Tem image and analysissoftware ver 4

We generated SEM images from 10
μL spore suspension drops and air-dried on a glass slide. Ahead
of imaging, we coated the sample with a ∼5 nm layer of platinum
using a Quorum Q150T-ES sputter coater. We then imaged spores using
a Carl Zeiss Merlin FESEM electron microscope using the InLens imaging
mode at magnifications of 7500–50 000×.
To
prepare spores for TEM, we fixed the spore suspensions using 2.5%
glutaraldehyde (TAAB Laboratories, Aldermaston, England) in 0.1 M
PHEM buffer and then postfixed them in 1% aqueous osmium tetroxide.
To further dehydrate the spores, we washed them in ethanol and acetone,
after which they were embedded in Spurr’s resin (TAAB Laboratories,
Aldermaston, England). The 70 nm spore sections were then post-contrasted
in uranyl acetate and Reynolds lead citrate ahead of imaging. We imaged
the spores using a Talos L120C (FEI, Eindhoven, The Netherlands) operating
at 120 kV. Micrographs were acquired using a Ceta 16M CCD camera (FEI,
Eindhoven, The Netherlands), equipped with TEM Image and Analysis
software ver. 4.17 (FEI, Eindhoven, The Netherlands).

Samples for TEM were prepared as liquid suspensions of spores after treatment with sodium hypochlorite and neutralisation with thiosulphate, while untreated control samples were suspended in water. After the incubation, samples are centrifuged and resuspended in MQ water twice to wash off any aqueous chemicals. Spores are fixed with 2.5% glutaraldehyde (TAAB Laboratories, Aldermaston, England) in 0.1 M sodium cacodylate buffer and further postfixed in 1% aqueous osmium tetroxide. They are further dehydrated through an ethanol concentration series and finally embedded in Spurr’s resin (TAAB Laboratories, Aldermaston, England) and polymerised overnight at 60 °C. 70 nm ultrathin sections are then post contrasted in uranyl acetate and Reynolds lead citrate. C. difficile spores were imaged using a JEM 1400 (JEOL Ltd.) using a Orius camera (Gatan Inc.). B. thuringiensis spores were imaged using a Talos L120C (FEI, Eindhoven, The Netherlands) operating at 120kV. Micrographs were acquired with a Ceta 16M CCD camera (FEI, Eindhoven, The Netherlands) using TEM Image and Analysis software ver. 4.17 (FEI, Eindhoven, The Netherlands). At least 10 spores were imaged for each experimental condition.