96 well plates

The growth curves were obtained using Brucella broth (BB) (Difco, Wokingham, UK) supplemented with different concentrations of FBS (1%, 5%, or 20%) or in the absence of nutrients using 0.89% saline solution (SS). To start the incubation to determine the growth curves, H. pylori and Candida strains were adjusted to an optical density (O.D.) of 0.1 at 600 nm at time zero. Then, 200 µL of each strain suspension was placed in 96-well plates (Thomas Scientific, Swedesboro, NJ, USA) and incubated under microaerobic conditions in Infinite M200 PRO equipment (TECAN, Männedorf, Switzerland). The microaerobic condition was obtained using CampyGen sachets (Thermo Scientific, Waltham, MA, USA). The absorbance of H. pylori cultures was measured every 8 h for 72 h, while the absorbance of yeast cultures was measured every 2 h for 50 h. Absorbances were measured at 600 nm using the same Infinite M200 PRO equipment. All assays were performed in triplicate.

In order to make sure to start the co-cultures of H. pylori and Candida strains when both microorganisms were in their respective exponential phase of growth, this assay was necessary to determine the incubation time required by each microorganism to reach the desired phase of growth. Each strain of either H. pylori or Candida spp. used in the present work was suspended at an optical density (OD) of 0.1 at 600 nm in Brucella broth (BB) (Difco, Wokingham, UK) supplemented with 5% FBS (BB-5%FBS), and 200 µL of each suspension were placed in a well of 96-well plates (Thomas Scientific, Swedesboro, NJ, USA) and plates incubated at 4 °C, 25 °C, 37 °C or 40 °C in an Infinite M200 PRO equipment (TECAN, Männedorf, Switzerland), in which the microaerobic conditions were obtained using CampyGen envelopes (Thermo Scientific, Waltham, MA, USA). The growth of each strain was monitored by OD at 600 nm every 8 h during 72 h for the bacterial strains and every 2 h during 50 h in the case of yeast strains. Additionally, in the case of H. pylori, 5 µL aliquots were obtained at each time OD was measured, and a Gram-staining was prepared to monitor possible morphological changes in bacterial cells caused by the incubation conditions.