Effect of fixation‐resin combinations and ruthenium red on elucidating outer envelope structure and surface morphology of two methanotrophic bacteria

Abstract

We examined the ultrastructure of the cell envelope in Type I, Methylomonas albus (BG8), and Type II, Methylosinus trichosporium (OB3b), methane-oxidizing bacteria by using different fixatives, ruthenium red (RR) combinations and resins. We compared LR White and Spurr embedments with the following fixations: glutaraldehyde/OsO4, two glutaraldehyde-paraformaldehyde, and two different en bloc ruthenium red procedures, one utilizing OsO4 and the other with glutaraldehyde/OsO4 in sequential fixation. These fixations were also studied by scanning electron microscopy (SEM). Unfixed cells prepared by freeze etch were used for comparison. Transmission electron microscopy of BG8 embedded in LR White resin (with or without red0 preserved a layer of cup-like structures that were not seen in Spurr resin-embedded cells unless ruthenium red was used. For OB3b, the second RR method preserved beads and filaments where only "spike-like" structures were seen in all other fixations in both resins. By SEM, all fixations preserved a capsular slime layer of BG8 that was removed from some cells by both RR methods. In all SEM fixations, a bead layer was preserved in OB3b that was enhanced by RR. Filaments seen by freeze-etch and thin-section techniques were not seen in SEM. Presence or absence of particular envelope structures in these methanotrophs is dependent on the combination of fixatives and/or resins employed and is species-specific. The chemical preparation methods used resulted in enhanced understanding of the structure and composition of the cell envelope.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture

“…The results presented in our study indicate that the visualization of the fine structure of the glycocalyx may be in a fundamental way influenced by the method of fixation and processing of the cell pellet for transmission electron microscopy. Similar problems arose in studies of bacteria, where a comprehensive elucidation of the surface morphology was based on the variable combinations of fixatives, ruthenium red employment, and resin formula (Fassel et al 1990). Our results are complicated by the fact that the material processed by the different methods originated from two distant phases of subculturing (passages Nos 6 vs. 21 and 22).…”

“…Similar problems arose in studies of bacteria, where a comprehensive elucidation of the surface morphology was based on the variable combinations of fixatives, ruthenium red employment, and resin formula (Fassel et al. ). Our results are complicated by the fact that the material processed by the different methods originated from two distant phases of subculturing (passages Nos 6 vs. 21 and 22).…”

Revised Taxonomy of the Methanotrophs: Description of Methylobacter gen. nov., Emendation of Methylococcus, Validation of Methylosinus and Methylocystis Species, and a Proposal that the Family Methylococcaceae Includes Only the Group I Methanotrophs

“…Thin sections of cells taken from 3-day-old liquid or plate cultures were used to determine intracytoplasmic membrane types. The ruthenium red staining procedures of Fassel et al (19) and Pierson et al (40) were used to process the samples.…”

Comparison of Alcian blue and ruthenium red effects on preservation of outer envelope ultrastructure in methanotrophic bacteria

“…Variation in preservation of these outer structures was seen with different specimen preparations involving fixation, embedding resin, and en bloc protocols with ruthenium red. Ruthenium red, applied in a protocol based on the work of Patterson et al (1975) and Mackie et al (1979), overcame a loss of an outer layer in the hydrophobic resin Spurr and enhanced ultrastructural preservation of outer envelope layers in both LR White and Spurr resins (Fassel et al, 1990).…”

“…In this study, we have compared RR with AB utilizing the same en bloc protocol that was successful in prior work (Fassel et al, 1990). Alcian blue and RR are applied en bloc to determine their effect separately and in comparison on the preservation of the outer structural layers of methanotrophic bacteria.…”