BIOL 2P96
Biology of Fungi
Lab 1
Aseptic Technique, Microscopy and Morphology I
Aseptic Technique
• To prevent contamination of oneself/co-workers, the
environment, and your work/culture.
• Label a 60mm agar plate with your name, lab day and time
and AT (for Aseptic Technique).
• Labels are written in Sharpie on the bottom/agar side (in
case you lose the lid), around the outside rim (to view the
culture/growth in the middle).
• Working near a flame, flame your loop starting from
handle end of wire towards the loop end. Then let cool for
10 seconds.
• Dip the loop into the sterile distilled water, and then
simple streak the agar of the plate with the water.
• Parafilm the lid and bottom to keep the lid on and prevent
contamination of the plate. Moisture and gases can still
Microscopy
1. Adjustable ocular lenses: these lenses have a 10X magnification and are adjustable
to the distance between your eyes.
2. Nosepiece/turret: this rotating piece holds the objective lenses.
3. Objective lenses: these lenses are on a rotating turret and there are usually 4
objective lenses of 3.5X, 10X, 40X and 100X magnification.
4. Course focus: this knob allows for larger focusing movements. This knob should
not be used when using the 40X or 100X objective lenses.
5. Fine focus: this knob sits on top of the course focus knob and allows for smaller
focusing movements. This should be the focus knob used while looking with the 40X
and 100X objective lenses.
6. Stage: this is where the slide is placed horizontally and held with the stage clips.
7. Light: this is the light source to illuminate the slide from below.
8. Condenser and iris diaphragm: these control how much light passes through to the
slide.
9. Stage control knob: this knob hangs down from the stage and controls the motion
of the slide from left to right and top to bottom.
Calibrating the Ocular Micrometer
(ruler in eyepiece)
• Line up the zero’s of the stage micrometer and the ocular micrometer
• Find another place father down the rulers where they both line up again
15 ocular
10 stage
10 stage divisions x 10 micrometer per stage division = 6.67 micrometers per ocular division
15 ocular divisions
Terminology and Morphology
• Hyphae: aseptate (coenocytic) or septate
(with cross-walls)
• Mycelium: network of hyphae
• Vegetative hyphae: absorb nutrients form
medium; many shapes and styles (favic
chambers, nodular organs, racquet or spiral)
• Aerial hyphae: support reproductive
structures (ex. conidia)
• Spores: sexual (zygospore, ascospore,
basidiospore) or asexual (sporangiospore,
conidiospore)
Kingdom Stramenopila
Phylum Oomycota “Water Moulds”
• Oomycete cell walls contain the polysaccharide cellulose; chitin is also
present in some species. In the true fungi no cellulose is found, only chitin.
• Wall proteins also differ with hydroxyproline found in Oomycota while
proline is present in the true fungi.
• Mitochondria the cristae are tubular while in true fungi the cristae are
plate-like.
• There are also differences in the zoospores which are biflagellate in the
Oomycetes (heterkont: one tinsel, one whiplash) and uniflagellate in the
Chytridiomycetes.
• Vegetative ploidy is 2n in the Oomycota; 1n or n+n in the fungi.
• There are a number of biochemical synthetic pathways (lysine
biosynthesis) and storage compounds in the Oomycetes that are very
different than those present in true fungi.
Phylum Oomycota
Order Saprolegniales
• Saprolegnia sp.
• Found in freshwater and soil
• Homothallic (single zoospore give
rise to a mycelium that forms
both oogonia and antheridia)
• Examine the prepared slide of
Saprolegnia (wm) and locate each
of the following: zoosporangia,
hyphae, oogonium, antheridium,
oosphere, and oospore.
Phylum Oomycota
Order Pythiales
• Phytophthora infestans
• Heterothallic (requires two strains for
sexual reproduction)
• Examine the Phytophthora infestans (leaf)
slide on the lower surface of the leaf and
locate the hyphae poking out of the
stomata.
• Locate the oval-shaped sporangia that
have fallen off. Are the zoospores visible?
• These zoospores have two flagella; a
whiplash flagellum faces the back and
pushes the spore through the water and a
tinsel flagellum points forward and pulls
the spores through the water.
extento.hawaii.edu
Phylum Oomycota
Order Pythiales
• Now examine the slide of
Phytophthora infestans tuber.
On this slide locate the
haustoria that have penetrated
into the cells of the host plant.
• Haustorium (specialized hyphal
invagination of plant cells that
digest the contents of these
infected cells and absorb the
nutrients).
Phylum Oomycota
Order Peronosporales
• Obligate biotropic pathogens (only
from living cells)
• Peronospora parasitica (pathogen of
Cruciferae {ex. mustard})
• Focus on the outer edge of the plant
stem and locate the branching
structures with the small round
spheres attached (sporangiophores).
• Now move into the host cells and
locate the branched lobed haustoria.
Phylum Oomycota
Order Peronosporales
• Plasmopara viticola (pathogen of
grape vines)
• Focus on the outer edge of the plant Grape leaf
stem and locate the branching
structures with the small round
spheres attached
(sporangiophores).
• Now move into the host cells and
locate the branched lobed
haustoria. Grape stem
400x & 40x
• Can you find any oospores
embedded in the plant tissue?
Phylum Oomycota
Order Peronosporales
• Albugo sp. (white rust on
flowering plants) differ by
sporangia and
sporangiophores.
• Locate an oospore in the
host plant tissue and then
sporangiophores with
sporangial chains where
the host epidermis tissue
has burst.
Kingdom Fungi
Phylum Chytridiomycota (The Chytrids)
• Most members of this group live in the soil they are predominantly
aquatic, and they all require water to complete their lifecycle.
• Chytrids (depending on the species) produce flagellated mitospores,
flagellated meiospores and flagellated gametes -- their reproductive
cells have a flagellum that allows them to swim. No other fungi have
flagella.
• Chytrids have chitin strengthening their cell walls.
• Order Chytridiales, Synchytrium endobioticum (black wart in potatoes)
• endobiotic (grows inside living organism) and holocarpic (whole
thallus is reproductive organ)
Kingdom Fungi
Phylum Chytridiomycota (The Chytrids)
• Order Chytridiales,
Synchytrium endobioticum
(black wart in potatoes)
• endobiotic (growns inside
living organism) and
holocarpic (whole thallus is
reproductive organ)
• Examine a prepared slide of
Synchytrium endobioticum
Phylum Blastocladiomycota
Order Blastocladiales
• Species of this phylum are found to
be saproptrophs and parasites of
other fungi, plants, algae and
invertebrates.
• Thallus has both broad true
hyphae and narrow rhizoids
(eucarpic).
• Allomyces arbusculus exhibits
alternation of generations
(lifecycles that include both
haploid and diploid stages that are
multicellular; i.e. undergoes Moore, et. al., 2020, p.110
mitosis of its haploid cells).
Phylum Blastocladiomycota
Order Blastocladiales
• Examine a prepared slide of
Allomyces arbusculus
sporothallus w.m (asexual
plant)
• observe the branched
mycelium, bearing rhizoids
and branched hyphae,
colorless, elongated
mitosporangia, and dark
pigmented, rounder, resistant
meiosporangia.
Phylum Blastocladiomycota
Order Blastocladiales
• Examine a slide of Allomyces
arbusculus gametothallus
w.m. (sexual plant).
• The larger female gametangia
occur at the end of the
hyphae, with the smaller
male gametangia
immediately below.
• Watch and read the Lab Biosafety presentation as some of the
Assignment Quiz questions are on lab safety.
• Complete page 15 and 16 of the Lab 1 MS Word document as aids to
the Assignment Quiz.
• Lab 1 Assignment Quiz is under Tests & Quizzes on Sakai. The quiz
must be submitted before 11:55pm on Sun Jan 24th.
• You have unlimited tries and no time limit while answering the
questions until the close/due date.
• Please ask questions by leaving a message in the Chat room, Forum or
email your TA, before the Quiz is due.
• References:
• D Moore, G D Robson and A P J Trinci (2020) 21st Century Guidebook
to Fungi, 2nd edition online. Cambridge University Press: Cambridge.
