Lanthanoid staining as a fast technology of preparing microbiological specimens for scanning electron microscopy

Abstract

The aim of the investigation was to demonstrate advantages of lanthanoid staining as a novel super-fast technology of preparing microbiological specimens for scanning electron microscopy, and to ground the potentials of using this technology in practical microbiology. Materials and Methods. Microbial cells (bacteria Acinetobacter baumannii, Corynebacterium diphtheriae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and fungi Candida albicans), and Staphylococcus aureusbased bacterial biofilms were stained with the preparation based on neodymium chloride and visualized using scanning electron microscopy. Observations were carried out in the mode of low vacuum, images were captured in the mode of detecting back-scattered electrons using a short working section of 4.5–3.0 mm. Specimens were compared to the untreated microorganisms or microbial samples obtained by means of traditional specimen preparation with dehydration and sputtering a gold-platinum coating. Results. The lanthanoid staining technology enabled visualization and evaluation of diagnostically relevant morphological structures of the following microbial cells: A. baumannii, C. diphtheriae, K. pneumoniae, P. aeruginosa, S. aureus, S. pneumoniae, C. albicans, including their relative position, shape, dimensions, presence of capsules and flagella, intracellular structures (polyphosphate inclusions, septum) as well as an extracellular bacterial matrix. The obtained images were characterized by a high contrast, which may be explained by the fact that lanthanoids are capable of providing a high density of electron back-scattering under the impact of the microscope electron beam, resulting in a great brightness of the biological object loci saturated with these elements. Conclusion. Lanthanoid staining of microorganisms is a perspective method of specimen preparation for scanning electron microscopy, which opens the possibility of practical application of this method in diagnostic technologies of clinical microbiology.