A novel semi-automated technique for measuring inhibition of intracellular mycobacterial growth by macrophage activating factor.

ABSTRACT

We have developed a rapid, in vitro method for measuring T lymphocyte-derived macrophage activating factor (MAF) which inhibits the proliferation of Mycobacterium microti within macrophages. This MAF may be important in the control of mycobacterial disease in vivo. Because MAFs are a heterogeneous group of factors with different activities there is a need for assays which are relevant to specific macrophage effector functions. The existing assays for MAFs which are relevant to killing or inhibition of replication of mycobacteria within macrophages are currently too cumbersome or time consuming for the large scale screening required for their detection and purification. In our assay, monolayers of mouse macrophages were infected with the murine pathogen M. microti and were cultured for 6 days with MAF or control medium. The intracellular bacteria were stained with auramine-O and were quantified using epifluorescence microscopy with television image analysis. Total assay time was one-seventh that of viable count methods, and image analysis estimates of bacterial loads are quicker and more objective than visual counts.