Figures & data
Figure 1. Light microscopy of control BCG-D colony (A), and typical “fried eggs” L-form colonies grown on Middlebrook 7H9 semisolid agar: colonies of filterable forms developed from control BCG-BG suspension (B) and (C) and .colonies obtained after starvation of BCG-BG (D) and (E) and BCG-D cultures (F). Magnification: (C)–200x; (A), (B), (D), (E) and (F)-400x; (F)-800x.
Figure 2. SEM of cells from culture of control strains: BCG-BG- (A), (B) and (E); and BCG-D- (C), (D) and (F). Typical single rod shaped bacilli (A) and (C), bacterial clusters (B) and (D), and filterable forms which size is similar or smaller than pore size of bacterial filter (E) and (F), arrows. Bar (white lines) = 10µm (A), (B) and (D); 1 µm (C), (E) and (F)
Figure 3. SEM of BCG subpopulation developed after filtration of control suspension: Clusters of granular forms and L-elementary bodies (A) and (B) or disperse single bodies of varied size (C) and (D), small rod shaped bacilli and filterable forms (E) and (F), arrows. Bar = 1µm (a, c, e); 10µm (b, d, f), Bar = 1µm
Figure 4. SEM of BCG cultures after starvation stress: BCG-BG – (A), (E), (F) and BCG-D- (B), (C), (D). Typical L-form cells: spherical large bodies (A) and (B) giant “mother” cells (C) and (E), elongated forms (A) and (D), small elementary bodies and granules (C), (E) and (F). Bar = 100 µm (A); 10 µm (b, c, e, f); 1 µm (D)
Figure 5. TEM of BCG-BG cell ultrathin sections (longitudinal and transversal) from: Control culture of dividing cells with cell walls and cytoplasm substructures of ribosomes and nucleoid (A) and (B); Stress starved culture- cells with separated cell walls (C), wall less bacterial cells (D), elongated L-forms with elementary bodies located inside (E) and single spherical L-bodies (F). Bar = 0.2µm
Figure 6.pncA allele-specific PCR. Amplifications were performed with chromosomal DNA from M. bovis (3); M. tuberculosis H37Rv (4); M. bovis BCG-BG, L-form (5); M. bovis BCG-D, L-form (6); M. bovis BCG-BG, filtered culture (7) and M. bovis BCG-D, filtered culture (8). PCR controls with water were also included, lines 2 and 9. All samples in gel A were tested with primers specific for M. tuberculosis, whereas all samples in gel B - with primers specific for M. bovis. DNA ladder (100–1000bp) was used in line 1 as size marker in both gels.
