Bacterial-Fungal Co-culture
Materials Required
Principle
Bacterial-fungal co-culture assays measure interkingdom biofilm formation, physical association, viable cell recovery, biomass accumulation, metabolic activity, and species-specific interaction outcomes. In classic Candida albicans-Staphylococcus aureus models, S. aureus preferentially associates with C. albicans hyphae, mixed biofilms can increase bacterial recovery compared with bacterial monoculture, and readouts include CFU counting, microscopy, crystal violet biomass staining, and XTT metabolic activity[1][2][3][4]. For C. albicans-Pseudomonas aeruginosa co-culture, the assay can also detect antagonistic or matrix-associated interactions, including phenazine-associated fungal toxicity on solid medium and increased dual-species biofilm biovolume or thickness in some biofilm conditions[5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use RPMI 1640 as the biofilm medium for C. albicans-S. aureus 96-well biofilm assays, phosphate-buffered saline for washing, Sabouraud dextrose agar with chloramphenicol for selective recovery of C. albicans, mannitol salt agar for recovery of S. aureus, methanol for biofilm fixation before crystal violet staining, crystal violet for total biomass staining, and XTT reagent for metabolic-activity measurement[3].
• Use serum-containing medium when reproducing the original C. albicans-S. aureus polymicrobial biofilm model in which S. aureus forms poor monoculture biofilms in serum but forms substantial polymicrobial biofilms with C. albicans[1].
• Use SYTO 9 for live microbial-cell fluorescence, calcofluor white for fungal cell-wall staining, and ConA-Texas Red for matrix staining when imaging C. albicans-S. aureus polymicrobial biofilms by fluorescence microscopy[1].
• Use XTT reagent when the experimental endpoint is total metabolic activity, and use crystal violet when the endpoint is total adherent biomass rather than viable-cell number or metabolic activity[3].
• Use sterile flat-bottom 96-well microplates for adhesion and biofilm formation, an incubator set to 37°C, an orbital shaker operated at 75 rpm for the Zago 96-well model, a spectrophotometer for inoculum standardization, a plate reader for absorbance-based XTT and crystal violet readouts, and fluorescence, confocal, or scanning electron microscopy when structural localization of bacteria on fungal hyphae is required[1][2][3].
• Use serum-containing medium when reproducing the original C. albicans-S. aureus polymicrobial biofilm model in which S. aureus forms poor monoculture biofilms in serum but forms substantial polymicrobial biofilms with C. albicans[1].
• Use SYTO 9 for live microbial-cell fluorescence, calcofluor white for fungal cell-wall staining, and ConA-Texas Red for matrix staining when imaging C. albicans-S. aureus polymicrobial biofilms by fluorescence microscopy[1].
• Use XTT reagent when the experimental endpoint is total metabolic activity, and use crystal violet when the endpoint is total adherent biomass rather than viable-cell number or metabolic activity[3].
• Use sterile flat-bottom 96-well microplates for adhesion and biofilm formation, an incubator set to 37°C, an orbital shaker operated at 75 rpm for the Zago 96-well model, a spectrophotometer for inoculum standardization, a plate reader for absorbance-based XTT and crystal violet readouts, and fluorescence, confocal, or scanning electron microscopy when structural localization of bacteria on fungal hyphae is required[1][2][3].
Experimental Procedure
• Prepare fresh C. albicans and bacterial cultures from maintained stocks; in the Zago model, C.albicans SC5314 was subcultured on Sabouraud dextrose agar with chloramphenicol at 37°C for 24-48 h, and S. aureus ATCC 25923 or ATCC 33591 was subcultured on mannitol salt agar at 37°C for 24-48 h[3].
• Wash cells and resuspend them in RPMI 1640; standardize C.albicans to OD540=1.0 and S. aureus to OD600=0.1, corresponding to approximately 10^7 cells/mL in the reported model[3].
• For the RPMI 96-well model, add 75 µL RPMI 1640 plus 75 µL of each single-species suspension for monoculture wells, and add 75 µL fungal suspension plus 75 µL bacterial suspension for dual-species wells; incubate adhesion assays for 90 min at 37°C with orbital shaking at 75 rpm[3].
• After adhesion, remove non-adherent cells by washing twice with 150 µL PBS, add 150 µL fresh RPMI 1640, and incubate biofilms for 12, 24, or 48 h; for 48 h biofilms, replace 75 µL spent medium with 75 µL fresh RPMI after the first 24 h[3].
• For viable-cell recovery, wash wells twice with PBS, resuspend the biofilm in 100 µL PBS, scrape adherent biofilm with a sterile pipette tip for 1 min, vortex the suspension, serially dilute in PBS, and plate on selective agar for species-specific CFU recovery[3].
• For XTT metabolic activity, prepare XTT at 1 mg/mL in ultrapure water, filter sterilize, and apply the assay after the selected adhesion or biofilm time point as reported for 90 min, 12 h, 24 h, and 48 h samples[3].
• For crystal violet biomass analysis, aspirate medium, wash wells with PBS, fix biofilms with 200 µL 100% methanol for 15 min, and stain fixed biofilms using the reported crystal violet method for total biomass measurement[3].
• For microscopy-based structure analysis, stain polymicrobial biofilms with SYTO 9, calcofluor white, and ConA-Texas Red, or process samples for SEM when the objective is to confirm bacterial association with fungal hyphae[1][2][3].
• Analyze co-culture outcomes using species-specific CFU counts, XTT absorbance, crystal violet absorbance, and microscopy; CFU counting measures viable recovered cells, XTT measures metabolic activity, crystal violet measures total biomass including cells and matrix, and microscopy shows spatial association between bacteria and fungal forms[1][2][3].
• Include single-species fungal and bacterial biofilms as controls, and include dual-species wells as the experimental condition; Zago et al. performed five technical replicates and repeated assays in three independent experiments, using two-way ANOVA with Tukey post hoc tests for CFU, XTT, and crystal violet data[3].
• Interpret increased bacterial CFU or biofilm biomass in dual culture cautiously, because C.albicans-S.aureus and C.albicans-P. aeruginosa interactions can be cooperative, antagonistic, or condition-dependent depending on medium, timing, species pair, and interaction chemistry[1][3][5][6][7].
• Wash cells and resuspend them in RPMI 1640; standardize C.albicans to OD540=1.0 and S. aureus to OD600=0.1, corresponding to approximately 10^7 cells/mL in the reported model[3].
• For the RPMI 96-well model, add 75 µL RPMI 1640 plus 75 µL of each single-species suspension for monoculture wells, and add 75 µL fungal suspension plus 75 µL bacterial suspension for dual-species wells; incubate adhesion assays for 90 min at 37°C with orbital shaking at 75 rpm[3].
• After adhesion, remove non-adherent cells by washing twice with 150 µL PBS, add 150 µL fresh RPMI 1640, and incubate biofilms for 12, 24, or 48 h; for 48 h biofilms, replace 75 µL spent medium with 75 µL fresh RPMI after the first 24 h[3].
• For viable-cell recovery, wash wells twice with PBS, resuspend the biofilm in 100 µL PBS, scrape adherent biofilm with a sterile pipette tip for 1 min, vortex the suspension, serially dilute in PBS, and plate on selective agar for species-specific CFU recovery[3].
• For XTT metabolic activity, prepare XTT at 1 mg/mL in ultrapure water, filter sterilize, and apply the assay after the selected adhesion or biofilm time point as reported for 90 min, 12 h, 24 h, and 48 h samples[3].
• For crystal violet biomass analysis, aspirate medium, wash wells with PBS, fix biofilms with 200 µL 100% methanol for 15 min, and stain fixed biofilms using the reported crystal violet method for total biomass measurement[3].
• For microscopy-based structure analysis, stain polymicrobial biofilms with SYTO 9, calcofluor white, and ConA-Texas Red, or process samples for SEM when the objective is to confirm bacterial association with fungal hyphae[1][2][3].
• Analyze co-culture outcomes using species-specific CFU counts, XTT absorbance, crystal violet absorbance, and microscopy; CFU counting measures viable recovered cells, XTT measures metabolic activity, crystal violet measures total biomass including cells and matrix, and microscopy shows spatial association between bacteria and fungal forms[1][2][3].
• Include single-species fungal and bacterial biofilms as controls, and include dual-species wells as the experimental condition; Zago et al. performed five technical replicates and repeated assays in three independent experiments, using two-way ANOVA with Tukey post hoc tests for CFU, XTT, and crystal violet data[3].
• Interpret increased bacterial CFU or biofilm biomass in dual culture cautiously, because C.albicans-S.aureus and C.albicans-P. aeruginosa interactions can be cooperative, antagonistic, or condition-dependent depending on medium, timing, species pair, and interaction chemistry[1][3][5][6][7].
Troubleshooting
Low S.aureus biofilm signal in serum monoculture:
Serum alone did not support strong S. aureus monoculture biofilm formation in the Harriott model.Solution
Include C. albicans-S. aureus dual-species wells and bacterial monoculture controls in rich medium or the same serum condition so that serum-dependent enhancement by C. albicans can be distinguished from baseline bacterial growth[1].
Crystal violet signal does not match CFU results:
Crystal violet stains total biomass, including matrix and dead cells, whereas CFU counts only viable recovered cells.Solution
Pair crystal violet with CFU and XTT rather than interpreting biomass as viable-cell abundance[3].
Weak bacterial recovery from dual biofilms:
Biofilm disruption may be incomplete before dilution and plating.Solution
Wash wells, scrape adherent biofilm with a sterile pipette tip for 1 min, vortex the suspension, and verify removal using crystal violet staining as described in the published protocol[3].
No visible bacterial-fungal spatial association:
The interaction is often localized to C. albicans hyphal elements rather than yeast-form cells.Solution
Use microscopy endpoints and conditions that permit hyphal development, and evaluate bacterial localization on hyphae rather than only bulk biomass[1][2][3][4].
References:
- [1]. Harriott MM, et al. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob Agents Chemother. 2009;53(9):3914-3922. [Content Brief]
- [2]. Peters BM, et al. Microbial interactions and differential protein expression in Staphylococcus aureus-Candida albicans dual-species biofilms. FEMS Immunol Med Microbiol. 2010;59(3):493-503. [Content Brief]
- [3]. Zago CE, et al. Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA). PLoS One. 2015;10(4):e0123206. [Content Brief]
- [4]. Kean R, Rajendran R, Haggarty J, Townsend EM, Short B, Burgess KE, et al. Candida albicans mycofilms support Staphylococcus aureus colonization and enhances miconazole resistance in dual-species interactions. Front Microbiol. 2017;8:258. [Content Brief]
- [5]. Gibson J, et al. Pseudomonas aeruginosa-Candida albicans interactions: localization and fungal toxicity of a phenazine derivative. Appl Environ Microbiol. 2009;75(2):504-513. [Content Brief]
- [6]. Phuengmaung P, et al. Coexistence of Pseudomonas aeruginosa with Candida albicans enhances biofilm thickness through alginate-related extracellular matrix but is attenuated by N-acetyl-l-cysteine. Front Cell Infect Microbiol. 2020;10:594336. [Content Brief]
- [7]. Dühring S, et al. Studying mixed-species biofilms of Candida albicans and Staphylococcus aureus using evolutionary game theory. PLoS One. 2024;19(3):e0297307. [Content Brief]
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