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Fig. 3. Phenazines modulate colony biofilm morphology. (A, B) P. aeruginosa PA14 wild type and Aphz1/2 cultures were spotted onto 1% agar plates (containing 1% tryptone, 40 g/ml Congo Red and 20 j1g/ml Coomassie Blue) and incubated at 20 °C for 7 days in the presence of 0.2 mM PCA, 0.2 mM pyocyanin (PYO), or in the absence of supplements. Each day, colonies from three independent experiments were scanned and their surface coverage was determined. Representative images of colonies at days 2, 4 and 6 are shown (A; scale bar is | cm). The data in (B) show the average surface coverage and standard deviation for the three independent experiments. (C) Phenazine titrations. Colonies were grown as above, supplemented with 0, 0.1, 0.2, 0.3 or 0.4 mM PCA or PYO. Surface coverage of colonies from three independent experiments was determined after incubation at 20 °C for 7 days. Bars indicate the standard deviation.

Figure 3 Phenazines modulate colony biofilm morphology. (A, B) P. aeruginosa PA14 wild type and Aphz1/2 cultures were spotted onto 1% agar plates (containing 1% tryptone, 40 g/ml Congo Red and 20 j1g/ml Coomassie Blue) and incubated at 20 °C for 7 days in the presence of 0.2 mM PCA, 0.2 mM pyocyanin (PYO), or in the absence of supplements. Each day, colonies from three independent experiments were scanned and their surface coverage was determined. Representative images of colonies at days 2, 4 and 6 are shown (A; scale bar is | cm). The data in (B) show the average surface coverage and standard deviation for the three independent experiments. (C) Phenazine titrations. Colonies were grown as above, supplemented with 0, 0.1, 0.2, 0.3 or 0.4 mM PCA or PYO. Surface coverage of colonies from three independent experiments was determined after incubation at 20 °C for 7 days. Bars indicate the standard deviation.

Related Figures (3)

Fig. 1. Aphzl/2 (Aphz ) has increased swarming motility. Images of three swarming plates for each strain or condition were captured and exported to Adobe Photoshop and the agar surface covered by the swarms was quantified using Analysis Tools. I. Ramos et al. / Research in Microbiology 161 (2010) 187-191
Fig. 1. Aphzl/2 (Aphz ) has increased swarming motility. Images of three swarming plates for each strain or condition were captured and exported to Adobe Photoshop and the agar surface covered by the swarms was quantified using Analysis Tools. I. Ramos et al. / Research in Microbiology 161 (2010) 187-191
Quantitative analysis of 4-day old biofilms formed by the wild type and a phenazine-defective mutant. Analysis was conducted using COMSTAT. Images from similar positions in the flow cell were acquired for all conditions in triplicate. * 25 uM pyocyanin (PYO) was added to the medium from the beginning of the experiment. > Minimum microcolony size at the substratum was set at 10 pm’. Table |
Quantitative analysis of 4-day old biofilms formed by the wild type and a phenazine-defective mutant. Analysis was conducted using COMSTAT. Images from similar positions in the flow cell were acquired for all conditions in triplicate. * 25 uM pyocyanin (PYO) was added to the medium from the beginning of the experiment. > Minimum microcolony size at the substratum was set at 10 pm’. Table |
Fig. 2. Phenazines support development of structured biofilms in a continuous flow cell system. Representative images of 4-day-old biofilms of the wild type (A and phz1/2 mutant without (B) and with addition of 25 uM pyocyanin (C) to the nutrient flow. Images of similar positions in the flow cell were taken in triplicate To determine whether the morphological trends we observed in flow cells might translate to a larger scale, we grew colony biofilms of the wild type and Aphz//2. As we reported previously (Dietrich et al., 2008), wild type PAI4 formed smooth colonies that developed concentric ridges only after prolonged incubation (4 days; Fig. 3A), while Aphz//2 formed wrinkled colonies that grew vertically within 2 days. Surface coverage by Aphz1/2 colonies was increased by up to To measure the impact of phenazines on biofilm formation directly, we first used a flow cell system to grow wild type and Aphz1/2 biofilms. In this system, the wild type formed heterogeneous biofilms that after 4 days developed large and abundant microcolonies (Fig. 2A). Biofilms of Aphz1/2 were flatter and consisted of fewer and smaller aggregates scattered throughout the field of view (Fig. 2B). The morphology of 4- day-old biofilms was analyzed using COMSTAT (Heydorn et al., 2000). A fixed threshold value and connected volume
Fig. 2. Phenazines support development of structured biofilms in a continuous flow cell system. Representative images of 4-day-old biofilms of the wild type (A and phz1/2 mutant without (B) and with addition of 25 uM pyocyanin (C) to the nutrient flow. Images of similar positions in the flow cell were taken in triplicate To determine whether the morphological trends we observed in flow cells might translate to a larger scale, we grew colony biofilms of the wild type and Aphz//2. As we reported previously (Dietrich et al., 2008), wild type PAI4 formed smooth colonies that developed concentric ridges only after prolonged incubation (4 days; Fig. 3A), while Aphz//2 formed wrinkled colonies that grew vertically within 2 days. Surface coverage by Aphz1/2 colonies was increased by up to To measure the impact of phenazines on biofilm formation directly, we first used a flow cell system to grow wild type and Aphz1/2 biofilms. In this system, the wild type formed heterogeneous biofilms that after 4 days developed large and abundant microcolonies (Fig. 2A). Biofilms of Aphz1/2 were flatter and consisted of fewer and smaller aggregates scattered throughout the field of view (Fig. 2B). The morphology of 4- day-old biofilms was analyzed using COMSTAT (Heydorn et al., 2000). A fixed threshold value and connected volume
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MicrobiologyColony Morphology
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