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.gifEukaryotehttp://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://users.rcn.com/jkimball.ma.ultranet/BiologyPages/Y/Yeast.jpg

http://www.theartisan.net/yeast_cell_final_resample.jpg

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

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

ReproductionConidiospores: are not enclosed within a sac

Sporangiospores: enclosed in a sac like head called a sporangium

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

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

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

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

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

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

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 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

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 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

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