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 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) The composition of rich media is NOT defined 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 psi Filter pore size of 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

Three strains are maintained in separate sectors on this plate Strains are maintained on plates containing medium and 1.5% agar Cultures are diluted with a series of streaks until single colonies are detectable A separate sterile toothpick is used for each of the 3 streaks For best results, use a light touch!

What do yeast require to grow in the laboratory? How is sterile technique used to culture microorganisms? What are the phases of yeast growth?

Lag Exponential "log" Stationary Death Number of population doublings Log (Cell density) 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 Freshly inoculated cultures adapt to the environment during lag phase – their metabolites "condition" the culture

Cells grow exponentially during log phase (also referred to as exponential or vegetative growth) Growth over the time interval t can be described mathematically: N t = N 0 2 t/DT (DT is the doubling time) Lag Exponential "log" Stationary Death Number of population doublings Log (Cell density)

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 Lag Exponential "log" Stationary Death Number of population doublings Log (Cell density)

OD 600 Cell number ( x 10 7 ) 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 Should be calibrated for each type of yeast What happens at the higher ODs? Data for haploid strain A364

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. Progressively more dilute suspensions 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 Can you calculate the number of cells in the original culture from this data? Colony size gives a rough idea about the growth rates of strains