Extraordinary growth phases of Nanobacteria isolated from mammalian blood

Nanobacteria, novel sterile-filterable coccoid bacteria inhabiting mammalian blood and blood products, have different growth phases depending on the culture conditions. These minute organisms produce biogenic apatite as a part of their envelope. This becomes thicker as the cultures age, rendering them visible in microscopy and resistant to harsh conditions. Mineral deposits were not formed without live nanobacteria. Apatite formation was faster and more voluminous in serum-free (SF) medium, and within a week, several micrometer thick 'castles' formed around each nanobacteria. These formations were firmly attached to the culture plates. Nanobacteria multiplied inside these thick layers by turning into D-shaped forms 2-3 mu m in size. After a longer culture period, tens of them could be observed inside a common stony shelter. The apatite shelters had a hollow interior compartment occupied by the organisms as evidenced by SEM and TEM. Supplementing the culture medium with a milk growth-factor product, caused the castles to grow bigger by budding. These formations finally lost their mineral layer, and released typical small coccoid nanobacteria. When SF cultures were supplemented with sterile serum, mobile D-shaped nanobacteria together with small 'elementary particles' 50-100 nm in size were found. Negative results in standard sterility testing, positivity in immunofluorescence staining and ELISA tests with nanobacteria-specific monoclonal antibodies, and 98% identity of 16S rRNA gene sequences proved that all of these unique creatures are nanobacterial growth phases.