Method for enumeration of 5-cyano-2,3-ditoyl tetrazolium chloride (CTC)-active cells and cell-specific CTC activity of benthic bacteria in riverine, estuarine and coastal sediments

Bacteria are the most abundant and active organisms in marine sediments and are critical for nutrient cycling and as a food source to many benthic and pelagic organisms. Bacteria are found both as free-living cells and as particle-associated cells, which can make investigations of these communities difficult. We found that common procedures for extracting bacteria from sediments leave the bacteria clay particle-associated and the clay particles clump, which reduce the reproducibility of direct counts. We optimized a sonication/surfactant method that produces a homogeneous suspension of bacterial cells against a uniform background of clay particles, which results in reproducible samples for epifluorescence microscopy. We developed a method to estimate CTC-positive cells and cell-specific CTC content in intact cores of surficial sediment communities from riverine, estuarine and coastal sites. Benthic bacterial abundances averaged 4.9x10(8) cells/g dry wt sediments in Apalachicola River, Florida sediments, 4.9-13.8x10(9) cells/g dry wt sediments in a variety of Apalachicola Bay sediments and 3.6x10(8) cells/g dry weight in shallow, anoxic Gulf of Mexico sediments. Percent CTC-positive cells ranged from low values of 9-10% CTC-positive cells in Apalachicola River and Apalachicola Bay sediments to high values of 25% CTC-positive cells in anoxic Gulf of Mexico sediments. After correction for abiotic CTC reduction and chlorophyll interference, estimates of cell-specific CTC reduction ranged from 0.15 to 0.55 fmol CTC(red)/active cell in the Apalachicola Bay sediments to 1.6 to 3.8 fmol CTC(red)/active cell in anoxic Gulf of Mexico sediments.