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 lima been agar slants stored in a refrigerator and transferred every three months.

 

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 lima been agar plate, stock plate B for further increase.  Incubate 7 days at room temperature (22° C) and ambient light.

 

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. Monticello, N.Y.

Mollicone,  M. R. N. 1999.Zoospore ultrstucture of Gonapodya polymorpha.Mycologia 91: 727-734.

Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd ed. Univ. Michigan Press, Ann Arbor, Michigan. 1187 pp.

 


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