DETECTION OF POLY(3-HYDROXYBUTYRATE) GRANULES BY ELECTRON-MICROSCOPY OF VIBRIO-HARVEYI STAINED WITH MALACHITE GREEN

Electron microscopy of potential morphological changes in Vibrio harve yi cells during development of luminescence revealed lipidlike poly(3- hydroxybutyrate) (PHB) granules at high cell density using the standar d glutaraldehyde fixation procedure. On staining with malachite green, much more definitive sponge-like granules with defined membranes were observed. Those spongelike granules were not detected in V. harveyi d ark mutants deficient in the production of the lux autoinducer, N-(3-h ydroxybutyroyl)homo serine lactone, which controls expression of lumin escence. Addition of autoinducer to the mutants during growth restored not only luminescence but also the synthesis of the spongelike granul es. In this study, the spongelike granules, produced on staining V. ha rveyi cells with malachite green, were measured by stereological analy ses. A linear relationship was found between the amount of material in the spongelike granules and the amount of PHB over a wide range of le vels modulated by the lux autoinducer and (or) growth conditions, prov iding strong evidence that staining with malachite green is a powerful technique for identification of PHB granules.