Hm20 resin

Exponentially growing cells were pelleted, loaded into 3 mm, 100 µm, or 200 µm carriers and high-pressure frozen using a Leica EM ICE. Samples were freeze substituted in a Leica AFS2 unit in 1% uranyl acetate +1% water in acetone. Samples were then maintained at − 45 °C for the remainder of the processing. Samples were washed with acetone and then ethanol for 15 min each, then infiltrated with HM20 resin (PolySciences) according to the following schedule: 25% resin in ethanol for 4 h, 50% resin in ethanol overnight, 75% resin in ethanol for 5 h, 100% resin for 6 h, 100% resin overnight, 100% resin for 8 h. Samples were then polymerized with UV light for 48 h, and gradually warmed to 0 °C after the first 24 h of polymerization. 90 nm ultranthin sections were acquired using a Diatome diamond knife with a Leica UC7 ultramicrotome and transferred to formvar-coated 100-mesh copper grids. Sections were post-stained for 10 min with 2% uranyl acetate, washed by passing over a series of warm water droplets, and then stained with Reynold’s lead citrate, washed, and dried. Grids were imaged in a FEI Tecnai 12 TEM operated at 120 kV using a Gatan OneView digital camera.

For light microscopy, samples of the fixed bacterial cell mix were stained with acridine orange (AO) (Sigma-Aldrich, UK) at 10 μg/ml for 10 min to stain nucleic acids. Samples were imaged using 63×/1.2 NA H2O immersion lens on the Leica SP8X confocal laser scanning microscope (CLSM) (Leica Microsystems, UK). Images presented are maximum intensity projections of confocal Z stacks, with DNA imaged at excitation 488 nm, and emission 500–550 nm and RNA imaged at excitation 488 nm and emission at 600–650 nm.
For electron microscopy, samples of the fixed cell mix pre and post lyophilisation were high-pressure frozen in a Leica HMP 010 high-pressure freezer (Leica, Austria) with 20% HMW dextran average M_r ~70,000 (Sigma-Aldrich, UK) acting as a cryoprotectant. These samples were freeze-substituted into HM20 resin (Polysciences, Inc., PA) containing 2% uranyl acetate. Sections of 100 nm thickness were cut using an UC6 (Leica Microsystems Ltd., UK) fitted with a diamond knife (Diatome, Switzerland) and mounted on carbon-coated copper grids (Agar Scientific, UK). Sections were imaged in a JEOL JEM 2100 electron microscopes (JEOL (UK) Ltd., UK) running at 200 kv at magnifications ranging from 400× to 25,000×. Images were captured on a Gatan US4000 CCD camera running Digital Micrograph software (Gatan Inc., CA).