SRV-CHX and SRV-placebo-coated stents were repeatedly incubated in 1 mL of TSBG medium with 10 µL of an overnight bacterial culture for 24 h for a total of 7 days. The OD at 600 nm was measured daily using the Ultraspec 10 Spectrophotometer (Amersham Biosciences, Buckinghamshire, UK). TSBG medium incubated with the stents without bacteria served as blank. The extent of biofilm formation on the stents was analyzed by different techniques as described below.
Ultraspec 10 spectrophotometer
Manufactured by Cytiva
Sourced in United Kingdom
The Ultraspec 10 spectrophotometer is a laboratory instrument designed for the measurement of light absorption or transmission in liquid samples. It is capable of analyzing the concentration of molecules or elements in a sample by detecting the amount of light absorbed or transmitted through the sample at specific wavelengths.
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3 protocols using ultraspec 10 spectrophotometer
1
Bacterial Biofilm Formation on Stents
2
Cultivation of Streptococcus mutans UA159
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S. mutans UA159 was cultivated in brain–heart infusion (BHI) broth (Acumedia, Lansing, MI, USA) [30 (link)]. The day before the experiment, 100 µL of a frozen (−80 °C) stock was inoculated in 10 mL BHI followed by incubation at 37 °C in a humidified incubator in the presence of 5% CO2 for 20 h. The chain formation and coccoid-to-ellipsoid morphology of the S. mutans culture were verified under light microscopy (Axio Lab.A1, Carl Zeiss GmbH, Jena, Germany) using the ×100 lens, and the optical density at 600 nm determined by Ultraspec 10 spectrophotometer (Amersham Biosciences, Amersham, UK), usually being in the range of 1.2–1.3. The purity of the culture was tested by seeding the bacteria on BHI-agar plates where they formed distinctive minute colonies that are strongly attached to the agar.
3
Characterizing sfGFP Expression in P. polymyxa
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For sfGFP fluorescence experiments, 3 mL of EPS medium supplemented with 50 µg mL -1 neomycin were inoculated with a single colony of the respective strains and grown over night at 30 °C, 200 rpm. After 18 h, each strain was sub-cultured 1:100 in 3 mL selective MM1 P100 medium and grown for 24 h at 30 °C, 200 rpm. After 24 h, 100 µL were transferred to a 96 well microtiter plate and OD600, GFP fluorescence (Ex. 488 nm Em. 515 nm) and mRFP fluorescence (Ex. 560 nm Em. 600 nm) measured in a Ultraspec 10 spectrophotometer (Amersham Biosciences, UK). Fluorescence values were normalized to OD600 in all experiments. In parallel, 1 mL of each culture was pelleted by centrifugation and used for qPCR experiments. Relative expression signals were calculated based on an off-target construct expressing gRNAs not binding to the genome of P. polymyxa DSM 365 or any plasmid used in this study (Table S 4).
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