Computational protocol: Methyl-Hydroxylamine as an Efficacious Antibacterial Agent That Targets the Ribonucleotide Reductase Enzyme

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Protocol publication

[…] To prepare biofilms developed under continuous flow, P. aeruginosa cells (5x105 cfu/ml) were cultured in LB medium at 25°C in flow chambers with channel dimension of 1x4x40 mm as described previously []. After 96 h of culture, formed biofilms were treated with 40 μg/ml of HA, HU or M-HA, and LB medium was used alone in the control sample. After 24 h of treatment at 25°C, biofilms were stained with 5 μM SYTO 9 at room temperature in the dark for 30 min, according to the specifications of the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes, Invitrogen).Confocal scanning laser microscopy of the biofilms was performed with a Leica TCS-SP5 confocal scanning laser microscope (Leica Microsystems, Wetzlar, Germany), with an excitation wavelength of 477 for SYTO9. To measure biofilm thickness, sections were scanned and Z-stacks were acquired at z step-size of 0.388 μm. Field size was 456 μm x 456 μm at 20X magnification. Microscope images were further processed with ImageJ analysis software (National Institute of Health, USA) and COMSTAT 2 software, specific for biofilm quantitative analysis []. […]

Pipeline specifications

Software tools ImageJ, Comstat
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mycobacterium bovis, Pseudomonas aeruginosa
Diseases Drug-Related Side Effects and Adverse Reactions
Chemicals Ciprofloxacin, Ribonucleotides