Observation of Apatitic Crystallite in Calcified E. coli K-12

Objectives: We chose the well-investigated bacteria, E. coli K-12 strain to study the initiation of biomineralization in bacteria. Methods: Calcified E. coli was obtained from the culture grown in calcification medium. After cultivation of bacteria for 2 weeks, they were collected by centrifugation, fixed with 2% glutaraldehyde in cacodylate-buffered solution, post-fixed with 1% osmium tetroxide in the same solution, and embedded in Araldite 502. Thin sections were prepared using a diamond knife. After staining, they were observed under a transmission electron microscope. Results: In the calcified E. coli precipitates, bacterial debris and electron dense material were observed. In higher magnifications, we observed lattice image in small crystallite, compatible with hydroxyapatite in the electron-dense area. Central dark line was observed in the central portion of some small crystallites. Furthermore, electron microscopic histochemistry of calcifying E. coli indicated that carbonic anhydrase (CA) activitiy located near the bacterial membrane and this activity was also observed near the calcification front of dental germ in murine mouth. Conclusions: These observations suggest that there is a relation existing between CA and the initiation of small crystallite formation.