A versatile and sensitive tritium-based radioassay for measuring hydrogenase activity in aquatic sediments

Abstract We present a method for the measurement of hydrogenase (H 2 ase) activity in aquatic sediments. The assay is based on the H 2 ase-mediated isotopic exchange between dissolved molecular hydrogen (H 2 ) and water. A slurry of sediment material is incubated with a tritiated hydrogen (HT) headspace in a glass syringe on a rotary shaker. The method includes a procedure for preparing HT from radiolabeled sodium borohydride, which is a useful alternative to purchasing HT directly. A method for measuring HT specific activity based on liquid scintillation counting is also presented. Validation tests were run using live and frozen cultures of Clostridium pasteurianum and Desulfovibrio vulgaris, and freshly collected marine sediments. Adherence to Michaelis-Menten kinetics was demonstrated. An interassay coefficient of variation of 15% was determined using frozen C. pasteurianum cultures as reference material. Serial dilutions of cultures and sediments showed that measured H 2 ase activity scales with cell concentration, and indicate that the method can detect C. pasteurianum cell concentrations of between 300 and 3000 cells/ ml. This technique allows measurement of H 2 ase activity in a variety of environmental samples, and will be particularly useful in the study of deep marine sediments with low microbial activity

Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates

Vibrio harveyi, which now includes Vibrio carchariae as a junior synonym, is a serious pathogen of marine fish and invertebrates, particularly penaeid shrimp. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions. With shrimp, the pathogen is associated with luminous vibriosis and Bolitas negricans. Yet, the pathogenicity mechanisms are imprecisely understood, with likely mechanisms involving the ability to attach and form biofilms, quorum sensing, various extracellular products including proteases and haemolysins, lipopolysaccharide, and interaction with bacteriophage and bacteriocin-like substances