Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane

Abstract

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.

Proteomic Analysis ofCampylobacter jejuni11168 Biofilms Reveals a Role for the Motility Complex in Biofilm Formation

“…Samples for scanning electron microscopy (SEM) were prepared as previously described (3,13). Mounted specimens were sputtercoated with 30 nm of platinum and viewed with a Hitachi S 3000 N scanning electron microscope at an accelerating voltage of 5.0 kV.…”

The Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

“…S. oneidensis wild type and mutant strains were grown similarly for scanning electron microscopy imaging. Cells were lifted off agar plates onto glass cover slips then fixed and treated as described previously [33]. Cover slips were coated with 2 nm iridium in an Emitech K575X sputter coater (Emitech, Ashford, England), and cells were viewed with a Hitachi S-4800 Field emission-scanning electron microscope (SEM; Hitachi, Pleasanton, CA) operating at 3 kV and a 4 mm working distance.…”

Evaluation of the Microanatomy of the Liver via a Rapid Sample Preparation Protocol and a Table-Top Scanning Electron Microscope

“…In line, drying of fenestrated liver endothelial cells via hexamethyldisilazane was found to be successful in the preservation of these fragile membrane-bound pores under in vitro conditions [7]. And drying by evaporation of hexamethyldisilazane has been described as a good alternative for a variety of biological samples [5,6]. It is well known that air-drying of biological samples in water is disastrous, because of the destructive action of the surface tension during drying.…”

“…Subsequent steps involve the immediate dehydration and air drying of the sample by evaporation of hexamethyldisilazane [5,6], which was immediately followed by sample mounting and sputter coating of the tissue blocks with a thin layer of gold [7]. This sample preparation method was combined with the newgeneration of table-top scanning electron microscopes.…”