Cultivating Predatory Bacteria Bdellovibrio

P. putida KT2440 and the evolved strain KT40AE were grown in lysogeny broth (LB) at 30°C with shaking at 250 rpm. Growth was monitored with a Shimadzu UV‐260 spectrophotometer at 600 nm (OD₆₀₀). Solid media were prepared with agar at 1.5% (w/v). The B. bacteriovorus HD100 strain was routinely grown in coculture in Hepes buffer (25 mM Hepes amended with 2 mM CaCl₂·2H₂O and 3 mM MgCl₂·3H₂O, pH 7.8) or DNB liquid medium (consisting of 0.8 g l⁻¹ NB supplemented with 2 mM CaCl₂ and 3 mM MgCl₂), with P. putida KT2440 as prey, as previously described (Martínez et al., 2016 (link)). Prey cultures were prepared from cells grown in NB for 16 h and diluted to OD₆₀₀ of 1 in Hepes buffer. After predation, the cocultures were filtered twice through a 0.45‐μm filter (Sartorius) and the B. bacteriovorus cells were centrifuged at 14 000 g, 4°C, 15 min. This pellet was subsequently suspended in 1–2 ml of MOPS buffer and used in the encapsulation protocol.