A new method for studying the binding and ingestion of zymosan particles by macrophages.

One of the major problems encountered during quantitative studies of phagocytosis is the discrimination without ambiguity between intracellular and extracellular particles. This difficulty is especially acute when zymosan particles are used because of their poor affinity for dyes. We show in this paper that zymosan particles may be stained by a mixture of a basic dye and tannic acid in water (or in an isotonic non saline solution). Crystal violet is one of the most suitable dyes and by combining this staining step with May-Grünwald Giemsa staining, it was possible to observe two populations of macrophage-associated particles. One comprises blue violet particles (BVP), and the second consists of particles with a purple-stained core (PPC). Treating macrophages in culture with various concentrations of cytochalasin B decreases the number of PPC and increases the number of BVP, in a dose dependant manner. Moreover, treatment with alpha-mannan or laminarin decreases the number of cell-associated particles, especially that of PPC. From these observations we concludes that BVP are extracellularly located and PPC are ingested.