Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis
<p>Viability and vitality staining of <span class="html-italic">C. parapsilosis</span> 11103595 overnight cultures. (<b>A</b>) Viability assessment of <span class="html-italic">C. parapsilosis</span> 11103595 planktonic cells using S9 (green) + PI (red) and TO (green) + PI (red) staining. Dyes were compared with CW (blue) merged image for cell visualization control. White arrows in the S9 + PI row highlight some of the cells that were not stained with S9 but were visible with CW. (<b>B</b>) Vitality evaluation using FDA (green) and FUN-1 (green and red) staining. CW (blue) merged image was used as a cell visualization control. White arrows in the FDA row indicate high intensity only in a dead cell. White arrow in the zoomed in image of the FUN-1 row shows a green non-metabolically active cell, while the red arrow shows a cell with red cylindrical intravacuolar structures (CIVSs), indicating metabolic activity. Confocal images were processed using ImageJ v1.54f. The scale bar of 10 µm is consistent for all cases, except in the FUN-1 zoomed-in image where it represents a 5 µm length. Images were taken at 100×. S9 = Syto9, PI = propidium iodide, TO = thiazole orange, CW = calcofluor white, FDA = fluorescein diacetate.</p> "> Figure 2
<p>Total biomass and metabolic activity of <span class="html-italic">C. parapsilosis</span> 11103595 biofilms treated with amphotericin B (AmB). (<b>A</b>) Total biomass quantification (cell biomass and extracellular matrix) with crystal violet (CV) assay and (<b>B</b>) metabolic activity evaluation by PrestoBlue assay after 20 h treatment with 2.5 μg/mL of AmB. Biofilm results experiments were conducted in triplicate. Numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Data are represented as mean ± standard deviation. Asterisks indicate statistically significant differences versus control in an unpaired <span class="html-italic">t</span>-test (*: <span class="html-italic">p</span>-value < 0.05, ***: <span class="html-italic">p</span>-value < 0.001). RFUs = relative fluorescence units.</p> "> Figure 3
<p>Viability staining of <span class="html-italic">C. parapsilosis</span> 11103595 biofilms treated with amphotericin B (AmB). (<b>A</b>) Viability assessment using S9 (green live cells) + PI (red dead cells) dye with the respective biomass quantification. (<b>B</b>) Viability assessment using TO (green live cells) + PI (red dead cells) dyes with the respective biomass quantification. Each dye combination was compared with the CW (gray) merged image and the respective biomass correction. Biomass quantifications were performed from images at a 10× magnification with the plugin COMSTA2 from ImageJ software v1.54f. Data are represented as mean ± standard deviation from <span class="html-italic">n</span> ≥ 3 replicates. Asterisks indicate statistically significant differences versus control (***: <span class="html-italic">p</span>-value < 0.001; ****: <span class="html-italic">p</span>-value < 0.0001). Numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Z-stack of biofilm top layers from confocal images at a 63× magnification were created using ImageJ v1.54f. The scale bar of 10 µm is consistent for all cases. S9 = Syto9, PI = propidium iodide, TO = thiazole orange, CW = calcofluor white.</p> "> Figure 4
<p>Vitality staining of <span class="html-italic">C. parapsilosis</span> 11103595 biofilms treated with amphotericin B (AmB). (<b>A</b>) Vitality assessment using FDA (green metabolically active cells) dye with the respective biomass quantification. (<b>B</b>) Vitality assessment using FUN-1 (green metabolically unactive cells and green/red metabolically active cells) with the respective biomass quantification. Each dye was compared with the CW (gray) merged image and the respective biomass correction. Biomass quantifications were performed from images at a 10× magnification with the plugin COMSTA2 from ImageJ software v1.54f. Data are represented as mean ± standard deviation from n ≥ 3 replicates. Asterisks indicate statistically significant differences versus control (*: <span class="html-italic">p</span>-value < 0.05). Numbers after the symbol ↑ indicate the percentage of increase in the mean value with respect to the control, while numbers after the symbol ↓ indicate the percentage of decrease in the mean value with respect to the control. Z-stack of biofilm top layers from confocal images at a 63× magnification were created using ImageJ v1.54f. The scale bar of 10 μm is consistent for all cases. CW = calcofluor white, FDA = fluorescein diacetate.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Viability and Vitality Staining of C. parapsilosis 11103595 Overnight Cultures
2.2. Total Biomass and Metabolic Activity of C. parapsilosis 11103595 Biofilms Treated with AmB
2.3. Viability and Vitality Staining of C. parapsilosis 11103595 Biofilms Treated with AmB
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Growing Conditions
4.2. Biofilm Formation on Silicon Coupons
4.3. Biofilm Total Biomass and Metabolic Activity
4.4. Dyes Evaluation
4.5. Quantification and Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Evaluated Dye | Non-Treated Biofilms | AmB-Treated Biofilms | Change (%) |
---|---|---|---|
%Viability (live cells biomass/(live cells biomass + dead biomass)) | |||
S9–PI | 96.6 ± 0.3 | 81.9 ± 2.9 | −15.2 ± 2.8 |
TO–PI | 94.9 ± 3.7 | 89.1 ± 1.4 | −6 ± 2.6 |
Metabolically active biomass (µm3/µm2) | |||
FDA | 23.1 ± 4.8 | 26.8 ± 3.1 | +17.1 ± 10.5 |
FUN-1 1 | 31.1 ± 0.9 | 19.5 ± 0.6 | −37.1 ± 1.1 |
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Arévalo-Jaimes, B.V.; Torrents, E. Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis. J. Fungi 2024, 10, 209. https://doi.org/10.3390/jof10030209
Arévalo-Jaimes BV, Torrents E. Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis. Journal of Fungi. 2024; 10(3):209. https://doi.org/10.3390/jof10030209
Chicago/Turabian StyleArévalo-Jaimes, Betsy Verónica, and Eduard Torrents. 2024. "Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis" Journal of Fungi 10, no. 3: 209. https://doi.org/10.3390/jof10030209