Uv 6300pc double beam spectrophotometer

Samples were processed as per Steinberg et al. (2021) (link). In summary, octocoral samples were homogenised with reverse osmosis water using an Omni TH Tissue Homogeniser. The resulting slurry was centrifuged and supernatant removed and saved, and this step was repeated. Supernatant protein was quantified against a bovine albumin standard using the Thermo Scientific Coomasie Plus (Bradford) kit and protocol. Symbiodiniaceae were counted using a Neubauer Improved haemocytometer with three haemocytometer fills, two grids per haemocytometer, and five counts per grid, which were averaged, for a total of 6 replicate counts (Steinberg et al., 2021 (link)). Chlorophyll was extracted in 100% acetone for 48 h and measured on a VWR UV-6300PC Double Beam Spectrophotometer (see Steinberg et al. (2021) (link) for more information).

Both PVDF400 and PMAA-PVDF membranes were convectively immobilized with subtilisin Carlsberg. For batch immobilization mode, 100 mL of 0.1 mg/mL solution of subtilisin was stirred in a water-filtration cell for 60 min. For convective immobilization mode, membranes were compacted once with a basic solution of sodium hydroxide in deionized water (pH 9) at 1 mL/min for 60 min and then again with deionized water at 1 mL/min for 60 min. The membranes were rinsed and immobilized with a 0.1 mg/mL solution of subtilisin Carlsberg at approximately 0.67 ml/min for 60 min. Mass of enzyme immobilized was determined by analyzing the subtilisin concentration of the functionalizing solution before and after the immobilization process with the UV-6300PC Double Beam Spectrophotometer (VWR) at a wavelength of 280 nm. Membranes were stored in “dry storage” conditions, meaning in a dry unsealed petri dish in a controlled-environment room (exposed to light) with a relative humidity of 40–60% and temperature of 15–21 °C.

Microtiter plate assay is the most common quantitative assay to detect biofilm as determined by Christensen et al. [39 (link)]. For this study, we inoculated all the bacteria from fresh agar plates into both LB and BHI media and incubated at 37 °C overnight in static conditions. The precultures were adjusted to OD₆₀₀ = 0.1 in the fresh medium using a UV-6300PC Double Beam Spectrophotometer (VWR, Radnor, PA, USA). Ninety-six-well flat bottom cell culture plate (hydrophilic) and 8-strips U-form bottom plate (hydrophobic) were filled with 200 µL diluted culture. The plates were incubated at 37 °C for 24 h without shaking. After two washes with the phosphate-buffered saline (PBS) (pH 7.2) to remove the planktonic bacteria, the wells were stained with 0.1% crystal violet (CV). Before staining with crystal violet, the plates were dried at 60 °C for two hours. The stained biofilm was resolubilized with 225 µL of 30% acetic acid and measured spectrophotometrically at 595 nm with a PowerWave microplate reader (BioTek, Winooski, VT, USA). All the samples were tested in triplicate. If OD values were above 3, a dilution was performed. The average score of three wells was calculated.