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
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

exhaust air
bubbles
remove cover
trapped
slip, place on
underneath
single streak clean glass
the coverslip incubate
of yeast slide with
place an plates at
culture at the droplet of
autoclave- 30C for
center of PDA water
sterilized 48 hrs
plate cover slip (inverted
on top of position)
streak

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

streak PDA incubate

plates at
for 30C for 48
yeast isolation AcA hrs
culture (inverted
position)

observe
cultural
characteristics
Procedure
Mode of Asexual Reproduction
suspend
yeast
culture for
wet
yeast mount
culture from technique
PDA plate
Procedure
Method of Sexual Reproduction
obtain a small amount air dry or heat
of culture, spread it on
fixed smear
the glass slide with
droplet of water
yeast culture
grow in MA at
25C
 Pseudomycelia True mycelia
-Formed after failed -Formed from the fission
separation of two of the ends, while the old
supposed to be daughter strands have thicken
cells resulting in an chitin cell wall for
elongated structure support.
 Pseudomycelia True mycelia
end becomes bud (bud - continuous
asexual spore - perpendicular to
blastospores)
walls
separate the 2 cells
side branches - away
(curved)
from main filament
septum that separates
the main filament
side branches - near to
each other
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
Organism Diameter Form Margin Elevation Color Texture
(mm)
Pichia 2 Round Entire convex white Smooth
Candida 4 L-form Entire raised Creamy Rough
white
Debaryom- 2 Round Entire Drop-like Creamy- Rough
yces white
Endomyco 4 L-form Undulate convex white Rough
psis
Hansenula 2 Complex Entire Drop-like Creamy- Rough
white
Results: Cultural Characteristics
Day 7:
PDA

Organism Diameter Form Margin Elevation Color Texture

(mm)
Pichia 2 Round Entire convex white Smooth

Candida 4 L-form Entire convex Creamy Rough

white
Debaryomyc 2 Round Entire Drop-like Creamy- Rough
es white
Endomycops 4 L-form Undulate convex white Rough
is
Hansenula 2 Complex Entire Drop-like Creamy- Rough
white
Result: Cultural Characteristics
Day 2:
ACA

Organism Diameter Form Margin Elevation Color Texture

(mm)
Pichia 1 round entire convex white Smooth

Candida 1.3 round entire raised Off- Smooth

white
Debaryomyc 1 Round entire Drop-like white smooth
es
Endomycops 1.5 Round entire convex white Smooth
is with
raised
Hansenula 1 round entire flat white smooth
Result: Cultural Characteristics
Day 7:
ACA

Organism Diameter Form Margin Elevation Color Textur

(mm) e
Pichia 2 Round Entire convex white Smoot
h
Candida 3 Wrinkled Entire convex Creamy- Rough
white
Debaryomyc 2 Round entire Drop-like Creamy- Rough
es white
Endomycops 2 Round Entire convex Creamy- Smoot
is white h
Hansenula 1 Round Entire Drop-like Creamy- smoot
white h
Results: Morphological Characteristcs and
Mode of Reproduction
Candida
Shape of Visible internal Mode of Asexual
vegetative cell structures Reproduction
Elliptical NONE Budding
Results: Morphological Characteristcs and
Mode of Reproduction
Debaryomyces
Shape of Visible internal Mode of Asexual
vegetative cell structures Reproduction
Spherical NONE Budding
Results: Morphological Characteristcs and
Mode of Reproduction
Endomycopsis
Shape of vegetative Visible internal Mode of Asexual
cell structures Reproduction
Ovoid NONE Budding
Results: Morphological Characteristcs and
Mode of Reproduction
Hansenula
Shape of vegetative Visible internal Mode of Asexual
cell structures Reproduction
Spheroidal, elongated NONE Budding
Results: Morphological Characteristcs and
Mode of Reproduction
Pichia
Shape of vegetative Visible internal Mode of Asexual
cell structures 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 Number of spores
Ascospores Ellipsoidal
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
Temperature
Moisture
Nutrient depletion (induces sporulation)
Time influences the age produce sexual spores
More resisitant to vegetative cells

Readily rupture
Highly refractile
Dimorphic fungi
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 35C to 37C
In general, dimorphic fungi produces mold
form at 25-30C and yeast form at 35-37C
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?

To avoid the objective lens of the microscope

from contamination and helps for focusing the
induce growth of mycelia
Provides the partial anaerobiosis condition
Discussion
How is
Schizosaccharomyces
morphologically
different from
Saccharomyces?

Saccharomyces are
globous or ellipsoidal in
shape; undergo
budding, divide
unevenly
Discussion
Schizosaccharomyces
are linear and rod-
shaped 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

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 water-
soluble 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?

Utilization of carbon compunds: Oxidative

(assimilation) utilization
Utilization of nitrogen compounds
Molecular identification
References: Websites
https://microbiologyinfo.com/potato-dextrose-
agar-pda-principle-uses-composition-procedure-
and-colony-characteristics/
https://assets.thermofisher.com/TFS-
Assets/LSG/manuals/IFU1565.pdf
http://microbeonline.com/endospore-staining-
principle-procedure-results/
https://www.kullabs.com/classes/subjects/unit
s/lessons/notes/note-detail/1534
http://biology.tutorvista.com/cell/asexual-
reproduction.html
References: Books
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.