Laboratory 2 : Yeasts Important in Foods: Microscopic and Macroscopic

Identification of Yeasts from Food.

Introduction :

Yeasts is a type of non motile fungi that contains only a single eukaryotic
cell.Some species may also develop multicellular characteristics by forming strings of
connected budding cells known as pseudohyphae or false hyphae .It has oval,
spherical or elongated shape.Most of them are in the phylum Ascomycota, only few in
Basdidiomycota..Like most of the fungi, yeasts also perform aerobic respiration, but
in the absence of air they derive energy by fermenting sugars and carbohydrates to
produce ethanol and carbon dioxide. Reproduction occur mostly through budding
formation, even though some species reproduce through binary fission.Some yeast
form ascospores,ascospore is a spore contained in an ascus or that was produced
inside an ascus. This kind of spore is specific to fungi classified as ascomycetes.

Objectives :

1) To allow the students to make pure culture studies of some of the common
yeasts found in foods.
2) To make isolates from naturally.
3) Become familiar with staining and observing yeasts morphology.

Materials :

1) Pure cultures (on Potato Dextrose Agar plates) of the following genera yeasts are
provided :

(a) Saccharomyces cerevisisae.

(b) Pichia spp.
(c) Rhodotorula gracilis

2) Metheylene blue staining

3) Microscope
4) Food sample
5) PDA plates.

Methods :

1) Macroscopic examination (Table 2.1)

The characteristics of given culture was studied on PDA and observed the
colonial morphology in the terms of :

(a) color of the colonies

(b) texture
(c) smell

2) Microscopic examination (Table 2.2)

1. The heat fixed film was prepared.
2. The film was covered with metheylene blue solution for 1 to 2 minutes.
 3. Then, the slide was tilted to allow the excess stain to run off into the staining
tub and washed gently with the water bottle.
4. Let the slide dry.
5. The stained yeasts was examined with microscope using 40x and oil immersion
respectively.
6. Observation should cover :

(a) shape typical yeasts

(b) budding cells
(c) ascospore
(d) etc.

3) A loopful of the food samples was streaked on the PDA. The plate was incubated
at room temperature for 2 to 3 days and observed microscopically as in Table 2.5.
Results :

1. Colonial Macroscopic Examination

Table 2.1: Colonial macroscopic examination

Yeasts Colonial macroscopic

color texture smell

Sacchromyces yellowish brown rough, shiny smelly

cerevisiae

Pichia spp yellowish shiny smelly,pungent

Rhodotorula brown rough,shiny smelly

gracilis
 2. Microscopic Examination

Yeasts Microscopic Illustration

shape budding cells acrospore

spherical round or oval globose (sphere)

Sacchromyces cerevisiae

round
ascospores
hat-shaped multilateral
Pichia spp

oval oval non-ascospores

Rhodotorula gracilis

Table 2.2: Microscopic examination

 3. Isolation of yeast from food sample
My food sample : tapai and bread
Table 2.5: Colonial macroscopic examination from food sample

My yeasts Colonial macroscopic

color texture smell

tapai creamy thread-like unpleasant

My yeasts Microscopic examination Illustration

shape budding cells ascospore

spherical round or oval globose

(sphere)

tapai
Discussion :

Based on the results obtained,only the spores can be seen in Rhodotorula sp.
under magnification 40x100. As for Saccharomyces cerevisiae and Pichia spp both
spores and buds cannot be seen clearly because it might be too thick while inoculating
the yeast culture.Rhodotorula sp. are pigmented basidiomycetous yeasts in the family
of Sporidiobolaceae. It also produce pseudohyphae that are rarely
present.Saccharomyces cerevisiae is a fungal group Ascomycota (the ascus-forming
fungi) and it supposed to have small bud at first, but enlarge progressively and
eventually separate from the mother cell by formation of a septum.Pichia spp also
ascus forming fungi that reproduce through budding at the end of the cells and it
supposed to form hyphae or pseudohyphae.

For acid production from sugar fermentation tests,Saccharomyces cervisiae can

ferment hexose sugar in both aerobic and anaerobic condition.Pichia spp is capable of
both aerobic and anaerobic fermentation by converting xylose sugar to
ethanol.Rhodotorula spp produce urease enzyme but it cannot ferment carbohydrates.
The gas production from sugar fermentation tests can be observed by the formation of
carbon dioxide bubble on top of fermentation mixture.

Potato Dextrose Agar (PDA) is mainly used for the cultivation of fungi but it is
also used for a general purpose medium for yeasts and moulds that can be
supplemented with acid or antibiotics to inhibit bacterial growth.Glucose-Yeast
Extract Agar is recommended for the selection and enumeration of yeasts and moulds
from foodstuffs.Therefore it is more advisable to use GYEA plates besides PDA to
obtain more accurate results.Yeasts have a great industrial and economic importance
and some of them are involved in food spoilage in beer and dairy products.
Fermentation of yeast can help in food making such as tapai and bread.

Conclusion :

In this experiment,students are able to make pure culture studies of some of common
yeasts found in foods. Students are also able to make isolates from naturally
contaminated foods such as tapai.Students also become familiar with staining and
observing yeasts morphology.

References :
1) http://www.antimicrobe.org/f16.asp
2) Fundamental Food Microbiology (Fifth Edition), Bibek Ray, Arun Bhunia,
2013,CRC Press.
3) Microbiology Principles And Explorations (9th Edition), Jacquelyn G. Black,
Laura
J. Black,2015,John Wiley & Sons.