Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms

Honraet, Kris; Goetghebeur, Els; Nelis, Hans

doi:10.1016/j.mimet.2005.03.014

Cited by 102 publications

(69 citation statements)

References 28 publications

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“…Alternatively, biofilms were produced in microtitre plates as follows. Saturated cultures in SD 2 % glucose were diluted to OD 600 0.05 in 100 ml RPMI 1640 medium in 96-well microtitre plates and incubated at 37 uC for 24 h. The microtitre plates were then washed with PBS using a HydroFlex platform (Tecan) and 100 ml of a 16 FDA solution (506 stock: fluorescein diacetate, 2 g l 21 in acetone; diluted to 16 in PBS) was added per well (Honraet et al, 2005). Plates were wrapped in aluminium foil and incubated for 1 h at 37 uC before measuring fluorescence in a Tecan Infinite M200 microplate reader using an excitation filter of 486±9 nm and an emission filter of 535±20 nm.…”

Section: Methodsmentioning

confidence: 99%

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

et al. 2009

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The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-walllocated, N-and O-glycosylated proteins. The characterization of C. albicans pga59D/pga59D, pga62D/pga62D and pga59D/pga59D pga62D/pga62D mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure. INTRODUCTIONThe cell wall is an essential component of fungal cells, preserving cellular integrity and playing a central role in the interaction of fungi with their environment. This is particularly the case for pathogenic fungi such as the opportunistic yeast pathogen Candida albicans, where the cell wall has been shown to play central roles in adhesion, virulence, biofilm formation, infection and immunomodulation (Albrecht et al., 2006;Douglas, 2003;Netea et al., 2006;Richard et al., 2002b;Sundstrom, 2002). Because of the essential role of the cell wall in cellular integrity and fungal specificity of some enzymes involved in its biogenesis, it is a recognized target for the development of novel antifungals (e.g. echinocandins that target the b-1,3-glucan synthase) (Latge, 2007).The organization of the fungal cell wall has been mainly characterized in the yeasts Saccharomyces cerevisiae and C. albicans and in the filamentous fungus Aspergillus fumigatus (Klis et al., 2006;Latge, 2007;Lesage & Bussey, 2006;Ruiz-Herrera et al., 2006). The yeast cell wall has a bilayered structure. The inner part is composed of a network of b-1,3-glucan molecules linked by hydrogen bonds. These chains can be bound covalently to b-1,6-glucan molecules and to chitin chains. The outer part of the cell wall is composed mainly of mannoproteins (Klis et al., 2006Lesage & Bussey, 2006). Most proteins in the cell wall of ascomycetous yeasts are glycosylphosphatidylinositolanchored proteins (GPI-modified proteins) that become covalently linked to b-1,6-glucan through a remnant of their GPI anchor. As the b-1,6-glucan moiety can be linked to b-1,3-glucan or chitin, the cell wall GPI-modified proteins are strongly linked to the cell wall Klis et al., 2001;Richard & Plaine, 2007).GPI-...

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Section: Methodsmentioning

confidence: 99%

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

et al. 2009

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“…1). We (and others) have previously shown that the XTT-reduction assay shows excellent correlation between cellular density and metabolic activity, thus providing a semiquantitative measurement of biofilm formation [22][23][24][25][26] . This colorimetric assay is non-invasive and non-destructive, requiring minimal postprocessing of samples as compared to other alternative methods (such as viable cell counts) and has the additional advantage that, in contrast to other methods such as crystal violet staining and dry mass measurements, it correlates with cell viability which is particularly useful for measuring the effects of drugs on biofilm cells.…”

Section: Introductionmentioning

confidence: 99%

“…For example, results using the XTT-colorimetric assay to compare biofilm-forming ability by different isolates need to be interpreted with caution since it has been reported that different fungal species and even strains from the same species show marked differences in their ability to metabolize the XTT substrate 27 . In addition, alterations in the metabolic states during the different phases of biofilm formation may lead to fluctuations in the ability of cells within the biofilms to metabolize this dye 23,27 . Some more recent articles indicate that the use of other vital stains (SYTO9, propidium iodide), fluorogenic assays or bioluminescence may represent alternatives and have some practical advantages over the use of XTT 23,24,28 .…”

Section: Introductionmentioning

confidence: 99%

“…In addition, alterations in the metabolic states during the different phases of biofilm formation may lead to fluctuations in the ability of cells within the biofilms to metabolize this dye 23,27 . Some more recent articles indicate that the use of other vital stains (SYTO9, propidium iodide), fluorogenic assays or bioluminescence may represent alternatives and have some practical advantages over the use of XTT 23,24,28 . This 96 well microtiter plate model for biofilm formation was originally developed for C. albicans and other Candida spp.…”

Section: Introductionmentioning

confidence: 99%

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A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

et al. 2008

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The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications since fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96 well microtiter-based method for the formation of fungal biofilms which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (XTT) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately two days to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step towards the standardization of antifungal susceptibility testing of biofilms.

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“…This methodology was based on a continuous flow model in which growth media flowed at a specified rate, and was baffled stirred in order to generate shear force. Biofilm accumulation was then quantified by harvesting the biofilm from coupons of a known surface area, disaggregating the cells and polymeric matrix, and subsequently performing viable plate counts [14][15]. In this experimental design, this procedure was repeated with the coupon system replaced with a tongue model previously cast in resin from a tongue impression taken from a patient suffering from halitosis ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Application of a Novel Tongue Resin Replication System for in vitro Biofilm Studies

Bernardi¹,

Grootveld²,

Tejpal³

et al. 2016

BMRJ

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Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms

Cited by 102 publications

References 28 publications

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

Application of a Novel Tongue Resin Replication System for in vitro Biofilm Studies

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