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. 2010 Jul;76(13):4136-42.
doi: 10.1128/AEM.03065-09. Epub 2010 Apr 30.

New device for high-throughput viability screening of flow biofilms

Michael R Benoit  1 , Carolyn G Conant, Cristian Ionescu-Zanetti, Michael Schwartz, A Matin
Affiliations

New device for high-throughput viability screening of flow biofilms

Michael R Benoit et al. Appl Environ Microbiol. 2010 Jul.

Abstract

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well ("static") biofilms are available, there are no methods for such screening of continuous flow biofilms ("flow biofilms"). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.

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Figures

FIG. 1.
FIG. 1.
BioFlux high-throughput system for screening of flow biofilm viability and other parameters. (A) Photograph of BioFlux system. The device consists of a pneumatic pump connected via an interface to the top of a BioFlux plate (shown on the microscope stage). (B) Schematic diagram of the BioFlux plate, showing 48 wells and 24 independent channels connecting pairs of wells. (C) Schematic diagram showing the inlet and outlet wells containing fresh and spent media, respectively. Pneumatic pressure delivered to the top of the inlet well pushes fresh medium through the microfluidic channel (containing the biofilm) and into the outlet well. The biofilm can be viewed with a microscope or scanned with a plate reader. (D) Close-up of two microfluidic channels (black lines). Each channel has a serpentine region (one serpentine region is enclosed in a box) to provide sufficient back pressure and a chamber for microscope viewing (arrow).
FIG. 2.
FIG. 2.
Biofilm formation and development in a BioFlux channel. The images are phase-contrast images obtained after flow began (the flow was from left to right; each channel was 370 μm wide) and show initial attachment (0.5 and 1 h), microcolony formation (2 and 3 h), and development (4, 5, and 6.5 h) into fully formed biofilms (21 h).
FIG. 3.
FIG. 3.
Comparison of static biofilm viabilities determined by colony counting and by using GFP-PI fluorescence ratios. (A and B) Representative fluorescence emission spectra of quadruplicate GFP-expressing UPEC (A) and PAO1 (B) biofilms following 1 h of treatment with bleach (in saline) at the concentrations indicated (as percentages) and PI staining. The absorption peaks on the left and right correspond to live and dead cells, respectively. (C and D) Mean levels of viability as determined by colony counting and GFP-PI fluorescence (GFP-PI Fluor.) for UPEC (C) and PAO1 (D) biofilms. The error bars indicate standard deviations. (E and F) Linear regression analysis of viability data for the two methods using pooled data from three independent experiments.
FIG. 4.
FIG. 4.
Estimates of the viability of GFP-producing P. aeruginosa PAO1 biofilms cultivated in the BioFlux device following treatment with bleach (A) or various antibiotics (B to E) and then stained with PI. Levels of viability were calculated by using the green fluorescence/red fluorescence ratios determined from quantified microscope images (black bars) and by using plate reader measurements (white bars). Isopropyl alcohol (70%) was used to ensure complete loss of viability. Insets show representative microscope images, including images of untreated (0) and isopropyl-alcohol treated (isop.) controls. Note that no biofilm remained after treatment with the highest dose of enrofloxacin (E) and the two highest doses of ciprofloxacin (D). Fluorescence signals were not detected (N.D.) with the plate reader, and no biofilms were observed using microscopy (insets). (F) Linear regression analysis for determinations of viability by the two methods.
FIG. 5.
FIG. 5.
Viability of PAO1 BioFlux biofilms determined using SYTO9-PI and GFP-PI. Treatment with low levels (0.01 and 0.1 μg/ml) of gentamicin increased SYTO9 uptake and green fluorescence, resulting in enhancement of false-positive viability (>100%), which did not occur with GFP-PI fluorescence. The Student t test P value is shown for each concentration of gentamicin; a P value of <0.05 indicates that there is a statistically significant difference.
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