Microbiology


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0 Q&A 1938 Views Aug 20, 2021

Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities. Current methodologies commonly involve static biofilm setups (such as biofilm assays in microplates, bead biofilms, or biofilms on glass-slides) and fluidic flow biofilm systems (such as drip-flow biofilm reactors, 3-channel biofilm reactors, or tubing biofilm reactors). Continuous flow systems take into consideration the contribution of hydrodynamic shear forces, nutrient supply, and physical transport of dispersed cells, which define the habitat for biofilm development in most natural and engineered systems. This protocol describes the assembly of 3 flow-system setups to cultivate Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 model biofilms, including the respective quantitation and observation approaches. The standardized flow systems promise productive and reproducible biofilm experimental results, which can be further modified according to specific research projects.

1 Q&A 10078 Views Mar 20, 2017
Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.



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