Pneumococcus type 2 serum

For visualization of the polysaccharide capsule, S. pneumoniae was grown in CY medium supplemented with or without 100 ng⋅μL ATc until OD (600 nm) 0.3, then rediluted into fresh CY medium and grown until OD (600 nm) 0.1. Cells were then harvested by centrifugation, washed, and resuspended in fresh prewarmed CY medium. The 1:1,000 pneumococcus type 2 serum (SSI Diagnostica) was added, and cells were incubated for 5 min on ice. After two washes with fresh CY medium, 1:1,000 of secondary Alexa Fluor 555-conjugated goat anti-rabbit antibody (Invitrogen; A27039) was added, and cells were incubated in ice for 5 min. The stained cells were then washed twice with fresh CY medium and once with ice-cold PBS. The pellet was then resuspended in PBS for imaging using epifluorescence.

Capsular polysaccharides of the D39s and D39ΔfabT mutant were detected by immunofluorescence microscopy as previously described (51 (link)). Briefly, pneumococcus type 2 serum (SSI Diagnostica, Denmark) was used as the primary antibody at a dilution of 1:50, and goat anti-mouse IgG-PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody at a dilution of 1:500. Finally, images were obtained with a Nikon Eclipse 80i microscope.