Maintenance and culturing baker's yeast

    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.

    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  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!