A rapid micro method for the simultaneous determination of phagocytic-microbiocidal activity of human peripheral blood leukocytes in vitro

A new, simple technique for simultaneously studying phagocytic and microbiocidal functions, using viable eukaryotic and prokaryotic microbes, is described. Fresh human venous blood from volunteers was placed on a coverglass and incubated to allow leukocyte adhesion to the coverglass. After clot removal, viable microbes in suspension were added and the coverglass preparation was incubated to allow phagocytosis. The excess microbes ( E. coli, S. aureus, L. monocytogenes, and C. albicans each have been used) were then rinsed off, and the vital fluorochrome, acridine orange (AO), was used for staining. A wet mount was prepared and examined by reflected fluorescence with an ultraviolet microscope. Intact (viable) polymorphonuclear (PMN) leukocyte nuclei and microbes appeared green (orthochromatic). Granules in the PMN cytoplasm were yellow or reddish. Nonviable PMN nuclei appeared yellowish or reddish and the nonviable microbes appeared bright red (metachromatic). Thus, phagocytized microbes may be counted and identified as viable or non-viable.