Abstract
Efficient, fast and new micro-analytical methods for characterization of ultrastructures of fungal spores with electron microscopy are very much required and essential. SEM analysis of biological materials, especially fungi, requires optimal preparation of the specimen and often requires the usage of dried samples which demands a challenging sample preparation. In the present investigation, we described a fast and improved method for the preparation of fungal specimen for scanning electron microscopy (SEM). The fungus, Curvularia lunata was grown on the surface of sterile Whatman No.1 filter paper which was overlaid on Potato Dextrose Agar (PDA) medium, gold coated immediately after removal from the growth medium and subjected to imaging. Generally, SEM imaging is done with samples that were fixed with chemical fixatives, dehydrated and gold coated specimens, but here we describe an easy and more efficient sample preparation for SEM which enabled enhanced image quality and precision visualization of fungal cells, especially the spores. The developed method has enabled the analysis of even the robust samples like fungal spores that to eliminating special temperature requirement. The ultimate goal was to develop an improved protocol/method applied to analysis of fungal spores with greater coverage about fungal specimen preparation. This method permits the use of rapid sample preparation and will allow us to imaging of individual spore or conidia structures in the context of fungal cell architecture which clarifies our understanding in fungal taxonomy/biology.
Highlights
Maximal structural preservation in their original form
The developed method involves growing the fungal sample on a small strip of Whatman No.l filter paper which was overlaid on potato dextrose agar (PDA) medium and drying under room temperature proved highly effective for visualisation of fungal spore in their native state without compromising the structural characteristics of the spores in a scanning electron microscope (SEM)
As molecular taxonomy moves toward studies of more complex systems, especially analysing the spores, the focus of interest has moved towards developing robust fungal samples preparation
Summary
Maximal structural preservation in their original form. These techniques comprise of cryofixation followed by freeze-drying, critical point drying and discrete types of chemical fixation treatments prior to dehydration of samples[5]. An innovative and modified technique are constantly being examined and refined for the design of preservation strategies of fungal spore sample optimum for SEM imaging, which includes hydrated, dehydrated/dried, chemical fixation, frozen hydrated, freeze drying, simple air drying and critical point drying of the biological sample[5,6,7]. We present a pipeline to generate reproducible high spatial fungal spore imaging sample preparation by imaging with Scanning Electron Microscopy (SEM). This pipeline is reliable to implement and preserves fungal spore and cell morphology without disturbing its native structure. We illustrate five successful approaches for this strategy, ranging from the different sample preparation and fixative to its subsequent SEM imaging
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