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,
REFERENCES -
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
Sparrow,
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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