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A Method for Quantitative Determination of Biofilm Viability

Welch, Ken, Cai, Yanling, Strømme, Maria·2012·Journal of Functional Biomaterials
DOI 10.3390/jfb3020418PMC PMC4047939
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Abstract

In this study we present a scheme for quantitative determination of biofilm viability offering significant improvement over existing methods with metabolic assays. Existing metabolic assays for quantifying viable bacteria in biofilms usually utilize calibration curves derived from planktonic bacteria, which can introduce large errors due to significant differences in the metabolic and/or growth rates of biofilm bacteria in the assay media compared to their planktonic counterparts. In the presented method we derive the specific growth rate of Streptococcus mutans bacteria biofilm from a series of metabolic assays using the pH indicator phenol red, and show that this information could be used to more accurately quantify the relative number of viable bacteria in a biofilm. We found that the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h−1, compared to 1.09 h−1 in planktonic growth. This method should be applicable to other bacteria types, as well as other metabolic assays, and, for example, to quantify the effect of antibacterial treatments or the performance of bactericidal implant surfaces.

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Figure 1 Schematic diagram of the method for determining the specific growth rate of biofilm bacteria. The specific growth rate μ is extracted from a plot of the time required for color switch of Metabolic Activity Assay (MAA) broth containing a set amount of biofilm bacteria as a function of MAA broth volume. Included is a laser scanning confocal microscopy image of a S. mutans biofilm using live/dead staining (L13152, Invitrogen, Eugene, UR, USA).
Figure 1 Schematic diagram of the method for determining the specific growth rate of biofilm bacteria. The specific growth rate μ is extracted from a plot of the time required for color switch of Metabolic Activity Assay (MAA) broth containing a set amount of biofilm bacteria as a function of MAA broth volume. Included is a laser scanning confocal microscopy image of a S. mutans biofilm using live/dead staining (L13152, Invitrogen, Eugene, UR, USA).CC BY · PubMed Central
Figure 2 Specific growth rate of planktonic S. mutans as determined from OD600 measurements. A fit to Equation (1) for the initial growth phase is included (solid line), showing that the initial growth rate is μ = 1.08 h−1.
Figure 2 Specific growth rate of planktonic S. mutans as determined from OD600 measurements. A fit to Equation (1) for the initial growth phase is included (solid line), showing that the initial growth rate is μ = 1.08 h−1.CC BY · PubMed Central
Figure 3 MAA measurements for planktonic S. mutans bacteria illustrating the time taken for the culture medium to switch color as a function of: (a) initial planktonic S. mutans concentration, and (b) assay volume for an initial planktonic S. mutans population of 5 × 105 cfu. Curve fits to Equation (6) (panel a) and Equation (7) (panel b) are included as solid lines giving specific growth rates of 1.09 h−1 (a) and 1.08 h−1 (b).
Figure 3 MAA measurements for planktonic S. mutans bacteria illustrating the time taken for the culture medium to switch color as a function of: (a) initial planktonic S. mutans concentration, and (b) assay volume for an initial planktonic S. mutans population of 5 × 105 cfu. Curve fits to Equation (6) (panel a) and Equation (7) (panel b) are included as solid lines giving specific growth rates of 1.09 h−1 (a) and 1.08 h−1 (b).CC BY · PubMed Central
Figure 4 Scanning electron microscopy (SEM) images showing two magnifications of a 16-hour-old S. mutans biofilm attached on a sample disk.
Figure 4 Scanning electron microscopy (SEM) images showing two magnifications of a 16-hour-old S. mutans biofilm attached on a sample disk.CC BY · PubMed Central
Figure 5 MAA measurements for biofilm S. mutans bacteria illustrating the time taken for the culture medium to switch color as a function of: (a) number of disks with S. mutans biofilm and (b) assay volume for an initial S. mutans biofilm on a single disk. Curve fits to Equation (6) (panel a) and Equation (7) (panel b) are included as solid lines giving specific growth rates of 0.687 h−1 (a) and 0.715 h−1 (b).
Figure 5 MAA measurements for biofilm S. mutans bacteria illustrating the time taken for the culture medium to switch color as a function of: (a) number of disks with S. mutans biofilm and (b) assay volume for an initial S. mutans biofilm on a single disk. Curve fits to Equation (6) (panel a) and Equation (7) (panel b) are included as solid lines giving specific growth rates of 0.687 h−1 (a) and 0.715 h−1 (b).CC BY · PubMed Central

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Research works printed but not linked: 27

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Metadata

Authors
Welch, K., Cai, Y., & Strømme, M.
Venue
Journal of Functional Biomaterials
Publisher
MDPI AG
Published
2012-06-01
Volume
3
Issue
2
Pages
418-431
ISSN
2079-4983
DOI
10.3390/jfb3020418
PMC
PMC4047939
Cited by
96
References
1
Language
en
Licence
creativecommons.org/licenses/by/3.0/ (vor)

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