This 1967 electron microscopy study examined the ultrastructure of Bacillus megaterium cell walls using shadowed preparations, negatively stained samples, ultrathin sections, and chemical treatments. The cell wall consists of two distinct layers: an inner rigid mucopeptide layer (90-110 nm thick) and an outer plastic teichoic acid layer. Hot formamide extraction removed approximately 50% of cell wall thickness, leaving behind the rigid mucopeptide "membrane." Total cell wall thickness ranged from 200-240 nm. Different staining methods produced variable results: uranyl acetate showed homogeneous walls, while osmium tetroxide, potassium permanganate, and lead citrate produced "triple-layered" appearances. The author argues this variation reflects staining artifacts rather than true structural organization. High-magnification sections revealed fine "channels" and electron-dense granules but no consistent ultrastructural pattern. The teichoic acid was located on the outer surface of the rigid mucopeptide layer and likely oriented perpendicular to the cell surface as short chains. The study demonstrates both the potential and limitations of electron microscopy staining for revealing cell wall architecture.

Key findings

• The B. megaterium cell wall comprises two separable layers: rigid mucopeptide (90-110 nm) and plastic teichoic acid (outer layer), totaling 200-240 nm thickness
• Different electron microscope stains produce artifacts; uranyl acetate provides the most reliable homogeneous representation, while permanganate and osmium create spurious "triple-layered" appearances
• Teichoic acid is positioned on the outer surface of the mucopeptide layer with probable perpendicular orientation as short polymer chains
• No conspicuous regular ultrastructure was visible despite high-magnification examination, suggesting the plastic layer's flexibility obscures organized patterning