Media
Yeast are commonly grown in the laboratory
using two general types of media, rich media and synthetic media.
Rich media is formulated to supplies yeast with ample metabolites, including
a nitrogen source and a carbon source, which can be varied. Synthetic
media can be formulated to be complete or selective and is more of a 'bare
bones' type media, containing known types and amounts of carbon and nitrogen
sources, essential minerals and vitamins as well as all or some of the
necessary amino acids and nitrogenous bases, depending on the particular
genetic background of the strain being used. To make media selective,
two approaches are taken; an 'add back' approach or a 'drop out' approach.
In the add back approach, the necessary amino acids and nitrogenous bases
for a particular strain are added to otherwise minimal media containing
only a carbon source, a nitrogen source, essential vitamins, and minerals.
The drop out approach starts with a minimal media base, and adds all the
potentially needed amino acids and nitrogenous bases except the one(s)
for which selection is desired. It is possible to have double-drop-out
media or to leave out even more components when desired. For this
laboratory, we use the drop out approach to made selective synthetic media.
Growth
Yeast are normally grown at 30oC
and can be grown on agar-containing plate media or in liquid media.
Yeast grown on plate media can be stored at 4oC as a for use
in ongoing experiments. If the plates contain rich media, the yeast
should be regrown every six weeks. If the plates contain synthetic
media, the yeast should be regrown every four weeks. Certain strains
bearing temperature-sensitive mutations should be regrown more often than
suggested by these guidelines. Liquid cultures are can be grown in
culture test tubes or flasks and are usually accompanied by rolling or
shaking to keep the yeast in suspension and the cultures well aerated for
optimal growth. Progress of growth us usually monitored by the absorbance
reading at 600 nm (principly a measure of light scattering) in a common
laboratory spectrophotometer (1 cm light path). When yeast are grown
in a media containing a fermentable carbon source (i.e. glucose), they
grow exponentially over time until the fermentable carbon source is depleated.
This point is the diauxic shift and marks a slower but still exponential
phase of growth that utilizes the non-fermentable carbon sources (ethanol)
produced by the yeast (see http://cmgm.stanford.edu/pbrown/explore/curve.html).
Once yeast stop growing, they have by definition entered stationary phase.
Most laboratory experiments are conducted using yeast grown and harvested
in early log-phase growth (OD600
= 0.5 - 1.0) although this can be altered to serve the purposes of the
experiment. If the intent of the experiment is to optimize yield
in the purification of proteins expressed in yeast during log-phase growth
or those induced after diauxic shift, harvesting cells at these periods
may make great sense.
Getting started
Yeast are normally cultured in liquid
for laboratory experiments. An overnight culture is started in the
appropriate media by innoculating the media with a 'glob' taken by a sterile
applicator stick from a petri plate stock of the desired yeast strain.
This liquid culture is placed in a roller or shaker overnight at the appropriate
temperature. The following day, this overnight culture is used to
innoculate liquid cultures for log phase growth. The amount of liquid
overnight culture used to innoculate the log-phase culture depends on the
conditions for growing the yeast, but generally the yeast should undergo
AT
LEAST two doublings or more before collection for the experiment (i.e.
they should start at OD600
0.1 if the cultures are to be collected at OD600
0.4). It is always easier to innoculate with less than needed and
add more innoculum than it is to deal with having innoculated the log-phase
culture too heavily at the on-set. Consider taking the OD600
reading after the first attempt to verify any 'guesses' you have about
how much to use, expecially when setting up multiple similar cultures.
The log-phase cultures are then placed in a roller or a shaker at the appropriate
temperature. The state of their growth can be monitored using OD600
readings until the desired reading is obtained. The yeast are then
collected by centrifigation for the purposes of the experiment.
Culture tips
Healthy laboratory yeast strains have a
dividing time of about 90 minutes in rich media and 140 minutes in synthetic
media (both with glucose) when grown at 30oC. The rate
of growth will be different if less easily metabolized carbon sources are
used.
The rate of the culture will be enhanced
with good aeration, so shaking vigorously can be of great help. The
best aeration (using normal microbial culture equipment) comes from using
an orbital shaker, where yeast are often shaken at 150-200 rpm, or even
250-300 rpm for very good aeration. Another way to enhance aeration
is to make sure the surface area of the interface of the media and the
air is maximal. Flasks optimize this when they are used with media
in volumes of only 10-20% of their total capacity. Full culture tubes
of course have the least aeration.
The media and stage of growth can
have a large influence on the organism, because of course this will determine
its environment and the organism will respond to this environment.
To optimize an experiment, some thought should be given to insure, even
testing, the ideal media and culture conditions. For example, different
carbon and nitrogen sources may influence the expression of the genes/proteins
under study, or the stage of the cell cycle or the growth stage may induce
or repress the genes of interest. Thus, use the literature, plan
your experiment well, and test and control for your assumptions!