Basal bodies of bacterial flagella in Proteus mirabilis. II. Electron microscopy of negatively stained material.

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored-often by means of a hook-in rounded structures approximately 50 mmicro wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.

Basal bodies of bacterial flagella in Proteus mirabilis. I. Electron microscopy of sectioned material.

Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been autolyzed in the cold, the observation was made that the flagella arose from basal bodies. However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated with actively developing cultures of Proteus mirabilis, the cell walls of which had been expanded slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy, and negative staining of whole mount preparations. This paper deals with the thin sections of bacteria after the usual fixation technique had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to be anchored in the cell by means of a hook-shaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 mmicro) and the lack of properties that would enable one to distinguish them from the ribonucleoprotein structures; in addition, their boundary appears to be delicate. However, when the cytoplasm is sparse in the cells, either naturally or as a result of osmotic shocking in distilled water, the flagella can be observed to emerge from rounded structures approximately 25 to 45 mmicro wide. Contrary to a previous suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite, i.e. the chondrioids. The observations in this part of the study agree with those described in the following paper (15) dealing with negatively stained preparations.