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Miniaturization and high-throughput screening is currently the focus of research activity in modern drug research facilities. Multiwell plates (or microplates) have been standard tools in analytical research and clinical diagnostic testing laboratories for several decades, as conventional test tubes were too cumbersome and slow (1,2). In the 1990s, microtiter plates were standardized, enabling more robotic and machine reading of their contents than ever before. This has made microplate readers more and more valuable. For standard enzyme-linked immunosorbent assay (ELISA) measurements and enzyme activity monitoring, stationary instruments with end point reading are sufficient (3). High-quality instruments also allow incubating of samples at a chosen temperature with shaking, enabling the cultivation of living cells and kinetic measurements of absorbance changes during specific time periods. Highly sophisticated readers specially designed (e.g., fluorescence assays or nephelometry) have been recently developed, some of them even equipped with an integrated pipeting system, and are able to read microplates of up to 1536 wells.
In yeast physiology and pathogenicity research, microplate readers have mainly been used for the identification of clinical yeast-like isolates (4) and serological diagnostics of Candida infections by immunocapture techniques detecting anti-Candida antibodies (5) and later also for antifungal susceptibility testing (6) or rapid detection of the effects of various chemicals (7).
This work is focused on the applications of a simple absorbance microplate reader (96-well format with temperature control and shaking option) in yeast physiology research. Its advantages and limitations for yeast growth curve measurements and the monitoring of media acidification are discussed. Sample experiments are taken mainly from an investigation of Saccharomyces cerevisiae cation homeostasis, regulated by the intracellular alkali-metal cation/H+ antiporters Kha1 and Nhx1 (8,9,10) and small GTPase Arl1 (11). A strain lacking all K+ exporting systems (ena1-4Δ, nha1Δ tok1Δ) was used for salt sensitivity tests.
Materials and Methods Yeast Strains and Cultivation MediaAll S. cerevisiae strains used in this study are derivatives of the W303-1A strain (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 mal10) (12). Additional mutations are listed in (Table 1).
Table 1. Saccharomyces cerevisiae Derivatives of W303-1A
The strains WAR0 and BWA0 were prepared by deletion of the ARL1 gene in W303-1A or BW31 (13), respectively. The deletion was performed by homologous recombination according to the protocol described by Güldener et al. (14). The TOB1 strain was prepared from the TOB strain (15) by excision of the KanMX deletion casette in the TOK1 locus according to the protocol also described in Reference 14,.
Cells were grown in YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose, 15 µg/mL adenine; 2% agar for solid media) or YNB medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose; 2% agar for solid media) with auxotrophic supplements (15 µg/mL each adenine, uracil, L-histidine, L-leucine, L-tryptophan) added after autoclaving. Media were sometimes supplemented with KCl or hygromycin B as indicated in the text. The pH was adjusted with KOH or HCl and buffered with 20 mM 2-[N-morpholino]ethane-sulfonic acid (MES, pH 5.5–6.5), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, pH 6.5–7.6), or N,N-bis[Hydroxyethyl]glycin (BICINE, pH 7.6–9.0). Media with a pH lower than 5.5 were not buffered.
Apparatus and Material Used for Absorbance MeasurementsAn ELx808 Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA) for 96-well ELISA plates, equipped with a shaker and a temperature control unit, was interfaced to a Dell computer running KC4 software (BioTek Instruments). The absorbance data were exported to MS Excel for further processing. A595 = 1.0 measured by this type of reader corresponds to approximately 8–9 × 107 cells/mL. It should be noted that this value is almost one order of magnitude higher than that of commonly used spectrometers, which is why the maximum absorbance reached seems much lower than that of manually measured growth curves ((Figure 1), A, D, and E, and 2A; cf. e.g., Reference 8,.
Figure 1.
Greinier Bio-One flat-bottom, 96-well PS microplates (Sigma-Aldrich, Prague, Czech Republic) were used for both growth curve measurements and acidification experiments. Plates were covered with a tightly attached sealing membrane (Breathe-Easy; Sigma-Aldrich) to prevent excessive evaporation and cross-contamination of the samples.
