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To each reaction tube, 10 µL of the following mixture was added: 4 µL of 5x First-Strand Buffer, 2 µL of 10 mM MgCl 2, 2 µL of 0.1 M DTT, 1.4 µL of RNase inhibitor, and 1 µL of Superscript II. Reactions were incubated at 65℃ for 5 minutes and cooled on ice. Each reaction contained 1 µg of total RNA, 1 µL of 50 µM hexamer, and 1 µL of 10 mM dNTP in a final volume of 10 µL. RNA was treated with amplification grade DNase I (Epicentre Biotechnologies) and used for cDNA synthesis with random hexamer primer (Invitrogen Life Technologies, Carlsbad, CA, USA) using Superscript II reverse transcriptase reagents (Invitrogen Life Technologies). albicans cells using the MasterPure Yeast RNA Extraction kit (Epicentre Biotechnologies, Madison, WI, USA). A colorimetric change resulting from XTT reduction was measured using a microtiter plate reader (EMax, Molecular Devices, Sunnyvale, CA, USA) at 490 nm.
![proteus vulgaris proteus vulgaris](https://live.staticflickr.com/7205/6962651021_8cb7de4ae0_b.jpg)
The plates were then incubated in the dark for up to 3 hours at 37℃. Louis, MO, USA) and menadione (0.4 mM, Sigma) solution was added to each well containing the prewashed biofilm and the control well. A 200-µL aliquot of XTT (1 mg/mL, Sigma, St.
![proteus vulgaris proteus vulgaris](https://image.slidesharecdn.com/proteuss-131030221447-phpapp02/95/proteus-4-638.jpg)
A quantitative measure of biofilm formation was calculated using the XTT reduction assay. After biofilm formation, the medium was aspirated, and non-adherent cells were removed by thoroughly washing the biofilm three times with PBS. A volume of 200 µL of medium was added to each well, and the plate was then incubated for another 72 hours. After the initial adhesion phase, the cell suspensions were aspirated, and each well was washed twice with phosphate-buffered saline (PBS) to remove loose adherent cells. The plate was incubated for 90 minutes at 37℃ in an orbital shaker at 75 rpm. Microorganisms were prepared for each condition and transferred to selected wells of a microtiter plate. 23 Biofilms were formed on commercially available pre-sterilized, polystyrene, flat-bottomed, 96-well microtiter plates (Costar, Cambridge, MA, USA). Biofilm formation was quantified using the method developed by Ramage, et al.