Immunoelectron Microscopy of <italic>Proteus vulgaris</italic> by the Plasma Polymerization Metal-Extraction Replica Method: Differential Staining of Flagellar (H) and Somatic (O) Antigens by Colloidal Golds

Abstract

Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.