EXERCISE 1: EXAMINATION OF MOLD AND YEAST CULTURES

Fungi
• eukaryotes - cell contains a true nucleus and many membrane-bound organelles
• unicellular or multicellular
• heterotrophic organisms (chemoheterotrophs) – organic compounds as sources
of energy and carbon
• mostly saprophytic - gets its energy from dead organic matter
• cannot photosynthesize because it cannot produce its own food
• sexual or asexual reproduction
• Some fungi are dimorphic, can exist as both mold and yeast
• aerobes to facultative anaerobes
• Importance:
• Industrial
• Beer and wine fermentation
• Chees and bread
• Antibiotics
• Source of food
• Truffles, mushrooms

Molds
• multicellular and filamentous
• Cottony or fuzzy appearance
• Hyphae and mycelium
• Presence of crosswalls (septa) – septated or non-septated (coenocytic)
• Function
• vegetative - nutrient uptake
• aerial – fertile; for reproduction
• Reproduction
• Sexual or asexual
• Asexual spores

Conidiospores Sporangiospores Arthrospores Chlamydospores

Yeasts
• Unicellular
• Reproduction: Sexual – ascospores
• Reproduction: Asexual – binary fission or budding
• Formation of pseudo or true mycelium

Enumeration of yeasts and molds

Note: Incubate the plates in upright position for 5 days at 25 – 30 deg C.

𝑐𝑓𝑢 Σcounts × DF
=
𝑔 [(1 × 𝑛1 ) + (0.1 × 𝑛2 )] × 𝑣𝑜𝑙. 𝑝𝑙𝑎𝑡𝑒𝑑
Note: Range for valid counts – 10-150 colonies

• Potato Dextrose Agar (PDA)

• Composition of PDA:
• Potato infusion - provides a nutrient base Potato infusion 200 gm
for luxuriant growth of most fungi
• Dextrose - carbohydrate source which Dextrose 20 gm
serves as a growth stimulant
• Agar - solidifying agent Agar 20 gm
• Commercial PDA: 39 grams per 1000 ml Distilled water 1 liter
• Chloramphenicol – inhibit the growth of
bacteria
• The recommended concentration of this antibiotic is 100 mg/liter
medium.
• PDA with TA (tartaric acid) is recommended for the microbial
examination of food and dairy products. pH 3.5 to inhibit the bacteria.
• Addition of chlortetracycline (antibiotic for skin infections) is
recommended for the microbial enumeration of yeast and mold from
cosmetics.
• 0.1 % Peptone Water
• Composition:
• 1 g of peptone in 1 liter
• Peptone - provides nutrients for survival of microorganisms
• minimal nutrient media designed to reduce multiplication of microorganisms
• useful as diluent or for making suspensions of non-fastidious microorganisms
for microbial enumeration procedures

Slide Culture Preparation

1. Petri dish was lined with paper
towel.
2. Place 2 toothpicks on top of the
filter paper.
3. Place a glass slide above the
toothpicks.
4. Aseptically, transfer a block of
agar to the center of the slide.
5. Inoculate the sides of agar block
with specimen then place the cover
slip on top of the agar block.
6. Incubate the setup at room temperature for 48 hours.
7. Observe the slide under LPO then HPO.

• Advantages of slide culture

• Rapid method of preparing fungal colonies for examination and
identification
• Permits fungi to be studied virtually in situ with as little disturbance as
possible

• Lactophenol Cotton blue

• mounting medium and staining agent
• Components of LPCB:
▪ Phenol – kills the organism
▪ Lactic acid – preserves fungal structures
▪ Cotton blue – stains the chitin in the fungal cell walls

Oxygen requirements for yeast

1. Obligate aerobes, those that must have
oxygen to extract energy from food, will gather
at the top of the test tube, near the broth’s
surface, to absorb maximum amount of oxygen.
2. Obligate anaerobes, those killed by
oxygen, will gather at the bottom of the test
tube, staying as far away from oxygen as
possible.
 3. Facultative organisms, those that can live with or without oxygen, will gather mostly
at the top, since aerobic respiration is the most energy efficient way to turn food
into energy; but since lack of oxygen does not hurt these microbes, they can
survive anywhere in the broth.
4. Microaerophiles gather at the upper part of the test tube but not at the top. These
microbes require oxygen, but at concentrations lower than those found in the
atmosphere.
5. Aerotolerant organisms are not affected at all by oxygen, so they can be found
evenly spread along the test tube.

Ascospore staining
• Structural staining
technique
• Primary stain: Malachite
green - water soluble and does
not adhere well to the cell wall
• Mordant: steam - helps the
malachite green to permeate the
low-permeability spore wall
• Decolorizer: water -
malachite green rinses easily
from the vegetative cells
• Safranin - counterstain
any cells which have been
decolorized
• Ascospores – green; vegetative cells – red/pink

1. Prepare the smear, air dry and fix by heat

2. Cover smear with paper towel to prevent accumulation of artifacts
3. Place the slide under the steam. Flood the smear with malachite green for 15 mins
4. Wash with distilled water
5. Counterstain with safranin for 30-60 sec
6. Wash and blot dry
7. Observe under OIO

MOLD CULTURES

Mucor

• White in color but turns a greyish-brown with aging

• From the reverse, it is white.
• Non-septate hyphae
• Sporangiophore and sporangium
• Columella
Rhizopus

• white initially and turns grey to yellowish brown

in time.
• Non-septate hyphae
• Rhizoids (resembles a root) and stolon (aerial
hyphae grow horizontally, connects rhizoids)
• Sporangiophore and sporangium
• Columella

Alternaria

• Wooly, color pale gray to olive brown on

surface
• Brown to black in reverse
• Septate, brown hyphae
• Conidiophore and conidium
• Macroconidia
• Brown conidia in chains

Fusarium

• white, lavender, pink, salmon or gray-

colored colonies on PDA
• Septate hyphae
• Phialides
• Macroconidia
• Microconidia
• Chlamydospores

Aspergillus

• Colonies on PDA black appressed

mycelium, becoming grayish on MYA.
• Septate hyphae
• Chains of conidia
• Vesicle
• Metula
• Phialide
• Foot cell
Penicillium

• blue-green or gray-green
• pale to yellowish on reverse
• Septate hyphae
• Conidiophore
• Metula
• Phialides
• Conidia

Trichoderma

• White colony, blue-green or yellow-green

• pale, tan, or yellowish on reverse
• Septate hyphae
• Conidiophores
• Phialides
• Conidia in ball shape

YEAST CULTURES

Saccharomyces cerevisiae

• Globose and ellipsoid in shape

• Flat, smooth and moist, glistening or dull
• Cream to tannish cream in color
• Asexual reproduction by budding
• No. of ascospores per ascus: 4 to 8
• Shape of ascospore – globose
• facultative anaerobe
• can ferment glucose and sucrose

Schizosaccharomyces pombe

• Rod-shape
• Colony appearance: venose; texture:
butyrous
• Asexual reproduction by fission
• No. of ascospores per ascus: 4-8
• Shape of ascospore - globose
• facultative anaerobe
• can ferment glucose and sucrose
Debaryomyces hansenii

• Globular
• White or yellowish in color
• Smooth, glistening or dull
• Asexual reproduction by budding
• No. of ascospores per ascus: 2
• Shape of ascospore – globose
• obligate aerobe
• variable fermentation on glucose and
sucrose

Pichia fermentans

• Ovoid to ellipsoid
• White or cream in color
• Asexual reproduction by budding
• No. of ascospores per ascus: 4
• Shape of ascospore – hat-shaped
• facultative anaerobe
• can ferment glucose

Rhodotorula spp.

• Orange to red in SDA

• Globose
• Smooth, glistening or dull
• Produce pink to red pigments
• Asexual reproduction by budding
• No. of ascospores per ascus: N/A
• Shape of ascospore – N/A
• Basidiospores
• obligate aerobe
• does not ferment carbohydrates