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Protocols  >  Laboratory protocol  >  Yeast culture and preparation

Yeast culture and preparation

Laboratory protocol

1 Saccharomyces cerevisiae cell stimuli
Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) was cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml. S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) were cultured in complete medium till exponentially phase and treated as before.

2 Spore stimuli
In order to test homogeneous yeast populations, pure spore cultures were obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells were grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation was assessed by optical microscopy. Zymolyase (2 mg/ml) was used to digest ascum and liberate spores. We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed to damage the ascus, without touching the spore cell wall. Zymoliase was inactivated by heat (65°C for 2 minutes) and washed away carefully together with the remainings of the empty ascus, by resuspending twice the spore culture in 500 µl of distilled water centrifuging and discarding the supernatant; the spores were then resuspended to a concentration of 108 cells/ml. In order to exclude damage on the spore cell wall or effects on spore viability 100 µl of a culture of 1000 spores per ml was plated on YPD in triplicate, the spores showed 100% viability. The procedure used allows exposing DCs to the pure spore culture and to analyze the response to the spores alone, eliminating confounding effects of the presence of the ascus. Although from a mycological point of view the term ascospores might seem more appropriate than spores, we will use the term spores to specifically indicate that the effects we are observing are only due to the spores and not to the ascus.
We observed that after 24 hours of culture in RPMI 1640 (GIBCO-BRL), spores were germinating and the exponentially growing cells were starting to divide. Therefore any analyses performed at later time points would be affected by this altered state, while no interference should be present until 24 hours.

3 Candida albicans hyphae stimuli
Serotype A C. albicans strain SC5314 was cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture was treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this was treated as for yeast cells. It should be noted that in all the experiments performed in this study live microrganisms were used.

Protocol Steps:
  • Saccharomyces cerevisiae cell stimuli Saccharomyces cerevisiae strain SK1 (MATa/alpha HO gal2 cupS can1R BIO, Kane SM and Roth J. 1974 Bacteriol. 118: 8-14) is cultured in complete medium (YPD, 2% yeast extract, 1% peptone, 2% glucose) for 18 hours, then collected, washed twice with sterile water and resuspended at 108 cells/ml.
    S. cerevisiae strains BY4741 (genotype, Mata his3delta1 leu2delta0 met15delta0 ura3delta0) and BY4741 och1 (Mata his3delta1 leu2delta0 met15delta0 ura3delta0 OCH1::kanMX4) are cultured in complete medium till exponentially phase and treated as before.

  • Spore stimuli In order to test homogeneous yeast populations, pure spore cultures are obtained by using this strain whose sporulation efficiency is 100%. To prepare spores, cells are grown on YPD plates, replicated on SPOIV medium (2% potassium acetate, 0.25% yeast extract, 0,1% glucose) and sporulation is assessed by optical microscopy. Zymolyase (2 mg/ml) is used to digest ascum and liberate spores. [We initially performed experiments to evaluate zymoliase sensitivity of asci for this specific strain and we assessed that 15 minutes incubation was the minimum time needed to damage the ascus, without touching the spore cell wall.] Zymoliase is inactivated by heat (65°C for 2 minutes) and washed away carefully together with the remainings of the empty ascus, by resuspending twice the spore culture in 500 µl of distilled water centrifuging and discarding the supernatant; the spores are then resuspended to a concentration of 108 cells/ml. In order to exclude damage on the spore cell wall or effects on spore viability 100 µl of a culture of 1000 spores per ml is plated on YPD in triplicate. [The spores showed 100% viability.] The procedure used allows exposing DCs to the pure spore culture and to analyze the response to the spores alone, eliminating confounding effects of the presence of the ascus. Although from a mycological point of view the term ascospores might seem more appropriate than spores, we will use the term spores to specifically indicate that the effects we are observing are only due to the spores and not to the ascus.
    We observed that after 24 hours of culture in RPMI 1640 (GIBCO-BRL), spores were germinating and the exponentially growing cells were starting to divide. Therefore any analyses performed at later time points would be affected by this altered state, while no interference should be present until 24 hours.

  • Candida albicans hyphae stimuli Serotype A C. albicans strain SC5314 is cultured overnight in Saboraud medium at 28°C and then shifted to RPMI medium for 18 hours at 37°C to allow hyphal growth. The culture is treated as for yeast cells. After microscopical inspection of the purity of hyphal culture, this is treated as for yeast cells. [It should be noted that in all the experiments performed in this study live microrganisms were used.]
Yeast culture and preparation Graph

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