Exosporial membrane plasticity of Clostridium sporogenes and Clostridium difficile.

This investigation examines the morphological alterations of the exosporial membranes of Clostridium sporogenes ATCC 3584 and Clostridium difficile ATCC 43594 and 9689 endospores in relation to their possible function during germination in the attachment/colonization process of these pathogenic bacteria. There is no reported function for the exosporial membrane, nor exosporial appendages, of clostridial endospores. Advances in high resolution, scanning electron microscopy (SEM) permit the examination of these delicate, morphological projections on intact spores in the process of attachment. The morphological plasticity of the exosporial membrane projections during activation and germination was examined to determine whether the appearance of these exosporial projections coincided with attachment of the spores to the nutritive substrate, and whether this attachment could be altered by physical agitation, cation competition with Ba2+, chelation with EDTA, or treatment with colchicine. Following incubation, activated spores could not be removed from the agar surface by agitation in water (pH 7.2 or 9.1), nor by agitation in buffer or colchicine, indicating that some form of adherence or attachment to the agar had taken place. When agitated in the presence of Ba2+ or EDTA in phosphate buffered saline or EDTA in water, all activated spores detached from the agar and exhibited decreased exosporial projections and minimal, if any, attachment structures to the agar surface. Activated clostridial spores were found to attach to agar by delicate extensions of the exosporium that could be disrupted by EDTA or Ba2+ exposure, but were unchanged when shaken in buffer or water.

Biological calcium absorption edge imaging using monochromatic synchrotron radiation.

Soft X-ray contact absorption edge images of unfixed, unstained biological specimens were made using monochromatic synchrotron radiation. X-ray contact replicas of unfixed, hydrated biological specimens at the nitrogen absorption edge and above and below the CaLIII absorption edge were compared to comparative conventional morphological and elemental high-resolution imaging methods (scanning and transmission electron microscopy, TEM-histochemistry and TEM-X-ray microanalysis). Soft X-ray absorption edge images made above the calcium absorption edge clearly revealed morphological detail and identified regions ladened with calcium as verified by TEM histochemistry of identical spores. Similarly, nitrogen absorption edge images identified residual nitrogenous material in the spore resuspension medium, and non-viable spores with nitrogen loss due to protoplast disaggregation.

Absorption edge imaging of bacterial endospores with synchrotron radiation.

This article describes a new method of viewing biological specimens by taking advantage of the absorptive characteristics of monochromatic X-rays above and below the absorption edge of a specific element. Bacterial endospores were imaged before and after treatment with an experimental vanadium-containing sporocide using monochromatic synchrotron radiation at the nitrogen absorption edge, and above and below the vanadium LIII absorption edge. This morphological study demonstrates a rapid, easy-to-use method of soft X-ray absorption edge imaging that can be used by the biologist to obtain morphological and elemental information that is not readily accessible using conventional microscopic and analytic techniques.

Cation shifts in human retina-choroid.

Human ocular tissues from 50 donor eyes were elementally and morphologically analyzed in order to correlate the elemental content and distribution of Ca, Ba, Cr, Cu, Zn, and Se with ocular morphology, sex, race, irideal pigmentation, age, time of death, birth weight, presence and severity of diabetes, and other pathologies noted at autopsy. Initially, to facilitate the transport of donor tissue to the laboratory, the eyes were fixed in glutaraldehyde. Because our preliminary data revealed alterations in elemental content following chemical fixation of ocular tissues, all of the subsequent samples were analyzed in their fresh, hydrated (unfixed) condition as soon after enucleation as possible. Samples were elementally analyzed by X-ray fluorescence spectrometry (XRF) and proton-induced X-ray emission spectrometry (PIXE) using high resolution Si(Li) X-ray detectors. Tissue was morphologiscally examined by scanning electron microscopy and light microscopy histoichemistry.

Contact microscopy with synchrotron radiation.

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.

Basic biological X-ray microanalysis.

As more scanning and transmission electron microscopes become equipped with x-ray detectors, increasing numbers of scientists and technicians will be trying to obtain elemental data using the myriad of specimen preparation methods and quantitative procedures that have been developed over the past 28 years. This paper is an introduction to the basic concepts of biological x-ray microanalysis for new investigators in this field. The general principles of specimen x-ray emission, x-ray nomenclature and specimen preparation techniques for x-ray microanalysis are explained with specific examples. A simplified approach to estimating the x-ray excitation volume in a biological bulk or sectioned matrix is given. Some of the common detector and microscope artifacts are described and illustrated. Included in this paper is a short discussion of the effective use of controls and standards and references for some of the current methods for making ones own standards.

High resolution SEM of bacterial virus T7.

High resolution "low-loss" scanning electron microscopy is a relatively new technique which permits an investigator to examine structures that were formerly visualized exclusively by transmission electron microscopy [1]. This paper presents some images of intact bacterial virus T7, viewed at the ultrastructural level. Due to the high resolution capibility of this technique, and the demanding physical prerequisites for visualization of the specimen, current specimen preparation techniques were modified in order to permit 1--2 nm resolution in surface mode. Using this method of microscopy, it is possible to view clearly this small bacteriophage (the smallest of the T-coliphages), adsorbed to its host bacterium, in a scanning mode at magnification (and resolution) comparable to TEM without resorting to the use of replicas, or reconstruction of a two-dimensional image.