MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Exercise 1 Study Guide

Examination of Mold and Yeast Cultures

Introduction

Molds and yeasts are intimately associated with our daily lives. Aside from causing
spoilage of food and deterioration of other organic matter, they may be very useful, too.
Some of the specific products made by yeasts and molds are antibiotics, enzymes,
vitamins, organic acids, beer, wine, whiskey and many other alcoholic beverages.

However, fungi may infect many agricultural crops, and one of the harmful effects
of fungal growth is mycotoxin production. When consumed, these secondary metabolites
can be injurious, even fatal, to man and animals. Examples of mycotoxins are aflatoxin,
fumonisin, penicillic acid, ochratoxin, patulin, to name a few.

Enumeration of fungi entails the use of culture media that will inhibit the growth of
bacteria. This is attained by addition of tartaric acid or antibiotics. Furthermore, the
restriction of mold colony spreading for improved enumeration is achieved through the
inclusion of dichloran.

Morphological as well as cultural characterization is important in the identification

of yeasts and molds. The slide culture technique and spore staining aid in the microscopic
observation of molds and yeasts, respectively. Moreover, the ability of yeasts to oxidize
different sugars is an important characteristic that is considered in the identification of this
microbial group.

Expected Learning Outcomes

At the end of the exercise, the student must be able to:

1. employ the different techniques in studying microscopic and cultural morphology

of yeast and molds (i.e. slide culture, wet mount, and ascospore staining
technique)

2. differentiate the metabolic properties of yeasts, in terms of sugar utilization

3. enumerate yeasts and molds in food samples

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Learning Activities:

1. Read the procedures for the examination of mold and yeast cultures listed below;

2. Watch the video demonstration uploaded in our Google classroom;

3. Post-lab presentation of the exercise leader and Q&A session/discussion forum;

and

4. Answer the study questions at the end of this exercise.

Procedures

Activity 1: Enumeration of yeasts and molds

Task: Watch short video on “Enumeration of yeasts and molds from food samples” (video
demonstration provided in our Google classroom)

1. Weigh 10g or mL of well-mixed (if liquid) or macerated (if solid) food samples and
transfer to 90mL 0.1% peptone water (1st dilution).

2. Transfer 1mL from (1) to 9mL 0.1% peptone water (2nd dilution). Prepare another
dilution by transferring 1mL from the 2nd dilution to 9mL 0.1% peptone water.

3. Inoculate 0.1mL of the three dilutions in duplicate onto solidified PDA with antibiotics.
Agar plates must be dried in a pre-disinfected laminar flow hood, for at least 15 min
before inoculation.

4. Spread the inoculum over the entire surface of the agar using sterile bent glass rod.

5. Incubate plates in an upright position for 5 days at 25-300C. Do not disturb until
colonies are counted.

6. Count colonies containing 10-150 colonies. If mold overgrowth has occurred, count
from the underside of the plate.

7. Report counts as colony-forming units (cfu) per g or mL of sample.

cfu/mL = average number of cfu x dilution factor if valid counts come from
volume plated one dilution

or
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

N= _______∑ C _________ if valid counts come from two

[ (1 x n1) + (0.1 x n2)] d consecutive dilutions
volume plated
where:
N = number of colonies per ml or gram of sample
∑ C = sum of all of the colonies in all plates counted
n1 = number of plates in the lower dilution counted
n2 = number of plates in the next higher dilution counted
d = dilution from which the first counts were obtained

Images:

Figure 1. Growth of yeasts on PDA obtained from bread sample

(left: 10-1, middle: 10-2, right: 10-3)
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 2. Growth of molds and yeasts on PDA from fruit juice sample
(left: 10-1, middle: 10-2, right: 10-3)

Activity 2: Morphological examination of mold reference cultures

Task: Watch the short video on using “Lactophenol cotton blue” (video demonstration
provided in our Google classroom)

Wet Mount Technique

Using a loop, transfer some mold mycelia and fruiting parts to a drop of lactophenol
cotton blue on a glass slide. Tease the mycelia with a coverslip and wire loop to separate
them into hyphae. Cover with a coverslip. Examine under the low power objective, then
under the high-power objective. Sketch the molds as seen under the microscope.
Describe the specimen and take note of certain characteristics or attributes as listed
below:
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Record the following:

⚫ Hyphae: septate or non-septate; types of branching of hyphae

⚫ Asexual spores: types of spores (conidia, sporangiospores, or arthrospores),

shape, color, arrangement

⚫ Special structures and location: stolons, rhizoids, foot cell, sexual spores

Task: Watch short video on how to perform the “Slide culture technique” (video
demonstration provided in our Google classroom)

Slide Culture Technique

Place a sterile glass slide in a sterile Petri dish lined with sterile paper towel.
Aseptically, cut pre-solidified PDA plate into 1cm2 squares. Carefully lift a small square
and place it on the sterile slide. Inoculate a small amount of the mold culture. Place a
sterile coverslip over the agar. Be sure to leave a space between the slide and the
coverslip for aeration. Moisten the paper towel with sterile water to ensure that the agar
will not dry out during the incubation period. Examine the slides at the next laboratory
period.

When you have finished with your slides and cover slips, deposit in a beaker for
sterilization. This prevents spreading of fungal spores which can contaminate the
laboratory.
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Images:

Figure 3. Appearance of Aspergillus niger on PDA slant

Figure 4. Wet mount of Aspergillus niger under HPO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 5. Appearance of Fusarium oxysporum on PDA slant

Figure 6. Wet mount of Fusarium oxysporum under HPO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 7. Appearance of Penicillium purpurogenum on PDA slant

Figure 8. Wet mount of Penicillium spp under EM (1000x)

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 9. Appearance of Rhizopus stolonifer on PDA slant

Figure 10. Wet mount of Rhizopus spp under HPO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 11. Appearance of Trichoderma on PDA plate

Figure 12. Wet mount of Trichoderma spp under OIO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Activity 3: Cultural and morphological examination of yeast reference cultures

1. Observe and take note of the following:

a. Pigment production

b. Surface growth (smooth or rough, dull or glistening)

2. Inoculate all specimens into glucose broth, lactose broth, and sucrose broth. Incubate
at 30oC for 3-5 days.

3. Observe which yeasts form pellicle. Also take note of the sugars which were utilized
by each of them.

Task: Watch a short video on “Schaeffer-Fulton staining”, which is the same procedure
performed for ascospore staining in yeast cells. (video demonstration provided in
our Google classroom)

4. Examine the yeast specimens by following the procedure below:

a. Prepare smears of the organisms, air dry and fix by heat.

b. Cover the smear with absorbent paper to prevent accumulation of artifacts during
staining.

c. Flood the smear with malachite green for 15 mins.

d. Wash thoroughly with tap water.

e. Counterstain with safranin for 30-60 sec. Wash slide and blot dry.

f. Examine the slides under the oil immersion objective. Spores are green, cells are
red.

5. Record the following:

a. Shape and size of cell

b. method of asexual reproduction (budding, fission or combination)

c. formation and number of ascospores

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Images:

Figure 13. Sugars used prior to inoculation of reference yeast cultures

Figure 14. Growth of yeast on broth characterized by turbidity and pellicle formation
(left) and sediment (right)
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 15. Growth of Saccharomyces cerevisiae on YDC plate

Figure 16. Saccharomyces cerevisiae under OIO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 17. Growth of Schizosaccharomyces pombe on YMA

Figure 18. Schizosaccharomyces pombe under OIO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Figure 19. Growth of Rhodotorula rubra on SDA

Figure 20. Rhodotorula spp under OIO

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Figure 21. Growth of Pichia fermentans on YMA

Figure 22. Pichia fermentans under OIO

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

References

Jay, J. M., Loessner, M.J. & Golden, D.A. 2005. Modern Food Microbiology. 7th ed. New
York, NY: Springer.
Matthews, K.R., Kniel, K.E. & Montville, T.J. 2017. Food Microbiology. 4th ed. Washington,
DC: ASM Press.
Ryu, D. & Wolf-Hall, C. 2015. Yeasts and Molds. In: Compendium of Methods for the
Microbiological Examination of Foods. 5th ed. Salfinger, Y. & Tortorello, M.L. (Eds.),
Washington, DC: APHA Press.
U.S. Food and Drug Administration. 2001. Chapter 18. Yeast, Molds, and Mycotoxins. In:
Bacteriological Analytical Manual. Retrieved from
http://www.fda.gov.food/foodscienceresearch/laboratorymethods/ucm071435.ht
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

EXERCISE 1
Examination of Mold and Yeast Cultures

Name: ______________________________

1. Sketch of mold cultures as seen under the microscope. Label the visible parts
(2 pts each: 1 pt drawing and label, 1 pt for description)

Specimen: Aspergillus

Drawing Description

(Total Mag.: ______x)

Specimen: Fusarium

Drawing Description

(Total Mag. : ______x)

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

Specimen: Penicillium

Drawing Description

(Total Mag.: ______x)

Specimen: Rhizopus

Drawing Description

(Total Mag.: ______x)

Specimen: Trichoderma

Drawing Description

(Total Mag.: ______x)

MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

2. Characteristics of mold specimen (0.25 pts per answer)

Asexual
Isolate Hyphae Special structure/s
Spores

Aspergillus

Fusarium

Penicillium

Rhizopus

Trichoderma

3. Appearance of surface growth of yeast cultures. (0.5 pts each)

Yeast Appearance
Saccharomyces
Schizosaccharomyces
Rhodotorula
Pichia
MCB 180 INTRODUCTORY FOOD MICROBIOLOGY

4. Microscopic observation of the yeast cultures (0.25 pts each)

Shape of
Yeast Method of reproduction
ascospores
Saccharomyces

Schizosaccharomyces

Rhodotorula

Pichia

Study Questions/Problem Solving

1. Explain why plates are incubated in an upright position. (2 pts)

2. The spread plate method is preferred over the pour plate technique when isolating
for yeasts and molds. Why? (2 pts)

3. What is the cfu/g or cfu/mL in each sample if the following counts were obtained after
spread plating on Potato Dextrose Agar with chloramphenicol? (3 pts each)

Dilution Mongo bread Melon juice

10-1 201, 257, 292 105, 89, 94
10-2 59, 41, 30 9, 7, 5
10-3 5, 3, 8 0, 1, 0