Cationic hydrous thorium dioxide colloids – a useful tool for staining negatively charged surface matrices of bacteria for use in energy-filtered transmission electron microscopy

Heinrich Lünsdorf,Ingeborg Kristen,Elke Barth

doi:10.1186/1471-2180-6-59

Abstract

BackgroundSynthesis of cationic hydrous thorium dioxide colloids (ca. 1.0 to 1.7 nm) has been originally described by Müller [22] and Groot [11] and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy.ResultsSynthesis of colloidal thorium dioxide has been modified and its use as a suitable stain of acidic mucopolysaccharides and other anionic biopolymers from bacteria, either as whole mount preparations or as preembedment labels, is described. The differences in stain behavior relative to commonly used rutheniumred-lysine and Alcian Blue™ electron dense acidic stains has been investigated and its use is exemplified for Pseudomonas aeruginosa adjacent cell wall biopolymers. For the first time thorificated biopolymers, i.e. bacterial outer cell wall layers, have been analysed at the ultrastructural level with electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI), leading to excellent contrast and signal strength for these extracellular biopolymers.ConclusionApplication of cationic hydrous ThO2 colloids for tracing acidic groups of the bacterial surface and/or EPS has been shown to be rather effective by transmission electron microscopy. Because of its high electron density and its good diffusibility it stains and outlines electro-negative charges within these biopolymers. In combination with ESI, based on integrated energy-filtered electron microscopy (EFTEM) Th-densities and thus negative charge densities can be discriminated from other elemental densities, especially in environmental samples, such as biofilms.

Highlights

Synthesis of cationic hydrous thorium dioxide colloids has been originally described by Müller [22] and Groot [11] and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy
The use of colloidal thorium dioxide, i.e. ThorotrastTM, as a high density stain for transmission electron microscopy began in the 1950s when colloids of 7 nm [10] were mainly used to trace the fate of ThorotrastTM in the tissues of mice and rats
In the present paper we describe the preparation of cationic colloidal hydrous thorium dioxide, based on modified methods from Müller [22] and Groot [11]

Summary

Introduction

Synthesis of cationic hydrous thorium dioxide colloids (ca. 1.0 to 1.7 nm) has been originally described by Müller [22] and Groot [11] and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy. In particular the significance of neutral and/or acidic EPS in biofilms, either 'in vitro' or 'ex vitro', is known [31,3] In this context it is of importance to obtain ultrastructural information on the amount and macromolecular architecture of the bacterial capsule and slime layers to allow morphological correlation with the physiological and pathogenic states in either 'in vitro' or (page number not for citation purposes). The use of colloidal thorium dioxide, i.e. ThorotrastTM, as a high density stain for transmission electron microscopy began in the 1950s when colloids of 7 nm [10] were mainly used to trace the fate of ThorotrastTM in the tissues of mice and rats. In 1981 Groot [11] performed comparative studies of positively charged thorium dioxide colloids versus rutheniumn red and colloidal iron as contrast agents for glycoaminoglycans, i.e. hyaluronanes

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