In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell

Abstract

Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

Marine Antifouling Behavior of Lubricant-Infused Nanowrinkled Polymeric Surfaces

“…Surfaces were exposed to Pseudoalteromonas spp. (OD 600nm = 0.25) in tryptic soy broth/artificial seawater, 3:1 26 for 1 h, then shaken at 150 rpm in fresh medium for 24 h at ambient temperature on an orbital shaker (Ratek). Addition of aqueous crystal violet (0.1%, Sigma-Aldrich) turned the biofilm purple, 26 before extraction into 30% acetic acid to measure absorbance (590 nm).…”

Use of confocal Raman microscopy to characterise ethyl cyanoacrylate adhesive depth curing

“…Courtecuisse et al demonstrated the effect of oxygen inhibition in an acrylate photopolymerization system 15 . Smith-Palmer et al used CRM to follow the initiation of biofouling by a marine bacterium in a flow cell 16 . Edwards et al have employed Raman spectroscopy to study curing of a cyanoacrylate system for the forensic enhancement of latent fingerprints 17 18 .…”

Antimicrobial Agent against Methicillin-Resistant Staphylococcus aureus Biofilm Monitored Using Raman Spectroscopy

“…The cross-validation analysis of variance (CV-ANOVA) representing the cross-validation result with p -value = 0. Reference [ 59 ] are cited in the supplementary materials.…”