Scd 050 sputter coater

Escherichia coli (strain W 3310) were grown overnight in Luria- Bertani (LB) medium. The overnight culture was adjusted to an optical density of 0.001 (OD₆₀₀) and incubated with the samples at 37 °C under gentle shaking (80 rpm). After 24 h the samples were fixed in paraformaldehyde, washed with PBS and MilliQ, and dried with nitrogen gas. After sputtering with a 10 nm gold layer (BALZERS SCD 050 Sputter Coater) the unstructured and hierarchically structured part of the titanium surface were imaged with SEM (FEI ESEM XL30 FEG). The experiments were performed three times with three samples respectively. Four images per structured and unstructured part of each sample were evaluated with ImageJ (v 1.52a Wayne Rasband, NIH USA) by measuring the area covered with bacteria. The results were normalized to the median of the coverage on the unstructured sample parts.

For SEM, five specimens were fixed either in glutaraldehyde (2.5% in 0.1 M sodium cacodylate buffer) for two hours and post-fixed overnight in OsO₄ (2% in 0.1 M phosphate-buffered saline [=PBS, Supplementary Protocol 2], pH 7.4) or in 4% paraformaldehyde (in PBS; Electron Microscopy Sciences, Hatfield, PA, USA) overnight. Samples were cut into smaller pieces using a razor blade, dehydrated in an increasing ethanol series, critical point dried in a CPD 030 (Bal-Tec AG, Balzers, Liechtenstein), glued onto standard aluminum stubs (diameter =12,5 mm; Plano GmbH, Wetzlar, Germany), sputter coated in a SCD 050 Sputter Coater (Balzers Union) and imaged using a field emission scanning electron microscope (Hitachi S-4000; Hitachi High-Technologies Europe GmbH, Krefeld, Germany) as described previously^{118 (link),119 (link)}.

The dried microcarriers were attached to the sample holder via carbon adhesive, and sputtered with gold for 60 s at 40 mA (SCD 050 Sputter Coater, Balzers). These samples were then imaged using a XL30 ESEM-FEG scanning electron microscope (Philips) using the secondary electron detector and acceleration voltages of 3.0–5.0 kV. A Dragonfly spinning disc confocal laser microscope (SPCLM) (Andor Technology Ltd, Belfast, Ireland) mounted on a Nikon Ti-E inverted microscope was used to visualize the Atto 647 labelled microcarriers in the hydrated state (phosphate-buffered saline solution (PBS)), with a 637 nm laser diode and images were taken with a 10x magnification objective (CFI Plan Apo Lambda, Nikon). Microcarrier diameter and pore size were determined using ImageJ software (NIH) as described previously [1 (link),2 (link)].

The optical and morphological properties of fibers are analyzed by polarizing optical microscopy (POM) and scanning electron microscopy (SEM). POM is done in transmission mode using an Olympus BX51 equipped with a Linkam (T95 series LTS120E, Surrey, UK) heating/cooling stage, an Olympus DP73 camera (Tokyo, Japan) and a Canon EOS760D camera (Tokyo, Japan). SEM imaging of the fibers are done using JEOL JSM-6010LA (Akishima, Japan) being operated in 12–15 kV range using an In-lens secondary electron detector. For SEM imaging fiber samples are gold coated (≈25 nm thickness) using a sputter coater (Balzers SCD 050 Sputter Coater) for 100 s. SEM image analysis is done using ImageJ^® software to calculate the fiber diameter [42 (link)]. The average fiber diameter is calculated by analyzing 50 fibers from different sample locations. Infrared spectroscopy (IR) is done using a Thermo Scientific^TM Nicolet^TM iS5 spectrometer with id5 ATR accessory.

To investigate the biocompatibility of the uptake and washing methods, colony forming units (CFUs) were counted after 48 hours of incubation at 30 °C on YPD plates (in triplicates) after a dilution series to prevent overcrowding. The respective controls for chemical and electrical transformation were produced with the exact same amount of pipetting and washing steps as for the transformation experiments. In order to check for cellular damage as a result of the used treatment, cells were imaged using SEM (FEI Magellan 400 XHR Scanning Electron Microscope, USA). In preparation, cells fixed in 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylic acid were dried on a cover slip and coated with a 4 nm gold layer for 30 seconds at 20 °C, 40 mA in a Balzers SCD050 sputter coater.

For scanning electron microscopy (SEM) imaging, the samples (springtails, replicates and exuviae) were dried and sputter-coated with 10 nm gold (BALZERS SCD 050 Sputter Coater). The animals and their exuviae could be dried under normal conditions, whereas the replicates had to be dried in an exsiccator under vacuum pumping for 16 h due to the strong uptake of water in the PEGda. Bacteria were counted in at least nine areas per sample type by the detailed analysis of all connected bacteria referred to as colonies. From these data the colony density and the size distribution of colonies were evaluated in order to illustrate more than the amount of cells with the intrinsic strong deviations due to partially inhomogeneous cell coverage, i.e. large areas without and large areas with many cells.