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Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

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Summary

In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.

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Abbreviations

cw:

cell wall

cy:

cytoplasm

FS:

freeze-substitution

FS-A:

GA/OsO4 freeze-substitution and Spurr's LV-embedding

FS-B:

GA freeze-substitution and Lowicryl K 4 M LT-embedding

FS-C:

GA freeze-substitution and Lowicryl HM 20 LT-embedding

go:

Golgi apparatus-like body

GA:

glutaraldehyde

g:

glycogen deposit

l:

lipid droplet

LT:

low temperature

Lowicryl LT-embedding:

Lowicryl low-temperature embedding

Lowicryl LT-resin:

Lowicryl low-temperature resin

MeOH:

methanol

mf:

microfilament

mt:

microtubule

m:

mitochondrion

mvb:

multivesicular body

ne:

nuclear envelope

np:

nuclear pore

npl:

nucleoplasm

nu:

nucleolus

n:

nucleus

OsO4 :

osmium tetroxide

pl:

plasmalemma

pr:

polyribosomes

Pb-citrate:

Reynolds' lead citrate

r:

ribosome

RT:

room temperature

rer:

rough endoplasmic reticulum

Spurr's LV-embedding:

Spurr's low viscosity embedding

Spurr's LV-resin:

Spurr's low viscosity resin

t:

tonoplast

Uac:

uranyl acetate

v:

vacuole

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  1. Institut für Biologie III (Pflanzenphysiologie), Rheinisch-Westfälische Technische Hochschule Aachen, Worringer Weg., D-5100, Aachen, Germany

    Sigrun Hippe & Monika Hermanns

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  1. Sigrun Hippe
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  2. Monika Hermanns
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Hippe, S., Hermanns, M. Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding. Protoplasma 135, 19–30 (1986). https://doi.org/10.1007/BF01277049

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