Stereo lumar

To study E. coli adaptation to the gut, we used a streptomycin-treated mouse colonization model40 . Briefly, SPF-raised mice (see E. coli and mouse strains above) were given autoclaved drinking water containing streptomycin (5 g l⁻¹) for 1 day. After 4 h of starvation for water and food, the animals were gavaged with 100 μl of a suspension of 10⁸ colony-forming units of a mixture of YFP- and CFP-labelled bacteria (ratio 1:1) grown at 37 °C in brain heart infusion medium to optical density (OD)₆₀₀ of 2. After gavage, the animals were housed in individual cages, and both food and water containing streptomycin were returned to them. Faecal pellets were collected for 24 days, diluted in PBS and plated in Luria Broth (LB) agar. Plates were incubated overnight and the frequencies of CFP- or YFP-labelled bacteria were assessed by counting the fluorescent colonies with the help of a fluorescent stereoscope (SteREO Lumar, Carl Zeiss). A sample of each collected faecal pellet was daily stored in 15% glycerol at −80 °C for further experiments.
For the evolution experiment, groups of five WT and 5 Rag2^−/− mice were gavaged and followed for 24 days. This procedure was repeated three times, such that a total of 15 mice of each genotype were analysed. While the results of E. coli evolution in WT mice were published earlier11 (link), experiments in both genotypes were conducted at the same time.

P. simonii × P. nigra pollen grains stored at −80°C were resuscitated at 5°C for 2 h, and 10 mg of the resuscitated pollen was evenly scattered in 10 mL liquid medium using a stainless steel mesh (50 μm). The liquid medium for PG and PTG consisted of 15% sucrose, 40 mg L⁻¹ CaCl₂, 20 mg L⁻¹H₃BO₃ (pH 6.0–6.3). Pollen grains were incubated in a 100 mL flask at 21°C in darkness for ~7 h, which resulted in ~75% germination and an average PT length of ~250 μm. The PTs were filtered using a 50-μm nylon mesh to remove the ungerminated pollen grains and collected from the nylon mesh for total RNA extraction. HP samples were collected and centrifuged at 27 (× g) rcf after resuscitated pollen grains were incubated for 1.5 h. HP viability was assessed with FDA staining. Resuscitated pollen grains were incubated for 1.5 h in liquid medium with 2 μg mL⁻¹ fluorescein diacetate (FDA). After washing out the FDA with liquid medium, the fluorescence of pollen grains was observed using a Zeiss SteREO Lumar. V12 fluorescence microscope.