Working with yeast
Microbiologists have developed techniques for isolating
and maintaining strains in the laboratory
Strains: microorganisms of the same species that are descended from
a single cell and possess the same genotype
What do yeast require to grow in the laboratory?
How is sterile technique used to culture microorganisms?
What are the phases of yeast growth?
What do yeast require to grow in the laboratory?
Carbon source – sugars supply energy
Nitrogen source –required for DNA, proteins, more
Salts – osmotic balance
Vitamins – needed for many cell processes
Minerals – components of enzymes
Laboratory strains are grown on either solid or liquid media that supply
their nutritional requirements
Yeast can be grown in either rich or defined media
The composition of rich media is NOT defined
The most commonly used rich media is YPD
Y = Yeast extract (water soluble fraction from autolysed, or
self-digested, yeast)
P = Peptone (animal proteins digested with proteases)
D = Dextrose (i.e. Glucose)
Most mutant S. cerevisiae strains are able to grow in YPD
We’ll use defined media in future experiments (more later)
Sterile media is required to prevent contamination of cultures
Autoclaves use pressurized steam
to kill microorganisms in media
Typical conditions:
121 ˚C
30-40 psi
Filter pore size of
0.2-0.45 µm
prevents passage of
micro-organisms in
medium
What do yeast require to grow in the laboratory?
How is sterile technique used to culture microorganisms?
What are the phases of yeast growth?
Microorganisms are transferred with sterile toothpicks or loops
Shake a sterile toothpick
from its container, taking
care to touch only the tip of
one toothpick
Platinum loops are
sterilized between each
transfer
NOTE: Cool loops by
touching them to sterile
medium before transfers
Strains are maintained on plates containing medium and 1.5% agar
Cultures are diluted with a series
of streaks until single colonies are
detectable
Streak 1 zig-zag
Streak 2 vertical
Streak 3 zig-zag
A separate sterile toothpick is
used for each of the 3 streaks
For best results, use a light touch!
Three strains are maintained in
separate sectors on this plate
What do yeast require to grow in the laboratory?
How is sterile technique used to culture microorganisms?
What are the phases of yeast growth?
What are the phases of the yeast growth?
Yeast growth curves are typical of microorganisms
Cell densities can be used to identify the growth phase; exact
numbers are characteristic of the organism and its genotype
Log (Cell density)
Stationary
Death
Exponential
"log"
Lag
Freshly inoculated cultures adapt to the
environment during lag phase – their metabolites
"condition" the culture
Number of population doublings
Cells grow exponentially during log phase
(also referred to as exponential or vegetative growth)
Growth over the time interval t can be described mathematically:
Nt = N02t/DT
(DT is the doubling time)
Log (Cell density)
Stationary
Death
Exponential
"log"
Lag
Number of population doublings
Stationary phase – Nutrients are limiting
Cells assume a smaller, more resistant form – can last a long time
Cell density doesn't change – rate of division is similar to rate of cell
death
Log (Cell density)
Stationary
Death
Exponential
"log"
Lag
Number of population doublings
Spectrophotometers give a rough idea of cell numbers
Works by principle of light scattering, NOT absorbance
Light scattering is affected by cell size and geometry
OD600
Data for haploid
strain A364
Cell number ( x 107 )
Should be calibrated for each type of yeast
What happens at the higher ODs?
The number of cells in a yeast culture can be determined by
making a series of dilutions and then "spotting" some of each
dilution on a nutrient agar plate.
1. Make a series of dilutions from the original culture using
sterile deionized water.
Hint: It’s very useful to draw pictures
or simple flow diagrams in your lab
notebook when you do serial dilutions!
2. Spot small volumes of each dilution in a row on an agar plate.
Place the plate on top of a grid while you spot the dilutions
Don’t touch the plate with the tip – let surface tension of the
drop spread the cells
3. Incubate the plate for several days at 30 ˚C.
Each row
corresponds to a
different yeast
mutant
5 µL are spotted
from each dilution –
the number of
colonies in the most
dilute suspension can
be counted
Progressively more dilute suspensions
Colony size gives a rough idea about the growth rates of strains
Can you calculate the number of cells in the original culture
from this data?