Laboratory 15:

Filamentous fungi and yeast

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
Eukaryote organisms
 Nuclei
 Membrane bound
organelle
 Also differences in
genetic material,
replication etc…
 No peptidoglycan

Eukaryotehttp://www.windows.ucar.edu/earth/Life/images/celltypes.gif
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
 Yeast
 Unicellular
 Non filamentous
 Spherical
 Membrane bound nucleus
 are eukaryote
 Facultative anaerobes

http://www.theartisan.net/yeast_cell_final_resample.jpg
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/Y/Yeast.jpg
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
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
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
 Reproduction
 Budding: Yeast

http://www.mansfield.ohio-state.edu/~sabedon/018yeast.gif
 Reproduction Conidiospores: are not
enclosed within a sac

Sporangiospores: enclosed
in a sac like head called a
sporangium
 Mold growth

http://ipm.ncsu.edu/current_ipm/colony.jpg http://www.inspect-ny.com/sickhouse/1263s.jpg
http://byebyemold.com/mold_images/images/penicillium/penicillium_c.jpg http://www.mould.ph/images/curvul3.jpg
Aspergillus niger


http://res2.agr.ca/winnipeg/storage/images/lo-res/mould/aspe06-l.jpg
http://www.ttuhsc.edu/SOM/Microbiology/mainweb/aiaq/Pictures/Aspergillus%20niger.jpg
http://wellino.de/aspergillus/images/aspergillus-niger-5.jpg
Penicillium frequentans

http://www.anselm.edu/homepage/jpitocch/genbios/31-14b-Penicillium.jpg
http://www.bioweb.uncc.edu/1110Lab/notes/notes1/labpics/Penicillium%20notatum.JPG
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
Objective 1A: Culturing yeast
Next lab (Wednesday)
Glucose Sucrose

Acid Gas Fermentation Acid Gas Fermentation

Organism
1. Baker’s yeast

2. Saccharomyces
cerevisiae

3. Candida albicans

4. Rhodoturula rubra
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)

•Baker’s yeast (1)

2 4
1 3 •Saccharomyces
cerevisiae (2)
•Candida albicans (3)
 Incubate at 37 C o
•Rhodoturula rubra (4)
 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
 Objective 2.A Culturing Molds
1. Streak 3 filamentous fungi in Sabouraud.
2. One mold per plate
3. 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
 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?
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
Slide culture technique
Day 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
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.
Slide culture

http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Illustrations/Slide_culture.jpg
Slide culture

http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Illustrations/Slide_culture.jpg
Slide culture

http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Illustrations/Slide_culture.jpg
Synthesis
 Culture 4 yeast : 2 Petri plates
 Culture 4 glucose fermentation tubes
 Culture 4 sucrose fermentation tubes
 Culture 4 molds
 Slide culture