This study aimed to determine optimal yeast growth conditions for undergraduate student schedules. Yeast were cultured under varying conditions of inoculation mass, media composition, and flask type. An inoculation of 0.01g yeast grown in rich media at 25°C and 100rpm reached an optimal OD600 of 0.5-0.7 within 14-24 hours, suitable for protoplasting. Preliminary results showed yeast with OD600 over 0.7 resisted protoplasting. The initial inoculation mass was critical to prevent overgrowth. Overall, an inoculation of 0.01g yeast in rich media at 25°C and 100rpm provided optimal growth conditions for student timelines and downstream protoplasting.

1. Determining Optimal Growth Conditions for Yeast Protoplasting to
Fit Undergraduate Student Schedules
Authors: Levi Brewer, Vanessa Chappell, Diana Mirceta, Dr. Roger Sauterer
Abstract: This study investigates yeast growth under different culture conditions to find optimal growth at an
OD600 of 0.5-0.7 within a 14-24 hour timeframe, suitable for undergraduate student schedules. Finding optimal
growth conditions is essential for the next step of the study, which will generate yeast protoplasts (cell wall-free
cells) using the enzyme mixture Lyticase. Generation of yeast protoplasts is essential for cell organelle fractionation,
specifically for the mitochondria and nuclei to determine potential histone- mitochondrial binding interactions that
may be involved in programmed cell death in these organisms. Yeast cultures were grown in 2-liter flasks with 1-liter
culture medium under different conditions, including varying initial inoculation mass, culture media, and flask type,
while keeping temperature and rotation rate constant. Overall, the initial inoculation mass of 0.01g of yeast, a
temperature of 25 degrees C, and a rotation rate of 100 rpm were optimal for growth. The initial inoculation mass
was especially critical to prevent over-growth of yeast cultures. Optimal culture media was either 100% “rich” media
or a 75%/25% “rich” to “poor” media ratio. Culturing in baffled vs. standard Erlenmeyer flasks showed no clear
effect on growth. Preliminary yeast protoplasting experiments indicated that culture OD600 must be less than 0.7 for
successful breakdown of the cell wall and that yeast from cultures with an OD600 higher than 0.7 are highly resistant
to the Lyticase enzyme mixture used for protoplasting.
I. Introduction
Saccharomyces cerevisiae, more commonly
known as yeast are eukaryotic microorganisms that
belong to the kingdom fungi. The fungal kingdom
has showed significant evolutionary changes over
the course of millions of years that has made them
distinct from their metazoan relatives, plants and
animals. Although yeast fall in their own kingdom,
many biological processes have remained
conserved between them and other eukaryotes.
Programmed cell death is a process in eukaryotes
that has conserved features and plays important
roles in embryonic development, organismal
maintenance, and elimination of cells with damaged
or irreparable DNA. The evolution of this process is
not completely understood among metazoans.
Although the details and mechanisms of the process
differ, it has been found that molecules involved in
apoptotic processes of animal cells have been
broadly conserved among the three eukaryotic
kingdoms. Previous studies have indicated that
histones released into the cytosol of animal and
plant cells bind to mitochondria and induce
cytochrome C release, which has been correlated
with cell death processes. The overall goal of this
project is to determine if histones released from
damaged nuclear DNA bind to yeast mitochondria
and induce cytochrome C release, as they do in
mammalian cells and angiosperm plants. Histone-
mitochondrial interactions of yeast cells will help
determine whether or not this is a conserved
mechanism among all eukaryotic kingdoms. The
first step in determining the histone-mitochondrial
relationship in yeast cells is finding optimal growth
conditions of yeast at an OD600 of 0.5-0.7 within a
14-24 hour timeframe suitable for student
schedules, indicating log phase growth of a healthy
culture that is suitable for protoplast generation.
Yeast cell walls must be removed for organelle
isolation, a process known as cell protoplasting.
Yeast protoplasts will be obtained through the use
of Lyticase, an enzyme mixture that will digest the
cell wall. Generation of protoplasts is required for
organelle fractionation, which will be critical for
the next step of the research. Upon obtaining
successful yeast protoplasts, future experiments
will work to isolate yeast mitochondria, purify
yeast histones, and test mitochondria for histone
binding and determine if cytochrome C is released.
We tested culture conditions and media mixtures to
optimize growth in the required time frame.
II. Methods
Saccharomyces cerevisiae: Fleischmann’s yeast was bought
at a retail store. All cultures were grown by incubating yeast
pellets in 1 L of culture media in a 2 L flask on a rotary
shaker at room temperature and 100 rpm. Three variations of
growth conditions were used to obtain the desired OD600:
initial inoculation mass, media composition, and baffled
versus un-baffled flasks. Two different types of media were
prepared. Rich media was made with 1% Bacto-Peptone, 2%
glucose, 3 mM potassium phosphate monobasic, 137 mM
sodium chloride, and 10 mM sodium phosphate dibasic and
autoclaved at 120 degrees Celsius for twenty minutes on the
liquid cycle. Poor media was prepared the same, except
glucose was at 1% and 312 mM ammonium nitrate took the
place of Bacto-Peptone as a nitrogen source. After
autoclaving at 120 degrees Celsius on the liquid cycle, 1%
100X mineral stock (3 mM calcium chloride, 4 mM ferrous
sulfate, 163 mM magnesium sulfate, 30 mM manganese
sulfate, and 2-3 drops concentrated sulfuric acid to prevent
precipitation) was added to all culture media. To determine
the effect initial inoculation mass had on yeast cultures, 1
liter cultures of rich media were inoculated with 0.01, 0.02,
0.05, 0.07, and 0.14 grams of Fleischmann’s yeast (see
Figure 1). To determine the effect media composition had on
yeast growth, 1 liter cultures of rich and poor media were
made, as well as 1 liter mixtures of 75% rich/25% poor, 50%
rich/50% poor, and 25% rich/75% poor were prepared (see
Figure 2). 0.01 grams of yeast were used to initially
inoculate the culture media. To determine the effect that the
type of flask had on growth, some cultures were grown with
baffled flasks while others were grown in normal flasks (see
Figure 3). Absorbance of the yeast at 600 nm was recorded
periodically, and when the yeast measured a 0.5-0.8
absorbance reading on a spectrometer the cultures were
harvested by centrifugation at 4600 x g in a GSA rotor. The
resulting pellet was stored in an equal volume of Lyticase
buffer (2% Bacto-Peptone, 0.7 M sorbitol, 10 mM Tris-base,
pH 7.5) at -20 degrees Celsius.
Protoplasting: To remove the cell walls of the yeast,
harvested yeast pellets were washed and suspended in
Lyticase buffer before being incubated with Lyticase (Sigma-
Aldrich) on a rotary shaker at 150 rpm and 37 degrees
Celsius. Prior to Lyticase addition, 60 mM 2-
mercaptoethanol was added to the cell suspension. Two
Lyticase units were added for every OD unit calculated from
the dilution by diluting the cell suspension before
multiplying OD units/mL by the total volume in mL of the
suspension in the tube. Absorbance was monitored on a
spectrometer alongside a control with no Lyticase added, and
the data recorded (see Figure 4).
III. Results
Figure 1: Yeast growth based
on initial inoculation mass.
All cultures incubated at 25
degrees Celsius and 100
rpms. OD in log-phase
growth should be less than
0.8 in 12-16 hours.
Inoculated with 0.01-0.02 g
allowed optimal growth in
desired time frame.
Figure 2: Yeast growth in
varying media composition. All
cultures were grown at 25
degrees Celsius and 100 rpm
with initial 0.01 g inoculation.
100% poor, 75% poor, and
50% poor media went out of
log-phase growth at low OD600.
Best culturing obtained with
75% rich and 100% rich media.
Figure 3: Yeast grown in
standard Erlenmeyer or
baffled culture flasks in 3
separate experiments. All
cultures used 0.01 g starting
inoculation mass and were
grown at 25 degrees Celsius
and 100 rpm. Data shows no
clear differentiation in
growth between flask types.
Figure 4: Effect of OD600
of culture at time of
harvest on yeast
protoplasting. Succesful
protoplasting should
reduce OD600 to 20% of
control. Yeast cultures
with initial OD600
exceeding 0.9 failed to
protoplast completely.
IV. Conclusion
In this experiment we attempted to grow yeast in time frames suitable for undergraduate schedules. We
performed growth curves on yeast using variations in culture media, initial inoculation mass and flask type. For all
experiments we kept incubation temperature and rotation rate constant. Poor media showed little signs of growth
regardless of variations in conditions and therefore is not suitable for growing large quantities of yeast. We saw no
significant or consistent differences when comparisons were made regarding yeast grown in baffled versus un-
baffled flasks. When cultures were grown with initial inoculation mass above 0.0 15g-0.0 2g the yeast over grew.
Preliminary trials of yeast protoplasting indicate yeast grown to OD600 beyond 0.7 proved to be impossible to
protoplast using the Lyticase enzyme mix. Overall, the initial inoculation mass of 0.01g of yeast grown in standard
conditions at a rotation rate of 100 RPM using rich media or a 75/25 Rich/Poor mixture were optimal for growth to
OD of 0.5 to 0.7 and eventual protoplasting. Investigation is still being done on variations in rotation rate and
temperature to accelerate yeast growth without causing complications with yeast protoplasting.