Maxq 4000 benchtop orbital shaker

Growth rates were estimated at different temperatures, pH, and NaCl concentrations. Only one parameter was changed each time and the two other parameter values were kept constant. Table 1 shows the different value combinations used. To prepare the preculture, approximately 20 mL of Thermus liquid medium were inoculated with strain OA30 and incubated overnight at 55 °C. The preculture was then transferred into a sterile 500 mL flask containing 100 mL of the same modified Thermus liquid medium to give an initial absorbance at 660 nm of at least 0.1. The culture was incubated in aerobic conditions using a Thermo Scientific MaxQ 4000 Benchtop Orbital Shaker (Thermo Scientific, Waltham, MA, USA) at 120 rpm for approximately 24 h. At different time intervals, the turbidity of the cultures was determined by measuring the increase in optical density at 660 nm with a Synergy H1 hybrid multi-mode microplate reader. At least 10 absorbance measurements were taken into account.

Helicobacter pylori NCTC 11637 = CCUG 17874 = ATCC 43504 (Epsilon-proteobacteria), infraspecific name, strain NCTC 11637, from human gastric antrum [45 (link)], and Helicobacter bilis (ATCC^® 49314™), originally from aborted sheep fetus, liver, and fluids, from Brookings, South Dakota [38 (link),99 (link),100 (link)], were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). Both were maintained on trypticase soy agar with 5% sheep blood for 72 to 96 h [101 (link)] (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) under a microaerobic atmosphere established with the BD GasPak^™ EZ container system and reagents (Becton Dickinson). The GasPak container with the plates inoculated with the bacteria was located within an orbital shake (MaxQ™ 4000 Benchtop Orbital Shaker, Thermo Fisher) and incubated at 37 °C for 96 h. At this point, each species of Helicobacter was harvested by scraping the colonies from the agar plate, and scrapings of the culture on the agar pale were resuspended in DMEM/F12 medium until an optical density at 600 nm of 1. 0 was reached, which corresponds to ~1 × 10⁸ colony forming units (cfu)/mL [102 (link),103 (link),104 (link)]. Viability of the bacteria was confirmed by visual inspection for movement of the flagellated bacteria.

The DAB (3,3′-diaminobenzidine) staining method was used to detect H₂O₂ accumulation^{94 (link)} in Bd plants in response to Hessian fly larval attack. The Bd plants were grown and infested as described above. DAB staining solution was prepared by dissolving DAB powder (Sigma-Aldrich, St. Louis, MO) in deionized water (1 mg/mL) and the pH of the solution was adjusted to 3.8 with 0.2 N HCl. The DAB solution was kept in dark by covering with aluminum foil. At 1 DAH, six infested Bd plants were dissected under a microscope by cutting below the root/crown junction and the leaf sheaths were removed to expose the larval feeding sites on the main stem. Four uninfested control Bd plants were dissected in the same manner. Each plant was placed in a 15 mL falcon tube covered with aluminum foil. To each tube 10 mL of DAB solution was added, and vacuum infiltrated for 5 min. Following vacuum infiltration, the tubes were shaken at 100 rpm in a MaxQ 4000 Benchtop Orbital shaker (ThermoFisher Scientific, Waltham, MA) for 18 h at room temperature under dark. The plants were then photographed with a DP21 camera system on SZX2 stereomicroscope (Olympus America Inc., Center Valley, PA).