MEASURING BACTERIAL ECTOENZYME ACTIVITIES IN MARINE WATERS USING MERCURIC-CHLORIDE AS A PRESERVATIVE AND A CONTROL

Investigations of bacterial ectoenzymes in seawater using fluorogenic substrate analogues such as 4-methylumbelliferone (4MUF) and beta-naphthylamine (BNAPH) are made more efficient and feasible in a greater range of environments if samples can be preserved and analyzed at a later date. Preservation by freezing has been shown to be feasible. However, activity will persist in the liquid phase until the samples are completely frozen and will commence again upon thawing. Addition of mercuric chloride (HgCl2) is an excellent means of terminating enzymatic activity, as well as providing a negative control for autofluorescence and autohydrolysis without the need to boil or autoclave seawater. HgCl2 causes relatively Little inhibition of fluorescence 4MUF (similar to 5%) and BNAPH (similar to 30%) in seawater. Furthermore, the effects are Linear over a wvide range of concentrations and consistent across the excitation and emission spectra. Inhibition of 4MUF fluorescence by Hg2+ in solutions of low ionic strength is reversible by addition of NaCl or other salts, so this method of preservation may be useful in fresh waters as well. 4MUF fluorescence in Hg2+-poisoned seawater is stable for long periods (>1 yr) when stored in the dark at -20 degrees C; BNAPH, however, loses fluorescence over time.