17. For devices of type 5 the original −80°C glycerol-stock was split into aliquots, overnight cultures were started by adding 6 uL from a thawed aliquot to a culture tube and were subsequently grown for 17 hours ± 3 min. After 1000× back dilution the cultures were grown for 210 ± 2 min (mean ± sd) to an OD600 of 0.34 ± 0.04 (mean ± sd). All initial cultures (of a given strain) used in the same experiment were started from the same −80°C aliquot. Imaging and data processing
Time-lapse fluorescence imaging of the bacterial populations was done using computer controlled microscopes. Three microscope setups were used: (i) an Olympus IX81 motorized inverted microscope controlled with the MicroManager 1.4.6 software [53], equipped with a 10× 0.25NA objective and Hamamatsu ORCA-R2 camera; (ii) a Ulixertinib datasheet Nikon Eclipse Ti+E inverted microscope controlled with the Nikon Elements AR software, equipped with a 10× 0.45NA objective and an Andor iXon 885 emCCD camera; and (3) an Olympus IX81 motorized inverted microscope controlled with the MicroManager 1.4.14 software [53], equipped with a 20× 0.75NA objective and Andor Neo sCMOS camera. Devices were scanned every 10 minutes for at least 20 hours. Fluorescence images were cropped, concatenated and rescaled using the software ImageJ 1.45 [54]. Sirolimus Further
analysis of the data was done using Matlab 2011b and statistical analysis was done using R 1.15.1 for Mac [55] and Matlab 2013a. Microfabricated devices Devices were fabricated from silicon as described in Keymer et al. [34] using either a one-step (device types 1,2,4 and 5) or two-step (device
type 3) process of photolithography and reactive ion etching. Inlet holes were hand drilled using a sandblaster and have a volume of approximately 200–500 nl (mean ± sd = 311 ± 65 nl, volumes estimated for 44 inlet holes on 6 devices by assuming a truncated-cylinder shape where the depth (=550 μm) is given by the thickness of the silicon wafer and the dimensions of the top and bottom surfaces were estimated from images PRKACG taken with a stereo-microscope). Devices were sealed with a polydimethylsiloxane (PDMS, SYLGARD 184) covered glass coverslips. Devices were used only once. Bacteria grow in 100 × 100 × 5 μm3 habitat-patches (patch for short, Figure 1C); habitat-patches are connected to form habitats, which consist of a linear array of 85 patches coupled by connectors of 50 × 5 × 5 μm3 (Figure 1C). Each microfabricated device (device for short, Figure 1A-B) consists of multiple habitats etched in the same piece of silicon and sealed with a common coverslip (see below). Habitats are connected to inlet holes using inlet channels (Figure 1A-B). Five types of microfabricated devices were used, in all cases the actual habitats are the same, however devices differ in the number of parallel habitats, the arrangement of the inlets and the inoculation procedure.
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