Capsule Induction and Quantification Protocol

Capsule induction was performed following the protocol described by Zaragoza et al. using capsule-inducing medium (10% SDB in 50 mM MOPS, pH 7.3) [34 (link)]. An inoculum was adjusted to 1 × 10⁸ cells/mL prior to plating. The cultures were incubated overnight at 37 °C with shaking (110 rpm). Following microscopy observation, a microscope slide containing one drop of India ink and one drop of each isolate was prepared and observed using a Leica DMI 3000B microscope (Leica Microsystems, Wetzlar, Germany). Images were acquired in bright field with 40× or 63× objectives, and capsule size and cell body (delimited by cell wall) were measured in 50 cells using ImageJ Software (Fiji) V2.1 [35 (link)]. The capsule area of 50 cells was measured for each isolate, and the procedure previously described was used to measure capsule size.