Exer 2 Morphology And Reproduction Of Yeasts 2c1557

Exercise 2: Morphology and Reproduction of Yeasts Christine Joy C. Manzanilla MCB 101 G-2L

Objectives

Characterize a culture of morphologically selected yeasts using Dalmau plate method  Characterize yeast through its mode of asexual reproduction, presence of sexual spores, formation of true or pseudomycelia  Properly perform wet mount technique. 

Introduction Yeasts are eukaryotic, non filamentous, unicellular fungi that are typically spherical or oval.  They do not reproduce as a unit unlike molds. Instead, the colony grows as the number of yeast cells increases by mitosis. 

Timeline of Activities  

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August 11 - Media Preparation (PDA, AcA, MA) August 16 - Inoculate media, poured media onto plates, streak for isolation (yeast cultures) August 18 - performed Dalmau plate method August 23 - Observation of Dalmau plate method, Spore staining, Observation of PDA and AcA plates, Wet mount from PDA August 25 - Spore staining

Yeast Cultures Candida parapsilosis  Debaryomyces hansenii  Hansenula anomala  Pichia fermentans  Endomycopsis burtonii 

Procedure 

Dalmau Plate Method

single streak of yeast culture at the center of PDA plate

place an autoclavesterilized cover slip on top of streak

exhaust air bubbles trapped underneath the coverslip

incubate plates at 30°C for 48 hrs (inverted position)

remove cover slip, place on clean glass slide with droplet of water

observe under microscope (HPO or LPO) for true mycelia or pseudomycelia

Procedure 

Cultural Characteristics

yeast culture

streak for isolation

PDA AcA

incubate plates at 30°C for 48 hrs (inverted position)

observe cultural characteristics

Procedure 

Mode of Asexual Reproduction

yeast culture from PDA plate

suspend yeast culture for wet mount technique

Procedure 

Method of Sexual Reproduction

yeast culture grow in MA at 25°C

obtain a small amount of culture, spread it on the glass slide with droplet of water

air dry or heat fixed smear

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Pseudomycelia

-Formed after failed separation of two supposed to be daughter cells resulting in an elongated structure



True mycelia

-Formed from the fission of the ends, while the old strands have thicken chitin cell wall for .

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Pseudomycelia

 end

becomes bud (bud asexual spore blastospores)  separate the 2 cells (curved)  septum that separates the main filament  side branches - near to each other

True mycelia  continuous  perpendicular to walls  side branches - away from main filament 

Results: Yeasts specimens grown on PDA using Dalmau plate method Candida

Pseudomycelium

Results: Yeasts specimens grown on PDA using Dalmau plate method 

Hansenula

Pseudomycelium

Results: Yeasts specimens grown on PDA using Dalmau plate method 

Pichia

Complex Pseudomycelium

Results: Yeasts specimens grown on PDA using Dalmau plate method 

Endomycopsis

Pseudomycelium

Results: Yeasts specimens grown on PDA using Dalmau plate method 

Debaryomyces

Pseudomycelium

Results: Cultural Characteristics 

Day 2: PDA Margin

Elevation Color

Texture Smooth Rough

Organism

Diameter Form (mm)

Pichia Candida

2 4

Round L-form

Entire Entire

convex raised

Debaryom- 2 yces

Round

Entire

Drop-like Creamywhite

Endomyco 4 psis

L-form

Undulate convex

Hansenula 2

Complex Entire

white Creamy white

white

Drop-like Creamywhite

Rough Rough Rough

Results: Cultural Characteristics 

Day 7: PDA Organism

Diameter (mm)

Form

Margin

Elevation

Color

Texture

Pichia

2

Round

Entire

convex

white

Smooth

Candida

4

L-form

Entire

convex

Creamy white

Rough

Debaryomyc es

2

Round

Entire

Drop-like

Creamywhite

Rough

Endomycops is

4

L-form

Undulate

convex

white

Rough

Hansenula

2

Complex

Entire

Drop-like

Creamywhite

Rough

Result: Cultural Characteristics 

Day 2: ACA

Organism

Margin

Elevation

Color

Texture

Pichia

Diameter Form (mm) 1 round

entire

convex

white

Smooth

Candida

1.3

round

entire

raised

Smooth

Debaryomyc 1 es Endomycops 1.5 is

Round

entire

Drop-like

Offwhite white

Round with raised round

entire

convex

white

Smooth

entire

flat

white

smooth

Hansenula

1

smooth

Result: Cultural Characteristics 

Day 7: ACA

Organism

Diameter (mm)

Form

Margin

Elevation

Color

Textur e

Pichia

2

Round

Entire

convex

white

Smoot h

Candida

3

Wrinkled

Entire

convex

Creamywhite

Rough

Debaryomyc es

2

Round

entire

Drop-like

Creamywhite

Rough

Endomycops is

2

Round

Entire

convex

Creamywhite

Smoot h

Hansenula

1

Round

Entire

Drop-like

Creamywhite

smoot h

Results: Morphological Characteristcs and Mode of Reproduction 

Candida Shape of vegetative cell

Visible internal structures

Mode of Asexual Reproduction

Elliptical

NONE

Budding

Results: Morphological Characteristcs and Mode of Reproduction 

Debaryomyces Shape of vegetative cell

Visible internal structures

Mode of Asexual Reproduction

Spherical

NONE

Budding

Results: Morphological Characteristcs and Mode of Reproduction 

Endomycopsis Shape of vegetative cell

Visible internal structures

Mode of Asexual Reproduction

Ovoid

NONE

Budding

Results: Morphological Characteristcs and Mode of Reproduction 

Hansenula Shape of vegetative cell

Visible internal structures

Spheroidal, elongated NONE

Mode of Asexual Reproduction Budding

Results: Morphological Characteristcs and Mode of Reproduction 

Pichia Shape of vegetative cell

Visible internal structures

Mode of Asexual Reproduction

Ovoid, ellipsoidal

NONE

Budding

Results: Sexual reproduction of yeasts 

Debaryomyces Sexual spore

Shape of spore

Number of spores

Ascospores

Ovoid

1-2

Results: Sexual reproduction of yeasts 

Candida Sexual spore

Shape of spore

Ascospores

Ellipsoidal

Number of spores

Results: Sexual reproduction of yeasts 

Endomycopsis Sexual spore

Shape of spore

Number of spores

Ascospore

Spheroidal

1-2

Results: Sexual reproduction of yeasts 

Hansenula Sexual spore

Shape of spore

Number of spores

Ascospore

Hat-shaped

1-4

Results: Sexual reproduction of yeasts 

Pichia Sexual spore

Shape of spore

Number of spores

Ascospore

Hat-shaped

1-4

Presence of sexual spores - stress     

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Temperature Moisture Nutrient depletion (induces sporulation) Time influences the age produce sexual spores More resisitant to vegetative cells

Readily rupture Highly refractile

Dimorphic fungi 

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Dimorphic fungi are fungi that have a yeast (or yeast-like) phase and a mold (filamentous) phase One of the characteristics common to dimorphic fungi is the ability to convert the mold forms to yeast forms by incubating subcultures in enriched media at 35°C to 37°C In general, dimorphic fungi produces mold form at 25-30°C and yeast form at 35-37°C Outside the host (mold), act as fungi (yeast) Example: Penicillium marneffei, Cryptococcus

YM Shift (Yeast-Mold Shift)   

CO2 Tension Temperature Redox potential

Discussion 

What is the purpose of the cover slip in Dalmau plate?

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To avoid the objective lens of the microscope from contamination and helps for focusing the induce growth of mycelia Provides the partial anaerobiosis condition

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Discussion 

How is Schizosaccharomyces morphologically different from Saccharomyces?

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Saccharomyces are globous or ellipsoidal in shape; undergo budding, divide unevenly

Discussion 

Schizosaccharomyces are linear and rodshaped cells; undergo fission, divide evenly to produce new cells

Discussion 

Discuss the principle of using PDA, AcA and MA plates in studying yeast cultures.

Media used:  PDA (Potato Dextrose Agar): is a general purpose medium for yeasts and molds that can be supplemented with acid or antibiotics to inhibit bacterial growth; contains dextrose, which is a carbohydrate, aids in the stimulation of growth

Discussion 

AcA (Acetate Agar) - nitrogen deficient medium, delays and depress growth and sporulation; media to be used in spore staining instead

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MA (Malt extract Agar) - designed to contain the proper formulation of carbon, protein and nutrient sources essential for yeast growth

Discussion 

Why is malachite green used in ascospore staining?

Malachite green (C23H25ClN2 (chloride)) is used for staining to identify the presence of ascospores. Malachite green dye is watersoluble and does not adhere well to the cell wall making it easily rinsed, leaving it for counterstain (usually Safranin)

Discussion 

What is the significance of having asexual and sexual modes of reproduction?

The higher fertility rate if both sexual and asexual modes of reproduction are present. Since cultures depend on favorable conditions Asexual reproduction - rapid growth, does not involve the process of meiosis and fertilization, hence the process does not require a mate for reproduction.

Discussion 

Sexual reproduction - allows adaptation to new environmental conditions and allows genetic recombination or genetic material from the parent. It also brings about variation so not two individuals are similar.

Discussion 

What other characteristics should be used to accurately identify yeasts?

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Utilization of carbon compunds: Oxidative (assimilation) utilization Utilization of nitrogen compounds Molecular identification

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References: Websites 

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https://microbiologyinfo.com/potato-dextroseagar-pda-principle-uses-composition-procedureand-colony-characteristics/ https://assets.thermofisher.com/TFSAssets/LSG/manuals/IFU1565.pdf http://microbeonline.com/endospore-stainingprinciple-procedure-results/ https://www.kullabs.com/classes/subjects/unit s/lessons/notes/note-detail/1534 http://biology.tutorvista.com/cell/asexualreproduction.html

References: Books 

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N.J.W. Kreger-van Rij. 1984. The Yeasts: A taxonomic study. Elsevier Science Publishers B.V. Amsterdam. Reed, G. 1984. Industrial Microbiology. Avi Publishing Company, Inc. Tortora, G. et al,. 1995. Microbiology: An Introduction. The Benjamin/Clummings Publishing Company. Inc.

Recommendations 

Prolong incubation period for better results especially in modifying the sexual reproduction (presence of ascospores) since the yeast cultures are still young.