Ruthenium red staining revealed both the long and short classes of cell surface fibril in thin sections of HB, indicating that the fibrils contained polyanionic polymers, probably polysaccharides. Also visible was a 16.2 ± 2.2 nm thick ruthenium red staining layer (RRL) outside the 16.7 ± 2.2 nm thick cell wall. The fibrils could not be seen after conventional glutaraldehyde and osmium fixation. The RRL was protease resistant and was not involved in septum formation. Loss of the fibrils after protease treatment coincided with a decrease of 54% in cell surface hydrophobicity, indicating that cell surface hydrophobicity was due partly to fibrils and partly to the RRL. There was no correlation between the lengths of fibrils as measured on whole cells after negative staining and on thin sections of ruthenium red stained cells. The thickness of the RRL was the same in three adhesion deficient mutants -strains HB-7, HB-V5 and HB-V51 - with various fibril lengths. However, a completely bald mutant, HB-B, had a significantly thicker RRL than HB, although it was unable to adhere to buccal epithelial cells, and it could not co-aggregate with V1. The RRL therefore did not contain adhesins.


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