Detection of poly(3-hydroxybutyrate) granules by electron microscopy of<i>Vibrio harveyi</i>stained with malachite green

Abstract

Electron microscopy of potential morphological changes in Vibrio harveyi cells during development of luminescence revealed lipidlike poly(3-hydroxybutyrate) (PHB) granules at high cell density using the standard 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 dark mutants deficient in the production of the lux autoinducer, N-(3-hydroxybutyroyl)homoserine lactone, which controls expression of luminescence. Addition of autoinducer to the mutants during growth restored not only luminescence but also the synthesis of the spongelike granules. In this study, the spongelike granules, produced on staining V. harveyi cells with malachite green, were measured by stereological analyses. A linear relationship was found between the amount of material in the spongelike granules and the amount of PHB over a wide range of levels modulated by the lux autoinducer and (or) growth conditions, providing strong evidence that staining with malachite green is a powerful technique for identification of PHB granules.Key words: PHB granules, malachite green, bioluminescence.