Delta vision epi fluorescence microscope

Air dried thin smears of P. berghei ookinete or schizont or sporozoites and oocysts, obtained from midgut of P. berghei infected Anopheles stephensi crushed in PBS, were fixed with 4% EM grade paraformaldehyde (Electron Microscopy Science) for 10 minutes. Permeabilisation, blocking and incubation with primary and appropriate secondary antibodies (S4 Table & S5 Table) was performed as described by [41 (link)] with additional last washes with 70% ethanol and absolute ethanol 1 min each, air-dried and mounted in VectaShield (Vectorlabs) containing DAPI (4', 6-diamidino-2-phenylindole) in glycerol for nuclear staining. Parasites were examined either under Delta Vision Epifluorescence microscope (Applied Precision). 100x objective, images were captured with CoolSNAP HQ camera (Photometrics) and deconvoluted using SoftWoRx software (Applied Precision) or under Axioplane2 (Zeiss) 100x objective, images were taken through HAMAMATSU ORCA_ER camera (HAMAMATSU) and Velocity software 4.1.0 (PerkinElmer). Images were processed using Fiji (NIH) as well as SoftWoRx explorer 1.3. Super-resolution images were captured through Elyra PS.1 super-resolution microscope (Zeiss) under 60x objective with sCMOS PCO camera and images were visualized with ZEN Black software (Zeiss) and processed with ZEN LITE software (Zeiss).

To determine whether toyoncin can inhibit B. cereus spore germination, an assay was performed as described previously (15 (link)). Briefly, 100 μl of 1.0 × 10⁷ spores/ml of B. cereus, 800 μl of LB medium, and 100 μl of toyoncin solutions at different final concentrations (0.1, 1, 5, and 10 μM) were mixed and incubated at 30°C for 3 h. Differential interference contrast microscopy images of vegetative cells or spores in the cultures were obtained with a DeltaVision epifluorescence microscope (Applied Precision, Issaquah, WA, USA), and the images were processed with the SoftWoRX Explorer Suite program (Applied Precision).