Dry anaerobic indicator strips

Mice were euthanized via CO₂ exposure at the humane endpoint, and up to five organs (brain, liver, spleen, kidney, and lung) were dissected. Whole organs were homogenized in 1 ml of sterile PBS through bead beating in a tube with two 5-mm steel beads (3 cycles of 1 min each). Organ homogenates were plated on YPD agar containing antibiotics (50 μg/ml ampicillin and 30 μg/ml chloramphenicol) for selective isolation of fungal colonies.
When hypoxic conditions were required, the GasPak EZ Container System was used (BD Diagnostics). YPD + antibiotic plates were placed in the GasPak Large Incubation Chamber, and three anaerobe sachets were added prior to sealing the chamber. Maintenance of hypoxic conditions (less than 1% O₂ and greater than 13% CO₂) was monitored using Dry Anaerobic Indicator Strips (BD).
If FK506 selection was required, 1 μg/ml FK506 was added to the YPD + antibiotic agar. Representative FK506-resistant (hyphal) and sensitive (yeast) colonies were picked for further analysis.

The cyanobacterium L. boryana strain dg5 (Fujita et al., 1996 (link); Hiraide et al., 2015 (link)) was used as the wild type. NK1 (ΔnifDK), NK2 (ΔnifZT), and NK8 (ΔnifX) isolated previously (Tsujimoto et al., 2014 (link)) were used as the control strains. For growth or induction of the nif genes under microoxic conditions, agar plates were incubated in an anaerobic jar (BBL GasPak anaerobic systems; BD Biosciences) with a sachet to create anaerobic conditions (Gas Generating Kit Anaerobic System, Oxoid, Basingstoke, Hants, United Kingdom or AnaeroPack-Anaero; Mitsubishi Gas Chemical; Tokyo, Japan). As described previously (Tsujimoto et al., 2018 (link)), Dry Anaerobic Indicator Strips (Dry Anaerobic Indicator Strips, BD Biosciences) were used to confirm anaerobic conditions in the jar.