THE ORDER MONOBLEPHARIDALES
GONAPODYA POLYMORPHA IN THE CLASSROOM
Isolate M# 36 of Gonapodya polymorpha
grows readily on lima bean agar and in liquid lima bean medium and illustrates
the following features of the genus: subumbellate
branching of the pseudoseptate mycelium, scalariform hyphal contents,
proliferation of sporangia and gametangia, oogonia containing 1—many oospheres,
which leave the oogonium propelled by the antheridial flagellum and form oospores
in the medium. Reproduction of G. polymorpha
seldom occurs in sterile conditions but when thalli are placed in unsterilized pond
water asexual and sexual reproduction result.
Species characters: G. polymorpha
has relatively robust hyphae, which may be 200—1000 µm
long when grown on seeds. This species
has fewer pseudoseptations than G. prolifera. The proliferating sporangia with rounded
bases and short, blunt tips occur in sub-umbellate clusters. The narrower antheridia and round oogonia occur together in small clusters. Oogonia contain 1—several
gametes and usually have more than one opening.
Female gametes usually leave the oogonium before
being fertilized and are propelled some distance by
the antheridial flagella before the zygotes settle. Mature oospores form
in the medium.
CAUTION - Use sterile technique at all times. Sterilize all glassware and media by autoclaving before
use.
LONG TERM MAINTENANCE - Cultures can be maintained on
PREPARATION
TIME - Start “stock plate A” 25 days
before scheduled class. Consider this Day 0.
DAY 0 - Transfer a piece of thallus
from stock culture to a lima been agar plate, stock plate A, and incubate 10
days at room temperature (22° C) and ambient light 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 - 10 slivers and
transfer the slivers to a new
DAY 17 - Using stock plate B, with a sterile transfer needle
move the slivers of old agar aside, cut agar containing each new thallus into small pieces and transfer the pieces into a 150
ml Erlenmeyer flask containing 50 mL of LB
broth. Incubate 5 days at room
temperature (22° C) and ambient lght; shake gently occasionally.
DAY 22 - Wash the thallus by
pouring it into a sterile wire basket and placing the basket in a Petri dish of
sterile 1/3 pond water (PW) for 10 minutes.
With sterile forceps lift the wire basket, empty the Petri dish, refill
with sterile 1/3 PW and replace the wire basket. Let stand in light.
DAY 24 - Using sterile technique check for presence of
zoospores in the liquid or for zoosporangia forming on the thallus. If zoospores are found,
zoospore production will probably continue until needed for class. If no zoospores or zoosporangia are found,
empty the wire baskets into Petri plates containing unsterilized
1/3 PW. To observe, place an agar block
on a microscope slide and cut the growth that extends beyond the edge of the
block with a razor blade, move block aside and apply cover slip to the cut
growth.
DAY 25 - Check thalli daily for
production of zoosporangia and gametangia that will
be present in 1—3 days. When unsterilized
PW is used, thalli may disintegrate quickly.
SCHEDULE ADJUSTMENTS - This schedule can be adjusted
by changing the incubation times of the agar plates. After the thallus
is washed the time in sterile PW and in unsterilized PW is adjustable.
RECIPES - Lima Bean agar is available from scientific supply
companies but if exactness of ingredient is not important
it can be made in the lab more cheaply.
Lima Bean agar
1000 mL distilled water
1 10 oz package frozen lima beans from the grocery
store
10 g agar for plates, 12 for slants
Boil
the beans in the distilled water for ˝ hour.
Discard the beans and filter the liquid to remove as much of the
resulting fibrous material as possible.
Replace the evaporated distilled water, add the agar and mix well before
and after autoclaving.
Lima bean liquid medium
1000 mL distilled water
1 10 oz package frozen lima beans from the grocery
store
Boil
the lima beans in the distilled water for ˝ hour. Discard the beans and filter the liquid to
remove as much of the resulting fibrous material as possible.
Replace the evaporated distilled water, mix well before and after
autoclaving, distribute to flasks and autoclave again.
T/2 broth for flasks and Petri dishes with
cover slips (Fuller and Jaworski, 1987)
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 the autoclave.
Keep unsterilized
water in refrigerator.
MATERIALS NEEDED - In addition to screw cap test tubes for agar slants
for long term storage if desired, you will need sufficient supplies of sterile plastic Petri dishes for agar plates, 150 ml
Erlenmeyer flasks (screw cap preferred), glass Petri dishes with wire
baskets for washing thalli,
microscope slides and cover slips.
REFERENCES -
Johns, Robert M. and R.K.
Benjamin. 1954. Sexual reproduction in Gonapodya. Mycologia 46: 201—208.
Karling, J. S. 1977.Chytridiomycetarum
iconographia. Lubrecht & Cramer.
Mollicone,
M. R. N. 1999.Zoospore ultrstucture
of Gonapodya polymorpha.Mycologia 91: 727-734.
Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd
ed. Univ.
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