Fluorescence histochemical double staining for the localization of intracellular hydrolases and tubercle bacilli

Fluorescence histochemical techniques are described to localize intracellular enzyme and tubercle bacilli in alveolar macrophages obtained from infected guinea pigs. The study demonstrated a double-staining technique using 4-methylumbelliferyl substrates for enzyme localization followed by auramine-O staining for intracellular tubercle bacilli. Upon enzyme substrate reaction, released 4-MU remains retained inside the cell and gives a green fluorescence, whereas tubercle bacilli give a yellow fluorescence of auramine staining. The technique is useful where a correlation of enzyme activity vs a presence of bacilli in vivo is required at the single cell level.