Since 1873, the waters at Helgoland Roads (sampling station "Kabeltonne") have been sampled daily to determine temperature and salinity. In 1962, microbiological parameters were determined for the first time to establish microbiological long-term studies on marine bacteria, starting with the colony-forming units (CFU). In the following years, several other microbiological parameters were integrated for different periods of time (e.g. activity parameters like ATP and ectoenzymatic activity, marine yeasts, oil-degrading bacteria, flagellates and molecular methods like PCR followed by denaturing gradient gel electrophoresis). To date, the total count of bacteria, flagellates and viruses have been acquired using fluorescent DNA dyes and epifluorescence microscopy. Here we present both a historical overview of the methods used and examples of results obtained over the past 40 years. Furthermore, we try to evaluate challenging new methods for marine microbial ecology, appropriate for long-term studies of marine bacteria.
a b s t r a c tThe alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.Ó 2008 Elsevier Inc. All rights reserved.The pyrrole-imidazole alkaloid ageladine A was first isolated and described by Fujita et al.[1] using bioassay guided fractionation of extracts of the marine sponge Agelas nakamurai.The alkaloid showed biological effects such as the inhibition of matrix metallo-proteinases and the inhibition of cell migration of bovine endothelial cells. In 2006 the total synthesis of ageladine A was completed by the groups of Weinreb and Karuso [2,3] and later optimized [4,5].Brominated pyrrole-imidazole alkaloids are known to be fish feeding deterrent against the reef fish Thalassoma bifasciatum [6,7] and demonstrate antibiotic activity [8] even against pathogens like Helicobacter pylori [9]. Especially, the degree of bromination and the guanidine moieties have shown to be important for the alkaloids efficacy to disturb cellular calcium ion entry via voltage operated channels in neuroendocrine cells [10][11][12], which possess mainly L-and N-type calcium channels common in neuronal cells. During these fluorescence measurements of cellular effects by pyrrole-imidazole alkaloids, ageladine A was noticed to show fluorescence during UV excitation [12], which was also described earlier by Fujita et al. [1]. We demonstrate here other surprising properties of ageladine A such as its sensitivity to pH changes covering a wide range and because of its high membrane permeability an easy whole animal pH sensitive staining.
Material and methodsCulture methods. PC12 cells from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were kept in culture medium containing RPMI 1640, 10% fetal calf serum, 5% horse serum, and 100 U penicillin/streptomycin per milliliter. Cells were cultivated in an incubator at 37°C, 90% humidity and 5% CO 2 with regular medium changes twice a week or when additionally necessary. Cells grew in culture flasks and 1-2 days prior to the experiments were seeded into petri dishes coated with coll...
Salinity is an important environmental control of aerobic methane oxidation, which reduces the emission of the potent greenhouse gas methane into the atmosphere. The effect of salinity on methane oxidation is especially severe in river estuaries and adjacent coastal waters, which are important sources of methane emission and, at the same time, are usually characterized by pronounced salinity gradients. Using methane oxidation rates determined by a radiotracer technique as a measure of methanotrophic activity, we tested the effect of immediate and gradual salinity changes on pure cultures of methanotrophic bacteria, and natural freshwater (Elbe River) and natural marine (North Sea) methanotrophic populations. According to our results, Methylomonas sp. and Methylosinus trichosporium are resistant to an increase in salinity, whereas Methylovulum sp. and Methylobacter luteus are sensitive to such an increase. Natural methanotrophic populations from freshwater are more resistant to an increase in salinity than those from marine water are to a decrease in salinity. In contrast to an immediate change of salinity, gradual change (1.25 PSU d −1) can attenuate salinity stress. Experiments with the natural populations revealed different reactions to changes in salinity; thus, we assume that the initial composition of the methanotrophic population, i.e. the ratio of sensitive versus resistant strains, also governs the community response to salinity stress.
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