Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform(TM) was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform(TM) were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Magnetosomal matrix: ultrafine structure may template biomineralization of magnetosomes

The organic matrix surrounding bullet-shaped, cubo-octahedral, D-shaped, irregular arrowhead-shaped, and truncated hexa-octahedral magnetosomes was analysed in a variety of uncultured magnetotactic bacteria. The matrix was examined using low- (80 kV) and intermediate- (400 kV) voltage TEM. It encapsulated magnetosomes in dehydrated cells, ultraviolet-B-irradiated dehydrated cells and stained resin-embedded fixed cells, so the apparent structure of the matrix does not appear to be an artefact of specimen preparation. High-resolution images revealed lattice fringes in the matrix surrounding magnetite and greigite magnetosomes that were aligned with lattice fringes in the encapsulated magnetosomes. In all except one case, the lattice fringes had widths equal to or twice the width of the corresponding lattice fringes in the magnetosomes. The lattice fringes in the matrix were aligned with the {311}, {220}, {331}, {111} and {391} related lattice planes of magnetite and the {222} lattice plane of greigite. An unidentified material, possibly an iron hydroxide, was detected in two immature magnetosomes containing magnetite. The unidentified phase had a structure similar to that of the matrix as it contained {311}, {220} and {111} lattice fringes, which indicates that the matrix acts as a template for the spatially controlled biomineralization of the unidentified phase, which itself transforms into magnetite. The unidentified phase was thus called pre-magnetite. The presence of the magnetosomal matrix explains all of the five properties of the biosignature of the magnetosomal chain proposed previously by Friedmann et al. and supports their claim that some of the magnetite particles in the carbonate globules in the Martian meteorite ALH84001 are biogenic. Two new morphologies of magnetite magnetosomes are also reported here (i.e. tooth-shaped and hexa-octahedral magnetosomes). Tooth-shaped magnetite magnetosomes elongated in the [110] direction are reported, and are distinct from arrowhead-shaped and bullet-shaped magnetosomes. Elongation of magnetite magnetosomes in the [110] direction has not been reported previously. A Martian hexa-octahedral magnetite particle was previously characterized by Thomas-Keptra et al. and compared with truncated hexa-octahedral magnetite magnetosomes. Hexa-octahedral magnetite magnetosomes with the same morphology and similar sizes and axial ratios as those reported by Thomas-Keptra et al. are characterized here. These observations support their claim that ALH84001 contains evidence for a past Martian biota.