation time on agar
plates is adjustable.
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. Autoclave
1/3 pond water in screw-capped bottles
Keep
unsterilized pond water in refrigerator
MATERIALS
NEEDED - In addition to screw cap
test tubes for agar slants (for long term maintenance), you will need sterile
plastic Petri dishes for agar plates, 150 mLErlenmeyer
flasks (screw caps preferred), glass Petri dishes and wire baskets for washing
cultures, glass Petri dishes for cover slip cultures, microscope slides and
cover slips..
REFERENCES - The following papers may provide useful
information.
Johns,
Robert M. and R.K. Benjamin. 1954. Sexual reproduction
in Gonapodya. Mycologia 46: 201—208.
Karling, J. S. 1977. Chytridiomycetarum iconographia. Lubrecht & Cramer.
Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd ed. Univ.
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