When normal chicken bone marrow is treated in vitro with the myeloblastosis virus, a cell population results (24), which is morphologically indistinguishable from that of cultures of circulating myeloblasts from the infected bird. Because of the very small number of cells available, it has not been possible effectively to sample the infected bone marrow cultures only a few days after infection. After growth sufficient to permit sampling, the viroplasts are chiefly of the form seen in older cultures. Occasionally, however, an early form is seen with the appearance of the cell in figure 2.

Adenosinetriphosphatase Activity of the Viroplast and Its Relation to Normal Bone Marrow Cells

A considerable advance in the technical aspects of the study of the myeloblast was realized when it was found, as already reported (14), that some of the structures designated as viroplasts in thin sections could be identified in the phase-contrast microscope and that they were thus accessible for study by cytochemical techniques. The results of investigations by these methods led to recognition of the property of certain of the viroplasts to yield a positive test for adenosinetriphosphatase activity with the Wachstein-Meisel reagent (30). This was of critical importance since the virus of myeloblastosis is rich in this enzyme (37). The presence of an enzyme in the viroplast similar to that of the virus was strongly suggestive of a relationship involved in the processes of synthesis of the virus. The possible significance of the observation was greatly heightened when it was found that certain structures, the granules, appearing in the normal cell in association with maturation of the myeloblast through the myelocyte to the adult granular white blood cell stage, likewise exhibit adenosinetriphosphatase activity. Observations by other investigators (82, 88, 89) have revealed a possible role of micro-bodies (gray bodies in the terminology used here) in the origin of mitochondria and, further, an indication (40, 41) that the granules of the myelocyte may be derived from mitochondria. Correlation of the results of these studies on the normal cell provides the basis for some clarification of the sites of myeloblastosis virus infection in the myeloblast, and the manner of cell response in the synthesis and disposal of the "mature" agent. Continued work with the problem has resulted in a marked refinement in the techniques for such light-microscope studies and in the unequivocal corroboration of the earlier findings, which are exemplified in the following illustrations.

The significant results of the study of bone marrow from a normal bird are illustrated in figures 8 and 9. In the positive phase-contrast picture of the living cells of figure 8, there are seen 3 myeloblasts (MB), portions of 2 myelocytes (MCL), and a heterophil (HP). The granules are revealed as dense bodies (GR) in the myelocytes and the heterophil, and none is present in the myeloblasts. In these myeloblasts, however, there are many vacuoles, the white areas, which can be identified either by supravital or fixed-preparation staining. The mitochondria show only as indefinite dark smudges. The same cells, indexed on the slide, quenched in liquid nitrogen, dehydrated, fixed with formalin vapor, and stained for adenosinetriphosphatase activity by the Wachstein-Meisel method, are shown in figure 9. It is evident that a large proportion, if not all, of the granules of the heterophil are strongly adenosinetriphosphatase-positive by this method of treatment. Similarly, many, but by no means all of the granules of the myelocytes are heavily stained. The spatial correlation between the stained granules of figure 9 and the granules seen by phase contrast in figure 8 is not perfect due to movement of the structures in the living cell during the period of observation prior to the quenching treatment in liquid nitrogen. Nevertheless, it is evident that the structures are identical. As noted before, no granules were discernible in the myeloblasts, and there is no evident positive staining for the enzyme in figure 9.

It is of much importance to emphasize the finding, which was regularly encountered, of the variation from strong to no staining of the granules of the normal myelocytes. From many studies, it seems clear that the granules seen by phase contrast by virtue of their optical properties are in variable stages of maturation. In this process, adenosinetriphosphatase is absent in the early stages of granule formation, but increases progressively to reach high concentrations in the mature granules, as can be seen by comparing the picture of the heterophil (HP) of figure 8 with HP of figure 9. Some stress should be given to the negative staining reaction of the normal myeloblasts after the process of fixation employed here. As will be discussed in a later report, improper handling of the cells during the fixation procedure results in damage to and consequent positive staining of mitochondria for adenosinetriphosphatase activity.

The findings with myeloblasts examined in this way immediately after removal from the diseased bird and those from cultures, in which the cells were not increasing, were essentially identical with those observed with the myeloblast from the normal bird. Thus, there was no evidence either of granule formation or of the presence of viroplasts identifiable either by electron- and phase-contrast microscopy or by the adenosine-triphosphatase reaction.

In striking contrast are the ultrastructural and chemical characteristics of the cells from growing cultures illustrated in figures 10 and 11. The negative phase-contrast picture of figure 10 shows 4 myeloblasts from a 61-day culture. Vacuoles (V) are seen as black circular areas. Mitochondria are numerous and scattered around the nucleus as irregular light images. A structure unique to such cells is the large bright body (PL), previously identified (14) as the viroplast, seen in variable numbers and sizes in thin sections in the individual myeloblasts. As seen in the same 4 cells, after fixation and treatment with Wachstein-Meisel reagent, figure 11, it is the property of these bodies, like many of the granules of the normal myelocyte and those of the heterophil, to react strongly with the adenosinetriphosphatase stain. Most of the black structures, PL of figure 11, can be identified in figure 10, particularly those in the upper regions of the upper 2 cells. Again, there has been some movement of the bodies during the period of observation. Clearly the mitochondria do not stain, and the vacuoles, which have also shifted position, are open areas.

A characteristic of the viroplasts not clearly shown in this series is, as in the granules of the myelocytes, the variation in degree of staining reaction of the structures identified by phase contrast. Thus, in this instance, not all the dense bodies of figure 11, especially those of the cell at the lower right, are clearly discerned in the phase-contrast picture of figure 10, due in part to movement of the structures and to change in cell contour while the myeloblast was in the living state. Nevertheless, it has been definitely established that, although many viroplasts possess the physical properties suitable for visualization in phase contrast, their chemical attributes differ broadly with respect to the content of the enzyme. This corresponds well to the findings in thin sections, which show a wide variation in the content of the viroplast of material staining with osmium tetroxide.