125 l gene frame

Appropriate culture or spore suspension dilutions were placed on agarose pads containing the appropriate medium and supplemented with 1.5% LSL-LE 8200 agarose (Lonza, Basel, Switzerland) on a microscopy slide and covered with a cover glass attached to a 125 µL Gene Frame (Thermo Fisher Scientific, Waltham, MA, USA). TLFM was performed on a Ti-Eclipse inverted microscope (Nikon, Champigny-sur-Marne, France) equipped with a 60× Plan Apo λ oil objective, a TI-CT-E motorized condenser and a Nikon DS-Qi2 camera. GFP was imaged using a quad-edge dichroic (395/470/550/640 nm) and FITC single emission filters. A SpectraX LED illuminator (Lumencor, Beaverton, OR, USA) was used as a light source, using the 470/24 excitation filter. Temperature was controlled at 30 °C (for sporulation) or 37 °C (for germination) with an Okolab cage incubator (Okolab, Ottaviano, Italy). Images were acquired using NIS-Elements software (version 4.51, Nikon) and the resulting pictures were further handled with the open source software ImageJ (version 1.54d) [24 (link)].

To generate agarose pads for the analyses, 2× BHIS containing 1%, 19 mM TA was mixed with molten 3.5% (wt/vol) Top-Vision Low Melting Point Agarose in sterile H₂O to a final concentration of 1.75% agarose and 0.5% (9.5 mM) TA. The resulting media was added to a 125 µL gene-frame (Thermo Fisher Scientific) adhered to a clean glass microscope slide and solidified. WT, ΔpdaA, and ΔcwlD spores were diluted to 0.2–0.5 OD₆₀₀ in sterile H₂O in biological triplicate and 1 µL was spotted on to a #1.5 coverslip and allowed to dry at 37°C. After the spores dried, the coverslip was inverted onto the agar pad and imaged immediately imaged via time-lapse phase-contrast microscopy at 37°C.

Agarose pads were made containing germination buffer (50 mM HEPES, 100 mM NaCl, pH 7.5), 1.75% (wt/vol) Top-Vision Low Melting Point Agarose, 0.2% (3.8 mM) TA (wt/vol), and either 10 mM Gly or 30 mM CaCl_2. The resulting media was added to a 125 µL gene-frame (Thermo Fisher Scientific) adhered to a clean glass microscope slide and solidified. WT, ΔdpaAB spores were diluted to 0.2–0.5 OD₆₀₀ in sterile H₂O in biological triplicate and 1 µL was spotted on to a #1.5 coverslip and allowed to dry at 37°C. After the spores dried, the coverslip was inverted onto the agar pad and imaged immediately imaged via time-lapse phase-contrast microscopy at 37°C.