THE ORDER MONOBLEPHARIDALES
PREPARATION OF MONOBLEPHARELLA SPP. FOR USE IN THE CLASSROOM
Cultures of Monoblepharella spp. can be grown on nutrient medium and demonstrate the characteristics of the genus. Hyphae are smaller and more delicate than Monoblepharis but show the right angle branching, the protoplasmic streaming and the ladder like appearance of the hyphae typical of Monoblepharidaceae. Monoblepharella hyphae soon develop great numbers of globose or spindle shaped nodules.
Inducement of reproduction may be difficult. If asexual reproduction is induced, look for zoosporangia, escaping zoospores, germination of zoospores and growth of young hyphae.
We have not succeeded in inducing sexual reproduction in pure culture; however, it should be possible if cultures are incubated at 30--32° C. If you succeed, look for oogonia with oospheres, antheridia, ferilization of oospheres, escape of the zygote, movement of the zygote powered by the antheridial flagellum and the mature oospore.
Species of Monoblepharella are identified by variations in the arrangement of the sexual structures.
CULTURES AVAILABLE - Cultures M# 15 and M# 33 usually produce zoosporangia.
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 2 months.
PREPARATION TIME - Start stock plate A 39 days before scheduled class. Call this Day 0.
DAY 0 - Transfer a piece of thallus from stock culture to an mPmTG agar plate, stock plate A, and incubate 14 days at room temperature (22° C) and light.
DAY 14 - Using stock plate A, with a sterile transfer needle cut the newly grown thallus into 8 or 10 slices and transfer them to a new mPmTG plate, stock plate B, for further increase. Incubate 14 days at room temperature (22° C) and light.
DAY 28 - Using stock plate B, with a sterile transfer needle move the slivers of old agar aside, cut the new thallus into small pieces and transfer the pieces into 2 250 ml Erlenmeyer flasks each containing 50 ml of T/2 broth. Incubate 1 in light and 1 in dark at room temperature (22° C) for 5 days.
DAY 33 - Wash the thalli by pouring into sterile wire baskets and placing 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 baskets. Leave both dishes in the light, noting those that were previously in the dark.
DAY 34 - If spores are present or developing in either plate, use sterile pipettes to transfer swimming spores into sterile Petri plates with cover glasses and carefully cover with T/2. Grow some in light and some in dark for 5 days at room temperature (22° C).
DAY 39 - Eight hours before scheduled class remove the T/2 from the cover glass plates with sterile pipettes and replace with sterile 1/3 PW. Keep in light or dark as indicated by results on Day 34. In class use sterile forceps to remove individual cover slips, wipe the under sides dry and invert on to microscope slides. Look for sporangia in all stages of growth and for swimming spores.
ALTERNATE PROCEDURES -
DAY 34 - If no spores are present or developing in either plate, replace the liquid with unsterilized 1/3 PW. If this procedure results in spore production it will probably happen within 24 hours. Creatures in the pond water will attack the fungus quickly as well.
To encourage sexual reproduction keep the inoculated Petri plates with cover glasses in the dark at temperatures of 30° C to 32° C.
SCHEDULE ADJUSTMENTS - Growing times of plates A and B can be adjusted slightly. Do not adjust growing times in flasks.
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 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, glass Petri dishes with 5 cover slips, microscope slides and cover slips.
WIRE BASKETS - Illustrated directions for making wire baskets for use in working with fungal cultures are given in the following reference:
Aronson, Jerome, and Melvin
S. Fuller. Pg 190. In:
Fuller, M. S., Ed. 1978. Lower Fungi In The Laboratory. Department of Botany,
Aronson, Jerome, and
Melvin S. Fuller. 1978. Pg 190. In: Fuller, M.
S., Ed. Lower Fungi In The Laboratory. Department of Botany,
Dolan, Thomas E. 1987. Monoblepharella sp. Pg 52. In: Zoosporic fungi in teaching and research. Eds. M. S.
Fuller and A. Jaworski.
Southwestern Publishing Corporation.
Fuller, M. S. and A. Jaworski, Eds. 1987. Zoosporic fungi in
teaching and research. Southeastern Publishing Corporation.
Karling, J. S. 1977. Chytridiomycetarum iconographia. Lubrecht & Cramer.
Shanor, Leland. 1942.
A new Monoblepharella from
F. K. 1960. Aquatic phycomycetes. 2nd ed. Univ.
Springer, Martha E. 1945. A monographic study of the genus Monoblepharella. American Journal of Botany. 32, No 5: 259-269.
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