Visualization of Clinically Relevant Bacteria During Exposure to Disinfectants

Swift, Dean; Re, Deanna Del; Dhyani, Kruti; Mair, Richard W.; Legner, M.; Cvitkovitch, Dennis G.

doi:10.1016/j.ajic.2013.03.089

Cited by 1 publication

(2 citation statements)

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“…Previous pilot experiments suggested that using nucleic-acid fluorescent probes helps visualize the dynamics of disinfectant action in bacterial biofilms [ 4 , 5 ]. In a more detailed study on hydroxyapatite discs using confocal laser scanning microscopy (CLSM), Xiao et al, 2012 [ 6 ] were able to quantify the impact of one agent in several consecutive 15-min intervals using specialized software.…”

Section: Introductionmentioning

confidence: 99%

“…As a starting point, we decided to use (1) a low-power, high Numerical Aperture (NA) objective to assess large (ca 0.4 mm 2 ) areas of biofilm that captures the emission of the entire thickness of the biofilm, (2) a high-sensitivity sCMOS camera that requires a fraction of the illumination energy normally needed for the CLSM (confocal) system and (3), the open source software package (FIJI ImageJ by the National Institutes of Health, USA) to binarize the entire time series of frames simultaneously. We also acknowledge that a substantial drawback of our initial experiments using time series of microscopic images [ 4 , 5 ] was the delivery (injection) of agents in a manual fashion that prevented standardizing the procedure and did not allow us to pinpoint the moment when the leading edge of injected fluid (disinfectant agent) arrives in the viewing area. The introduction here of a precise Linear Pump solves those issues.…”

Section: Introductionmentioning

confidence: 99%

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Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy

Legner¹,

Jonkman

Swift³

2020

Microorganisms

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A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell’s inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%