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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/images/celltypes.gifEukaryotehttp://www.windows.ucar.edu/earth/Life/images/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://users.rcn.com/jkimball.ma.ultranet/BiologyPages/Y/Yeast.jpg
http://www.theartisan.net/yeast_cell_final_resample.jpg
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Filamentous fungi Multicellular
organism Long branched
filament called hyphae
Hyphae aggregate to form a mass mycelium
Aerobeshttp://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.jpg
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Reproduction Sexual and asexual Asexual
Budding:The 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/018yeast.gif
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ReproductionConidiospores: are not enclosed within a sac
Sporangiospores: enclosed in a sac like head called a sporangium
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Mold growth
http://byebyemold.com/mold_images/images/penicillium/penicillium_c.jpg http://www.mould.ph/images/curvul3.jpghttp://www.inspect-ny.com/sickhouse/1263s.jpghttp://ipm.ncsu.edu/current_ipm/colony.jpg
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Aspergillus niger
http://wellino.de/aspergillus/images/aspergillus-niger-5.jpghttp://www.ttuhsc.edu/SOM/Microbiology/mainweb/aiaq/Pictures/Aspergillus%20niger.jpg
http://res2.agr.ca/winnipeg/storage/images/lo-res/mould/aspe06-l.jpg
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Penicillium frequentans
http://www.bioweb.uncc.edu/1110Lab/notes/notes1/labpics/Penicillium%20notatum.JPG
http://www.anselm.edu/homepage/jpitocch/genbios/31-14b-Penicillium.jpg
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Objective 1: Culturing yeast
A. Fermentation tubesInoculate each yeast into a glucose and a sucrose fermentation tube
2 tubes per yeast, 8 totalIncubate at 37oC
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Objective 1A: Culturing yeastNext lab (Wednesday)
Organism
Glucose Sucrose
Acid Gas Fermentation Acid Gas Fermentation
1. Baker’s yeast
2. Saccharomyces cerevisiae
3. Candida albicans
4. Rhodoturula rubra
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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)
12
34 •Baker’s 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
1. Streak 3 filamentous fungi in Sabouraud. 2. One mold per plate3. Do one straight line in the middle of the plate
Aspergillus niger, Penicillium frequentans Rhizopus nigricans
4. Next lab (Wednesday) Observe both bottom and top of the plate
Characteristics Aspergillus niger
Macroscopic: Colony appearance
Hyphae color
Spore color
Underside color
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Objective 2.B Environmental sample Molds
1. Each team also does an environmental sample (expose to the air). Incubate at 27oC.
2. 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/MallochLab/Malloch/Moulds/Illustrations/Slide_culture.jpg
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Slide culture
http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Illustrations/Slide_culture.jpg
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Slide culture
http://www.botany.utoronto.ca/ResearchLabs/MallochLab/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