Yeast Planktonic Culture
Materials Required
Principle
The protocol is based on established methods for cultivating Saccharomyces cerevisiae in planktonic (free-floating) culture, focusing on growth kinetics, high-throughput phenotyping, and quantitative assessment of cellular responses. Key principles include the use of defined media for controlled growth conditions, monitoring of optical density (OD) over time to assess exponential growth phases, and application of microplate reader-based assays for sensitive and scalable measurements of yeast growth and chemical sensitivity.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• YPD or synthetic complete medium
• Sterile water
• 96-well microplates
• Pipettes and tips
• Microplate reader (for OD measurement)
• Incubator set at 30°C
• Vortex mixer
• Centrifuge (optional for cell harvesting)
Experimental Procedure
• 2.Dilute the overnight culture to an initial OD600 of ~0.05-0.1 using fresh medium.
• 3.Transfer 100 μL of diluted culture into each well of a 96-well plate.
• 4.Place the plate in a microplate reader pre-warmed to 30°C and monitor OD600 every 15-30 minutes for 12-24 hours.
• 5.Record growth curves and calculate growth rates during the exponential phase.
• 6.For chemical sensitivity assays, add test compounds to wells at desired concentrations prior to inoculation and compare growth inhibition to control wells.
Troubleshooting
If no growth is observed:
• Ensure proper inoculum density, check viability via plating, verify media sterility and nutrient content.If inconsistent OD readings:
• Ensure consistent mixing before reading, avoid bubbles in wells, calibrate microplate reader regularly.If growth curves are not reproducible:
• Standardize starting OD, use fresh media batches, maintain consistent temperature and shaking conditions.References:
- [1]. Jung PP, et al. Protocols and programs for high-throughput growth and aging phenotyping in yeast. PLoS One. 2015;10(3):e0119807. [Content Brief]
- [2]. Olivares-Marin IK, et al. Saccharomyces cerevisiae exponential growth kinetics in batch culture to analyze respiratory and fermentative metabolism. J Vis Exp. 2018;(140):58192. [Content Brief]
- [3]. Toussaint M, et al. High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. Nat Protoc. 2006;1(4):1922-1928. [Content Brief]
- [4]. Marešová L, et al. Applications of a microplate reader in yeast physiology research. Biotechniques. 2007;43(5):667-672. [Content Brief]
- [5]. Gulati M, Lohse MB, Ennis CL, Gonzalez RE, Perry AM, Bapat P, et al. In vitro culturing and screening of Candida albicans biofilms. Curr Protoc Microbiol. 2018;50(1):e60. [Content Brief]
- [6]. Weerasekera MM, et al. Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development. Mem Inst Oswaldo Cruz. 2016;111(11):697-702. [Content Brief]
- [7]. Hung CW, et al. A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae. Sci Rep. 2018;8(1):11919. [Content Brief]
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