Laboratory 15: Filamentous fungi and yeast

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Laboratory 15 Filamentous fungi and yeast
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Fungi

• 70 000 species described
• Macroscopic or microscopic
• Heterotrophic organism
• Unicellular or multicellular
• Mostly terrestrial but some adapted to aquatic
  life
• Aerobic or facultative anaerobe
• Prefer cool and damp niches

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Eukaryote organisms

• Nuclei
• Membrane bound organelle
• Also differences in genetic material, replication
  etc
• No peptidoglycan

• Eukaryotehttp//www.windows.ucar.edu/earth/Life/im
  ages/celltypes.gif

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Dimorphism

• Fungus that can present 2 forms depending of
  conditions
• Yeast
• Filamentous fungi
• Could be opportunistic pathogens

http//gsbs.utmb.edu/microbook/images/fig75_3.JPG
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Yeast

• Unicellular
• Non filamentous
• Spherical
• Membrane bound nucleus ? are eukaryote
• Facultative anaerobes

http//www.theartisan.net/yeast_cell_final_resampl
e.jpg
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/Y/Yeast.jpg
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Filamentous fungi

• Multicellular organism
• Long branched filament called hyphae
• Hyphae aggregate to form a mass ? mycelium
• Aerobes

http//www.anselm.edu/homepage/jpitocch/genbios/31
-01-FungalMycelia-L.jpg
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Sabouraud agar

• Selective media
• Contain sugars and peptone
• Low pH (5) which is inhibitory for most other
  microorganisms
• Invented by a French Doctor (Dr. Sabouraud)
  specialist in scalp disease

http//www.bium.univ-paris5.fr/sfhd/img/gd/sabou.j
pg
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Reproduction

• Sexual and asexual
• Asexual
• BuddingThe parent cell can divide into two equal
  or unequal daughter cells
• Spore prodution
• Sporangiospores
• Conidiospores

http//www.anselm.edu/homepage/jpitocch/genbios/31
-01-FungalMycelia-L.jpg
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Reproduction

• Budding Yeast

http//www.mansfield.ohio-state.edu/sabedon/018ye
ast.gif
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Reproduction
Conidiospores are not enclosed within a sac
Sporangiospores enclosed in a sac like head
called a sporangium
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Mold growth
http//www.inspect-ny.com/sickhouse/1263s.jpg
http//ipm.ncsu.edu/current_ipm/colony.jpg
http//byebyemold.com/mold_images/images/penicilli
um/penicillium_c.jpg
http//www.mould.ph/images/curvul3.jpg
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Aspergillus niger

http//res2.agr.ca/winnipeg/storage/images/lo-res/
mould/aspe06-l.jpg
http//www.ttuhsc.edu/SOM/Microbiology/mainweb/aia
q/Pictures/Aspergillus20niger.jpg
http//wellino.de/aspergillus/images/aspergillus-n
iger-5.jpg
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Penicillium frequentans
http//www.anselm.edu/homepage/jpitocch/genbios/31
-14b-Penicillium.jpg
http//www.bioweb.uncc.edu/1110Lab/notes/notes1/la
bpics/Penicillium20notatum.JPG
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Objective 1 Culturing yeast
A. Fermentation tubes Inoculate each yeast into a
glucose and a sucrose fermentation tube 2 tubes
per yeast, 8 total Incubate at 37oC
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Objective 1A Culturing yeastNext lab (Wednesday)
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Objective 1B Culturing yeast

• Streak yeast
• Divide 2 sabouraud plate in a half
• We have 4 different yeast
• Inoculate one per half (As you did for bacteria)

• Bakers yeast (1)
• Saccharomyces cerevisiae (2)
• Candida albicans (3)
• Rhodoturula rubra (4)

• Incubate at 37oC

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Objective 1B. Culturing yeast(Next lab Wednesday)

• Put a drop of methylen blue on a slide and mix a
  loopful of yeast. Put a coverslip on and observe
  under the microscope
• Draw your observations of your 4 yeast

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Objective 2.A Culturing Molds

• Streak 3 filamentous fungi in Sabouraud.
• One mold per plate
• Do one straight line in the middle of the plate
• Aspergillus niger,
• Penicillium frequentans
• Rhizopus nigricans
• Next lab (Wednesday)
• Observe both bottom and top of the plate

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Objective 2.B Environmental sample Molds

• Each team also does an environmental sample
  (expose to the air). Incubate at 27oC.
• Next lab (Wednesday)
• Observe both bottom and top of the plate
• Can you identify any of the contaminating molds
  based on comparisons to the known molds?

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Objective 3 Culturing 1 mold under slide culture
technique

• Pick up one mold to this part
• Some are very fragile and hyphae can break as
  they are mounted onto a slide
• To overcome this ? slide culture which is an in
  situ culture of the fungi

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Slide culture techniqueDay 1

• Using aseptic technique, place a block of agar on
  a slide in a Petri dish
• Inoculate the centers of the four sides of the
  agar block with the study fungus
• Cover the inoculated agar block with a steril
  cover slip.
• Using aseptic technique add 8 ml sterile water to
  the bottom of the Petri dishes
• Incubate at 25 C until sporulation occurs. Do
  not invert the plate

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Slide culture technique Next lab

• Carefully lift off the cover slip and lay aside
  with the fungus growth upward
• Lift the agar square from the slide and discard
• Place a drop of lactophenol cottom blue on the
  slide and cover with a clean cover slip
• With a clean slide place a drop of lactophenol
  cottom blue near one end and cover with the
  original cover slip. Make sure you can
  distinguish between Sporangiospores and
  Conidiospores.

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Slide culture
http//www.botany.utoronto.ca/ResearchLabs/Malloch
Lab/Malloch/Moulds/Illustrations/Slide_culture.jpg
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Slide culture
http//www.botany.utoronto.ca/ResearchLabs/Malloch
Lab/Malloch/Moulds/Illustrations/Slide_culture.jpg
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Slide culture
http//www.botany.utoronto.ca/ResearchLabs/Malloch
Lab/Malloch/Moulds/Illustrations/Slide_culture.jpg
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Synthesis

• Culture 4 yeast 2 Petri plates
• Culture 4 glucose fermentation tubes
• Culture 4 sucrose fermentation tubes
• Culture 4 molds
• Slide culture