Figure 4. Morphology of unstained serum pellet particles observed by TEM shows similarity to NB.

Serum pellets were prepared from untreated serum (A and E) or after addition of either CaCl₂ (B and F), Na₂HPO₄ (C and G), or both (D and H) to the indicated serum, followed by preparation for TEM. Samples were viewed without fixation or staining. The serum pellets displayed various morphologies including round particles (A, C, F, and G) while other samples harbored spindles with more crystalline appearance (B, D, E, and H). In general, the combination of calcium and phosphate tended to produce more readily spindles with needle-like crystalline projections (D and H). The various NB controls shown in the bottom two rows were displayed to show comparable morphologies with predominant round particle shapes (3^rd row) or more crystalline aggregates (4^th row). However, morphological variations can still be seen within each row. Thus, NB cultured directly from 10% HS (I) or 10% FBS (J) displayed predominantly chain-linked ovoid or spherical shapes resembling dividing bacteria. In contrast, the NB strains “nanons”(K) and DSM 5820 (L) shown here appear further along in their crystallization and while they have retained ovoid shapes, they also show more pronounced fusion and aggregation. In the case of “nanons” (K), there are noticeable thick walls that appear less electron-dense than the core, presumably formed of minerals. For DSM 5820 (L), crystalline bridges can be seen connecting the fused ovoid particles. (M–P) show further progression in aggregation and crystallization, with the appearance of coalesced films (more noticeable in N and P). Film-like aggregation is generally seen with longer periods of incubation or by reducing the amount of serum in the culture medium (to less than 3%). Scale bars: 50 nm (C, G, H, J); 100 nm (A, D, F, N, P); 200 nm (B, E, I, K–M, O).