Low Initial Cell Density Research Articles

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.

Acid production, acid tolerance and growth rate of Lotus rhizobia in laboratory media

Twenty-seven strains of Lotus rhizobia were tested for acid tolerance in yeast-extract mannitol broth (pH 4.6) by multiplication from low initial cell densities. Acid production on unbuffered yeast-extract mannitol agar slopes incorporating bromothymol blue indicator was also determined. All slow-growing, alkali-producing strains were acid sensitive and four of the six fast-growing, acid-producing strains were acid tolerant as was one fast-growing, alkali-producing strain. The method of testing for acid tolerance proved suitable for fast-growing strains and results are discussed in relation to the ecological implications of acid and alkali production by rhizobia.

Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane.

The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.

Morphological transformation of [formula omitted] cells subcultured at low cell densities

Morphological transformation in C3H/10T 1 2 cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm 2. These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm 2) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm 2) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the C3H/10T 1 2 transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.

EXPERIMENTAL MANIPULATION OF ALTERNATIVE PATHWAYS OF DIFFERENTIATION IN CULTURES OF EMBRYONIC CHICK NEURAL RETINA.

Embryonic chick neural retina cells can transdifferentiate during long-term cell culture into either pigmented epithelium or lens fibres. We have found that some culture conditions influence the choice between these pathways. Pigment cell development is promoted by low initial cell densities and by the use of a medium based on Earle's salt formulation rather than Hank's, while lens fibre development is encouraged by high initial cell densities and by folding the cell sheet into multilayered regions. Some differences in in vitro cell properties of neural retina are reported for two genotypes previously found to exhibit differences in in vitro cell properties of lens epithelial cells.

CYTOKININS AND THE GROWTH OF CULTURED SYCAMORE CELLS

SummaryExternally supplied cytokinins can induce cell division in sycamore cultures at cell densities at which, in the absence of a supply of cytokinin, they fail to grow. When cultures are initiated at cell densities where growth occurs in the absence of added cytokinins only after a long lag phase, the addition of cytokinins reduces the length of the lag phase. In these effects zeatin is fully effective at much lower concentrations than kinetin.As the duration of stationary phase is increased in the stock cultures, the ability of the cells to grow from a low initial cell density declines. This appears to be due to their declining endogenous cytokinin activity and declining ability to take up added cytokinin from their culture medium. It is postulated that, during the lag phase, the cells develop a critical level of cytokinin activity by an excess of endogenous synthesis over loss to the culture medium and internal degradation. When the phase of cell division has been initiated, the subsequent growth of the cultures is unaffected by low external levels of cytokinins.