Microscopy of fungal biofilms

Lagree, Katherine; Desai, Jigar V.; Finkel, Jonathan S.; Lanni, Frederick

doi:10.1016/j.mib.2017.12.008

Cited by 32 publications

(24 citation statements)

References 16 publications

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“…Biofilms were prepared and imaged as previously described in detail [61]. Overnight cultures were diluted to an OD 600 of 0.2 in 2 ml RPMI media in 6 well culture plates containing 1.5 cm x 1.5 cm sized medical-grade silicone squares.…”

Section: Biofilm Microscopy and Quantificationmentioning

confidence: 99%

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality

et al. 2020

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Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

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Section: Biofilm Microscopy and Quantificationmentioning

confidence: 99%

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality

et al. 2020

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“…Therefore, in comparison with other conventional large specimen imaging techniques, the Mesolens stands as a novel and improved method for in situ imaging of live bacterial communities. Additionally, recent advances in light sheet microscopy 33 and mutli-photon microscopy 34,35 have been applied to biofilm imaging. However, these methods currently cannot resolve sub-micron information over multi-millimetre scales, as with the Mesolens.…”

Section: Discussionmentioning

confidence: 99%

Functional channels in matureE. colicolonies

Rooney

Amos

Hoskisson

et al. 2019

Preprint

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17Biofilms are important in medicine, industry and the natural environment, however their structure 18 is largely unexplored across multiple spatial scales. We have studied the architecture of mature 19Escherichia coli macro-colony biofilms by means of the Mesolens, an optical system which 20 uniquely allows simultaneous imaging of individual bacteria and over one hundred cubic 21 millimetres of its biofilm milieu. Our chief finding is the presence of intra-colony channels on the 22 order of 10 µm in diameter in E. coli biofilms. These channels have a characteristic structure and 23 reform after total mechanical disaggregation of the colony. We present evidence that the channels 24 transport particles and function to assist the absorption of nutrients. These channels potentially 25 offer a new route for the delivery of dispersal agents or antimicrobial drugs to biofilms, ultimately 26 lowering their impact on public health and industry. 27

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“…However, a large degree of clarification can be achieved by infiltrating fixed biofilms with a high refractive index liquid that approximately matches the refractivity of major cellular constituents. After clarification, imaging at submicron resolution can be carried out by confocal microscopy through the entire thickness of almost any C. albicans biofilm 13 . In wild type specimens, it is easy to see that biofilms consist of long entangled hyphae, clusters of budded yeast-form cells or pseudohyphal cells, void spaces, and some amount of extracellular matrix.…”

Section: Introductionmentioning

confidence: 99%

Clarifying and Imaging <em>Candida albicans</em> Biofilms

Lanni

Lagree

Huang

et al. 2020

JoVE

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The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 µm biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

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Microscopy of fungal biofilms

Cited by 32 publications

References 16 publications

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality

Functional channels in matureE. colicolonies

Clarifying and Imaging <em>Candida albicans</em> Biofilms

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