Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Abstract

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

In vitro Evaluation of Photodynamic Effects Against Biofilms of Dermatophytes Involved in Onychomycosis

“…In particular, two types of peculiar extracellular matrix (ECM) architecture could be observed: (i) an extremely thin “blanket-like” layer covering the areas between hyphae (Figure 1B); (ii) very fine “mesh-like” layer wrapping the filaments of hyphae (Figure 1C). The biofilm morphology of high resolution and magnification was investigated and imaged by SEM technique to confirm the biofilm-formation in this study and the SEM images obtained are similar to those reported previously (Brilhante et al, 2017; Vila et al, 2017; Guzel Tunccan et al, 2018).…”

Anticandidal activity of bioinspired ZnO NPs: effect on growth, cell morphology and key virulence attributes of Candida species

“…These biofilms are major virulence factor and play an important role in C. albicans pathogenesis. The structural complexity and polymeric extracellular matrix make biofilms more resistant to antifungal drugs than planktonic cells [64,65]. It has been reported that C. albicans can form biofilms on almost any kind of medical device such as vascular and urinary catheters, joint prostheses, cardiac valves, artificial vascular bypass devices, pacemakers, ventricular assist devices and central nervous system shunts [66].…”

Characteristics of Biofilms Formed by C. parapsilosis Causing an Outbreak in a Neonatal Intensive Care Unit

“…However, we observe few pseudohyphae. Regarding the biofilms of this study compared with those of past reports, we should note that the biofilms in this study formed very large masses with dense ECM, and they seemed quite sticky [ 12 , 21 , 22 , 23 ]. We theorized that C. parapsilosis in invasive fungal infections likely forms unusual biofilms with very dense ECM.…”