Omnilog plate reader

Biolog Phenotype Microarrays were performed as previously described^{36 (link)}. In short, PAO1 and PAO1 ΔmexZ strains were streaked on LB agar plates and incubated at 37 °C. Cells were swabbed from the plates and suspended in IF-0 GN Base (inoculation fluid) at a density corresponding to 42% transmittance in the Biolog turbidimeter. The cell suspensions were diluted in IF-0 minimal medium containing Biolog redox dye mixture D (tetrazolium), and aliquots were added to the two different carbon-source plates; PM1 and PM2A. The plates were incubated at 37 °C in an OmniLog plate reader (Biolog) for 48 h, and growth/respiration was measured kinetically by determining the colorimetric reduction of the tetrazolium dye. Export of OmniLog data was performed using OmniLog OL_FM/Kin 1.20.02 software (Biolog). The average area beneath each kinetic curve was used for analysis. Total catabolic function was calculated as previously described^{37 (link)}.

Phenotype MicroArray (Biolog, Hayward, CA) experiments were performed in duplicate according to the manufacturer’s instructions [61 (link), 62 (link)]. P. aeruginosa strains were streaked on LB agar plates and incubated at 37 °C until colonies appeared on the plates (16–30 hours (h)). Cells were swabbed from the plates and suspended in IF-0 GN Base (inoculation fluid) at a density corresponding to 42 % transmittance in the Biolog turbidimeter. The cell suspensions were diluted 1:6 in IF-0 minimal medium containing Biolog redox dye mixture D (tetrazolium), and 100-μL aliquots were added to carbon-source plates (PM1 and PM2A). For the nitrogen-source plate (PM3B), inoculations were supplemented with 30 mM glucose and 2 μM ferric citrate. The plates were incubated at 37 °C in an OmniLog plate reader (Biolog) for 72 h, and growth/respiration was measured kinetically by determining the colorimetric reduction of tetrazolium dye. Export of OmniLog data was performed using OmniLog OL_FM/Kin 1.20.02 software (Biolog). The average area beneath each kinetic curve was used for analysis. OD600 was measured after 60 h of growth at 37 °C, and relative catabolic capacities of individual substrates were determined as previously described [63 (link)].

C. auris strains were pregrown on YPD agar plates incubated at 30°C for 24 h. The high-throughput phenotypic screen was performed using Biolog Phenotype MicroArrays for microbial cells (PMs) according to the manufacturer’s protocol for yeasts (Biolog, Inc., Hayward, CA, USA), and as reported previously (39 (link)). The following plates were used: PM1, PM2 (carbon sources), PMs 3, 6, 7, and 8 (nitrogen sources), PM4 (phosphorus and sulfur sources), PM9 (osmolytes), and PMs 21 to 25 (chemical sensitivity). The plates were incubated at 37°C for 24 to 48 h in an OmniLog plate reader (Biolog, Inc., Hayward, CA, USA), and metabolic activity was recorded automatically every 15 min at an OD of 750 nm.