A novel method for estimating killing ability and digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro

We describe a novel method by which phagocytosis, digestion and killing of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated simultaneously. Suspensions of P. brasiliensis (yeast-like phase) were sonicated, counted and incubated at 37 degrees C with known numbers of phagocytes. Control preparations contained no phagocytic cells. At given intervals samples were incorporated into gelatin nutrient medium and droplets of the mixtures were incubated at room temperature. Live yeast-like P. brasiliensis germinate in vitro and produce filaments. After incubation, droplets may be melted and examined under phase contrast optics, or the cells may be washed and stained by a variation of Papanicolaou's method. Digested P. brasiliensis, intact but non-germinating yeasts and filamented (viable) yeasts may be identified and counted. Killing and digestive abilities of phagocytes may be estimated by the difference between values obtained from phagocyte-containing and control preparations.