Eclipse t12

Axenic cultures (5 ml) of each strain were grown to OD_600nm = 0.6–0.7. Cells were harvested and resuspended in 7H9 (100 μl) and labelled with alk-DADA (2.5 μl) at 37°C for 60 min. Cells were centrifuged, washed in 1X PBS (Lonza) and fixed overnight in 250 μl of 2.5% glutaraldehyde (Sigma) at room temperature. Fixed cells were centrifuged at 13 000 rpm for 2 min and resuspended in PBS-Tween-BSA (PBSTB) buffer (250 μl) and centrifuged at 13 000 rpm for 2 min. Pellets were resuspended in PBSTB (230 μl), to which CuACC reaction components were added in the following order: 10 mM CuSO₄ (5 μl), 6.4 mM TBTA (5 μl), 60 mM Sodium Ascorbate (5 μl), 1 mM azido-probe (5 μl). The CuACC reaction was incubated at room temperature for 30 min and harvested by centrifuging the cells at 13 000 rpm for 2 min. The cells were washed three times in 1X PBS and added to an agarose pad as described previously. Images were captured using the Nikon Eclipse T12 and PE4000 LED light source, in the TL DIC channel (200 msec exposure) and pre-configured azo488 channel (excitation 490 nm, 50% power; emission 512 nm; 3 sec exposure).

Axenic cultures of each strain were grown to OD_600nm = 0.6–0.7 and TADA (2.5 μl) was added to a 500 μl aliquot, followed by incubation at 37°C, for 30 min. Cells were harvested and washed in 1X PBS (Lonza) twice before fixing overnight in 250 μl of 2.5% glutaraldehyde (Sigma) at room temperature. Fixed cells were centrifuged at 13 000 rpm for 2 min and resuspended in PBS before spotting onto 1% agarose on glass slides, covered with a cover slip and sealed. Images were captured using the Nikon Eclipse T12 and PE4000 LED light source, in the TL DIC channel (200 msec exposure) and pre-configured TADA channel (excitation 550 nm, 50% power; emission 598 nm; 2 s exposure).