A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Abstract

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

Mild Heat Stress Affects on the Cell Wall Structure in <i>Candida albicans</i> Biofilm

“…Ultrathin sections were cut to a thickness of 80 nm with a diamond knife (Diatome Co. Ltd., Switzerland) on an Ultracut S ultramicrotome (Leica Microsystems, Vienna) and picked up on 300-mesh grids. They were stained with uranyl acetate and lead citrate in a staining tube, covered with Super Support Film (Nisshin EM Co. Ltd., Tokyo), and observed in a JEM-1400 electron microscope (JEOL, Tokyo) at magnifications of 2,500 to 40,000 at 100 kV 24) .…”

Nuclear behavior and roles indicate that Codiolum phase is a sporophyte in Monostroma angicava (Ulotrichales, Ulvophyceae)

“…After treatment with 100% acetone for 10 min, the samples were gradually substituted with 30% and 60% epoxy resin (Quetol 651; Nissin EM, Tokyo, Japan) in acetone at room temperature for 1 h each, 90% epoxy resin in acetone at 60°C for 1 h and 100% epoxy resin at 60°C for 60 h. The samples were embedded in 100% epoxy resin with a polymerization accelerator (DMP‐30; Nissin EM) at 60°C for 48 h. The specimen blocks in which the samples were embedded were sectioned with a diamond knife using an Ultramicrotome (Ultracut S; Leica, Wetzlar, Germany). For efficient observation of nuclei in Codiolum phases, we cut 70 nm‐thick serial sections (Yamaguchi and Chibana ). The sections were mounted on slot meshes (Nissin EM) and a formvar support film.…”

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination

“…The serial ultrathin sections were then transferred onto a formvar support film mounted on an aluminum rack with 20 pores of 4-mm diameter. Then, the grids with the sections were then dried, detached from the rack with the support of the formvar, and stained with uranyl acetate and lead citrate (Yamaguchi et al, 2009 ; Yamaguchi and Chibana, 2018 ). Of the 10 grids prepared, serial 3 grids were subjected to TEM examination.…”