Hts xt

25 µL of the incubation mixture with the bacteria and antimicrobials was placed on IR-transparent Si-microtiter plates with 96 wells (Bruker Optics, Ettlingen, Germany) and dehydrated for 2.5 h using a vacuum desiccator (ME2, Vacuubrand, Wertheim, Germany). The FTIR spectra were acquired using an HTS-XT associated with a Vertex-70 spectrometer (Bruker Optics, Ettlingen, Germany) at a resolution of 2 cm⁻¹ between 400 to 4000 cm⁻¹. Each final spectrum results from the average of 40 spectra acquisitions.

Briefly, 25 μL of pre-diluted serum (1 to 10 in Milli-Q water), from each serum sample, was pipetted to a 96-well Si plate and then dehydrated in a desiccator for 150 min under vacuum (Vacuubrand, ME 2, Wertheim, Germany). Spectral data were collected using an FTIR spectrometer (Vertex 70, Bruker, Billerica, MA, USA) equipped with an HTS-XT (Bruker) accessory. Each spectrum represented 64 coadded scans, with a 2 cm⁻¹ resolution, and was collected in transmission mode, between 1500 and 1700 cm⁻¹. The first well of the 96-well plate did not contain a sample, and the corresponding spectra were acquired and used as the background, according to the HTS-XT manufacturer. All spectra used in the following sections were submitted to atmospheric compensation.

Of each sonicated fungal suspension, 8 μl was transferred to an IR-light-transparent Silicon 384-well microtiter plate (Bruker Optik GmbH, Germany). The samples were dried at room temperature for 45 min to form films suitable for FTIR analysis. FTIR measurements were performed using a High Throughput Screening eXTension (HTS-XT) unit coupled to a Tensor 27 spectrometer (both Bruker Optik GmbH, Germany). The spectra were recorded in the region between 4000 and 500 cm⁻¹ with a spectral resolution of 6 cm⁻¹ and an aperture of 5.0 mm. For each spectrum, 64 scans were averaged.

Prior to sample loading, a ‘96 well’ silicon transmission plate (Bruker, www.bruker.com) was pre-washed in analytical grade methanol three times followed by dH₂O three times, and the plate dried. To 30 mg (±1 mg) of ground leaf tissue 1.5 ml of sterile ultra pure dH₂O was added and the sample thoroughly mixed. Thirty microlitre homogenates of each biological replicate were loaded onto the pre-washed sample plate to generate technical replicates, and three readings were taken from each sample spot to serve as analytical replicates. The plate was oven dried at 50 °C until samples were completely dry prior to loading into the motorised high-throughput stage (HTS-XT; Bruker) attached to a Bruker Equinox 55 FT-IR (Winder et al., 2004, 2006 ). The FT-IR transmission mode protocol was based precisely on the method previously described by Harrigan et al. (2004) (link). Spectra were collected over the wavelength range of 4000–600 cm⁻¹ with a resolution of 4 cm⁻¹. To improve signal-to-noise ratio, the resulting spectra were co-added and averaged. Spectra were displayed in terms of absorbance as calculated using Opus 4 software, which uses the background spectrum of the reference well subtracted from the spectra recorded from the sample wells.