title: Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis.
abstract: Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells. Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis. This layer is less apparent with traditional staining methods used for electron microscopy. Renografin gradients were used to purify B. subtilis spores. These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine. For B. anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification. In addition to demonstrating a previously undiscovered layer surrounding the spores of B. subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms. Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species.
authors.affiliation: Department of Pathology and Microbiology, University of South Carolina School of Medicine, 6439 Garners Ferry Rd., Columbia SC, 29208, USA.
authors.forename: Lashanda N
authors.initials: LN
authors.lastname: Waller
authors.forename: Nyssa
authors.initials: N
authors.lastname: Fox
authors.forename: Karen F
authors.initials: KF
authors.lastname: Fox
authors.forename: Alvin
authors.initials: A
authors.lastname: Fox
authors.forename: Robert L
authors.initials: RL
authors.lastname: Price
mesh_terms: D001408:Bacillus anthracis / Q000378:metabolism / Q000648:ultrastructure*; D001412:Bacillus subtilis / Q000378:metabolism / Q000648:ultrastructure*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000648:ultrastructure*; D008854:Microscopy, Electron; D012430:Ruthenium Red / Q000737:chemistry*; D013171:Spores, Bacterial / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D012430:Ruthenium Red