Enhanced method for High Spatial Resolution surface imaging and analysis of fungal spores using Scanning Electron Microscopy

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.

Figure 1

SEM analysis of Curvularia lunata spores processed by growing the fungus on Whatman No.1 filter paper strips overlaid with PDA medium with simple air drying (Native state). (a) Condiospores showing smooth wall and uniform density (scale bar = 30 μm). (b) A bunch of conidia without any perturbation (scale bar = 20 μm). (c) Spore dimension analysis in their native preserved form (scale bar = 20 μm). (d) Morphological characteristics of a single spore (scale bar = 10 μm).

Figure 2

SEM analysis of Curvularia lunata spores processed by chemical fixation with glutaraldehyde followed by dehydration in ethanol and simple air-drying. (a) Fungus spore severely distorted and extensively shrinked by glutaraldehyde fixation (Scale bar = 30 μm). (b) The dimension analysis of damaged spores (Scale bar = 30 μm).

Figure 3

SEM analysis of Curvularia lunata spores processed by fixation with osmium tetroxide followed by dehydration in ethanol and simple air-drying. (a) Fungal spores were severely zapped out by the treatment of osmium tetroxide (Scale bar = 30 μm). (b) Spore deformed in structures (Scale bar = 20 μm). (c) Spore dimension analysis after treatment (Scale bar = 20 μm).

Figure 4

SEM analysis of Curvularia lunata spores processed by flash freezing with liquid nitrogen (a) The surfaces of condiospore are little blurred as compared with native state preservation (Scale bar = 30 μm). (b) A semi-crytalline film formation on the outer surface of the spore (Scale bar = 20 μm). (c) Spore Dimension analysis of the spore (Scale bar = 20 μm). (d) Morphological characteristics of a single spore showing mild damage by slush nitrogen (Scale bar = 10 μm).

Figure 5

SEM analysis of Curvularia lunata spores processed by flash freezing with liquid nitrogen followed by freeze drying (a) Formation of crystal structures on the surface of conidiospore (Scale bar = 30 μm). (b) Spore dimension analysis (Scale bar = 20 μm). (c) Morphological characteristics of a single spore showing extensive damage on the structure (Scale bar = 10 μm).

Figure 6

A 3D graph depicting the fungal spore dimensions imaged under SEM based on its length, thickness and inward groove in micometer scale (μm) when processed with various sample preparations treatments.[Violet dot-Native, Red dot- Chemical fixation, Orange dot- Post fixation with Osmium tetroxide (O₂O₄), Blue dot-Flash freezing and Green dot-Flash freezing followed by freeze drying].

Figure 7

Schematic representation of sample preparation of Curvularia lunata spores for Scanning Electron Microscopy. (1) Isolation of Curvularia lunata from black kernel disease infected rice plants. (2) Morphological visualization of the fungal spores under light microscope (40x). (3) Maintenance of the fungal inoculums in PDA slant. (4) Inoculation and growth of the fungus on PDA medium overlaid on sterile Whatman No.1 filter paper strips, (5) Removal of the strips containing the fungal spores with sterile forceps. (6) The strips were placed on top of the stub for ion sputtering. (7) SEM images of the fungal spores at native state. (8) Flash freezing. (9) Flash freezing and freeze drying. (10) Chemical fixation with glutaraldehyde and (11) Chemical fixation with osmium tetroxide.

Figure 8

Overall design of fungal spore sample preparation for Scanning Electron Microscopy. Infected rice seedling caused by Curvularia lunata and its microscopic view (1, 2), Fungal grown on whatman No.1 filter paper (3), gold coating (4), The observed imaging includes; Native state (4a), flask freezing (4b), flask freezing and freeze dried (4c) glutaraldehyde (4d) and osmium tetroxide (4e).