Fluorescent Acid-Fast Microscopy for Measuring Phagocytosis of Mycobacterium avium , Mycobacterium intracellulare , and Mycobacterium scrofulaceum by Tetrahymena pyriformis and Their Intracellular Growth

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Abstract
Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium , Mycobacterium intracellulare , and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis . There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium , M. intracellulare , and M. scrofulaceum . Phagocytosis was rapid and reached a maximum in 30 min. M. avium , M. intracellulare , and M. scrofulaceum grew within T. pyriformis , increasing by factors of 4- to 40-fold after 5 days at 30°C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis . Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium , M. intracellulare , and M. scrofulaceum .

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