Quarter strength ringer s solution

The soil samples (20 g) were shaken for 30 min in 180 mL of quarter-strength Ringer's solution (Oxoid, Milan, Italy) containing 0.324 g of tetrasodium pyrophosphate to detach the bacteria from the soil particles. After the soil particles were allowed to settle for 15 min, the solution was diluted tenfold in series. Potential PAH-degrading microorganisms were isolated by spreading the serial dilutions on a minimal selective solid medium (MSSM), containing 1% natural nutrients (soil extract obtained from freshly collected meadow soil) [27 (link)], 0.5% contaminated soil aqueous extract (CSAE) obtained as above reported, and 1.5% agar bacteriological (Oxoid). The plates were incubated at 28°C for 15 days.
The isolated colonies that were able to grow by streaking on a minimal solid medium containing 1.5% CSAE as the sole carbon source were further selected.
A serial enrichment strategy was carried out that inoculated the selected microbial isolates in a minimal selective liquid medium (MSLM) containing increasing concentrations of CSAE (up to 50%), with or without the addition of nutrients (1% soil extract). The growth of the isolates in the liquid culture was determined by a spread plate count method using MSSM containing the same amount of CSAE (up to 50%).

Rhizosphere samples were collected from two different sites in the northwest of Morocco (33°32′ 00″N, 7°35′00″W) in November 2018. In each field, five different oat plants were randomly selected for sampling, collected and stored at 4°C before analysis (Romano et al., 2020 (link)). The main physical and chemical properties of the rhizosphere samples are summarized in Table 1. For bacterial isolation, 10 g of the samples was shaken for 30 min in 90 ml of quarter strength Ringer’s solution (Oxoid, Milan, Italy) containing tetrasodium pyrophosphate (16% w/v) as previously described (Ventorino et al., 2014 (link)). Dilutions were performed from each sample followed by streaking in modified Pikovskaya’s (MPVK) without yeast extract (Nautiyal, 1999 (link)) and containing CaHPO₄ as the only inorganic phosphate source. After incubation for 7 days at 30°C, colonies distinguished based on phenotypic features such as morphology and biochemical characteristics (Gram reaction and catalase activity) were picked from plates and purified by repetitive streaking on plate count agar (PCA, Oxoid). The isolates obtained were stored at 4°C as slant cultures for further analysis.

S. aureus strains SA04, SA18, SA20, SA46, and SA51, harbouring the classical and some newly described se genes (namely sea, seb, sed, seh, and ser), were chosen to assess the ability to produce SEs. Bacterial cultures were prepared as described by Schubert et al. [136 (link)]. Briefly, 100 mL of brain heart infusion supplemented with 1% yeast extract (BHI + YE, Biocorp, Warsaw, Poland) were inoculated with pre-culture to reach an optical density of 0.02 at 600 nm (OD₆₀₀). Prior to inoculation, the pre-cultures were washed twice with phosphate-buffered saline (PBS) to remove residual BHI broth and enterotoxins (repeated centrifugation at 12,000× g for 5 min and resuspension in PBS). Cultures were incubated at 37 °C with constant agitation at 230 rpm. The bacterial cell concentration of the broth cultures at 0, 24, and 48 h of incubation was determined by plating serial tenfold dilutions, prepared in quarter-strength Ringer’s solution (Oxoid, Basingstoke, UK), onto BHI agar. The pH of the broth cultures was measured using a FE20-FiveEasy™ pH-meter (Mettler-Toledo, Greifensee, Switzerland).