Application of a rapid direct viable count method to deep-sea sediment bacteria.
ABSTRACT For the first time, a Live/Dead (L/D) Bacterial Viability Kit (BacLight ) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250-5600 m) and sediment depth (0-5 cm). Percentage of viable (and thus potentially active) cells ranged between 20-60% within the first and 10-40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3+/-5.4 nmol cm(-3) h(-1)) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations (p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.
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ABSTRACT: There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. The nomenclature regarding "viability" and "vitality" should be used carefully.The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literatureResults obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.As microbiological parameter the Plating Efficiency should be used for comparison.Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.BMC Oral Health 01/2014; 14(1):2. · 1.34 Impact Factor
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ABSTRACT: The deep-sea represents a substantial portion of the biosphere and has a major influence on carbon cycling and global biogeochemistry. Benthic deep-sea prokaryotes have crucial roles in this ecosystem, with their recycling of organic matter from the photic zone. Despite this, little is known about the large-scale distribution of prokaryotes in the surface deep-sea sediments. To assess the influence of environmental and trophic variables on the large-scale distribution of prokaryotes, we investigated the prokaryotic assemblage composition (Bacteria to Archaea and Euryarchaeota to Crenarchaeota ratio) and activity in the surface deep-sea sediments of the Mediterranean Sea and the adjacent North Atlantic Ocean. Prokaryotic abundance and biomass did not vary significantly across the Mediterranean Sea; however, there were depth-related trends in all areas. The abundance of prokaryotes was positively correlated with the sedimentary concentration of protein, an indicator of the quality and bioavailability of organic matter. Moving eastwards, the Bacteria contribution to the total prokaryotes decreased, which appears to be linked to the more oligotrophic conditions of the Eastern Mediterranean basins. Despite the increased importance of Archaea, the contributions of Crenarchaeota Marine Group I to the total pool was relatively constant across the investigated stations, with the exception of Matapan-Vavilov Deep, in which Euryarchaeota Marine Group II dominated. Overall, our data suggest that deeper areas of the Mediterranean Sea share more similar communities with each other than with shallower sites. Freshness and quality of sedimentary organic matter were identified through Generalized Additive Model analysis as the major factors for describing the variation in the prokaryotic community structure and activity in the surface deep-sea sediments. Longitude was also important in explaining the observed variability, which suggests that the overlying water masses might have a critical role in shaping the benthic communities.PLoS ONE 08/2013; 8(8):e72996. · 3.53 Impact Factor
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ABSTRACT: Knowledge on spatial scales of the distribution of deep-sea life is still sparse, but highly relevant to the understanding of dispersal, habitat ranges and ecological processes. We examined regional spatial distribution patterns of the benthic bacterial community and covarying environmental parameters such as water depth, biomass and energy availability at the Arctic Long-Term Ecological Research (LTER) site HAUSGARTEN (Eastern Fram Strait). Samples from 13 stations were retrieved from a bathymetric (1,284-3,535 m water depth, 54 km in length) and a latitudinal transect (∼ 2,500 m water depth; 123 km in length). 454 massively parallel tag sequencing (MPTS) and automated ribosomal intergenic spacer analysis (ARISA) were combined to describe both abundant and rare types shaping the bacterial community. This spatial sampling scheme allowed detection of up to 99% of the estimated richness on phylum and class levels. At the resolution of operational taxonomic units (97% sequence identity; OTU3%) only 36% of the Chao1 estimated richness was recovered, indicating a high diversity, mostly due to rare types (62% of all OTU3%). Accordingly, a high turnover of the bacterial community was also observed between any two sampling stations (average replacement of 79% of OTU3%), yet no direct correlation with spatial distance was observed within the region. Bacterial community composition and structure differed significantly with increasing water depth along the bathymetric transect. The relative sequence abundance of Verrucomicrobia and Planctomycetes decreased significantly with water depth, and that of Deferribacteres increased. Energy availability, estimated from phytodetrital pigment concentrations in the sediments, partly explained the variation in community structure. Overall, this study indicates a high proportion of unique bacterial types on relatively small spatial scales (tens of kilometers), and supports the sampling design of the LTER site HAUSGARTEN to study bacterial community shifts in this rapidly changing area of the world's oceans.PLoS ONE 01/2013; 8(9):e72779. · 3.53 Impact Factor