Preparation of Biological Samples for Transmission X-Ray Microanalysis: A Review of Alternative Procedures to the Use of Sectioned Material

Although transmission X-ray microanalysis of biological material has traditionally been carried out mainly on sectioned preparations, a number of alternative procedures exist. These are considered under three major headings - whole cell preparations, analysis of cell homogenates and biological fluids, and applications of the technique to microsamples of purified biochemicals. These three aspects provide a continuous range of investigative level - from the cellular to the molecular. The use of X-ray microanalysis with whole cell preparations is considered in reference to eukaryote (animal) cells and prokaryotes - where it has particular potential in environmental studies on bacteria. In the case of cell homogenates and biological fluids, the technique has been used mainly with microdroplets of animal material. The use of X-ray microanalysis with purified biochemicals is considered in relation to both particulate and non-particulate samples. In the latter category, the application of this technique for analysis of thin films of metalloprotein is particularly emphasised. It is concluded that wider use could be made of the range of preparative techniques available - both within a particular investigation, and in diverse fields of study. Transmission X-ray microanalysis has implications for environmental, physiological and molecular biology as well as cell biology

Copper Toxicity in Erwinia amylovora : An X-Ray Microanalytical Study

The effect of Cu2+ ions on the growth and elemental composition of the phytopathogenic bacterium Erwinia amylovora was investigated by in vitro culture in nutrient broth supplemented with CuSO4. No detectable inhibition in bacterial growth occurred with medium supplemented at 10-4M Cu (compared to control medium with no added Cu), but partial inhibition occurred at 10-3M Cu (limiting toxicity) and complete inhibition at 10-2M Cu. Under conditions of limiting toxicity, incubation in Cu2+ ions leads to a reduction in the general synthesis of cell materials and the formation of abnormally large cells with a small dry mass. X-ray analysis revealed the uptake of small levels of Cu2+ under these conditions, plus wide-ranging changes in other major elements present in the bacterial cells. These changes included a reduction in the level of K and increased levels of the divalent cations Ca and Fe. The results obtained are consistent with the toxic effect of Cu being mediated via its effect on the cell membrane, with internal elemental changes resulting from a perturbation of membrane permeability

X-Ray Microanalysis with the Environmental Scanning Electron Microscope: Interpretation of Data Obtained Under Different Atmospheric Conditions

X-ray microanalysis of non-biological and biological specimens was carried out in an environmental scanning electron microscope (ESEM) over a range of atmospheric conditions. Introduction of water vapour into the specimen chamber lead to direct X-ray contribution from oxygen atoms, an increase in extraneous background (causing reduced P/B ratios of other elements), X-ray absorption (also reducing P/B ratios) and broadening (skirting) of the electron beam. Similar results were obtained after introduction of an argon atmosphere. These effects were reduced under conditions of minimal chamber atmospheric pressure and maximal accelerating voltage. Because of beam skirting, quantitative X-ray microanalysis of biological specimens in a water vapour atmosphere was only valid where the sample was spread over a wide area (leading to mean elemental values for the whole preparation). Unless appropriate correction factors or changes in instrumentation can be implemented, quantitative analysis of wet specimens in ESEM cannot be applied to discrete specimens or to limited areas within a mixed sample

Studies on the Occurrence and Elemental Composition of Bacteria in Freshwater Plankton

The occurrence and cation content of bacteria in a eutrophic freshwater lake (Rostherne Mere, Cheshire, UK) were investigated over a one year sampling period in relation to cation changes in the lake surface water and phytoplankton. Scanning electron microscope examination of trawl-net and filtered samples demonstrated bacterial association with Anabaena, Aphanizomenon and diatoms. Direct counts of associated and unassociated bacteria showed that increases in bacterial population relate to population decline of major algal constituents. Spectrophotometric determination of selected cation levels in the lake water demonstrated wide fluctuations throughout the sampling period, with elevated levels of transition metals before and at the end of Summer stratification. Zn and Pb also showed increased levels in relation to episodic events. Mass fractions of spectrophotometrically-determined selected cations (Fe, Cu, Zn and Pb) in phytoplankton also varied considerably during the sampling period, with major increases apparently following peaks in water level. X-ray microanalysis of whole, unassociated bacterial eel ls demonstrated high levels of soluble and bound cations, including K, Ca, Fe, Cu, Ni, Zn and Pb. Changes in the cation levels of bacteria did not follow a similar pattern to the general phytoplankton - probably due to differences in uptake or adsorption or to cycling of bacterial cells in the water column

Scanning X-Ray Microanalysis of Microcarrier Cultured Endothelial Cells: Elemental Changes During the Transition to Confluency and the Effect of Ionophore A23187

Porcine endothelial cells were grown on microcarrier beads and examined by scanning electron microscopy (SEM) at various times after initiation of culture. Total cell coverage on the bead surface varied from mean values of approximately 7% (3h) to 80% (96h). Beam penetration into the subcellular matrix presents a major problem with SEM X-ray microanalysis of microcarrier cultured cells and necessitates the use of an accelerating voltage not exceeding 10kV. At this voltage and below, X-ray contribution from elements present in the microcarrier bead has minimal effect on the determination of cell elemental levels. Washing the cells with 0.15M sucrose was the least perturbing of the rinsing techniques investigated. removing surface culture medium but not internal diffusible ions. X-ray microanalysis revealed detectable levels of Na, P, S, Cl, K and Ca in the cells, with well-marked changes from initial attachment to confluency. The level of K decreased from approximately 1.0% at 3h to 0.4% at 24h, with a corresponding decrease in the K/Na ratio. This unexpectedly low level of K was invariably observed after 24h, and is a genuine feature of established microcarrier culture. The effect of ionophore A23187 was determined at the 3h culture stage, and resulted in significant increases in the concentration of divalent cations (Mg2+, Ca2+), monovalent ions (Na+, Cl-) and a decrease in the level of K+

The Electron Microscope Detection and X-Ray Quantitation of Cations in Bacterial Cells

Electron microscope autoradiography and X-ray microanalysis have been used for the detection and quantitation of cations in the bacterium Pseudomonas tabaci. These techniques differ in the information they provide (relating to either cation uptake or in situ levels), their applicability to different cations, their sensitivity and their spatial resolution. With uptake of 63Ni2+, high resolution autoradiography (involving gold latensification and physical development) demonstrated a high degree of cation localisation to the central nucleoid area (glutaraldehyde-fixed cells) and within this to the constituent chromatin (acetic-alcohol preparations). X-ray microanalysis of whole bacterial cells revealed the presence of substantial levels of K (mainly soluble cation}, Ca, Mn, Ni, Cu and Zn (mainly in soluble cations) and Fe (present as major soluble and insoluble components). The use of whole cells provided a particularly useful experimental system to demonstrate the importance of cell preparation technique in relation to element detectability. The application of X-ray microanalysis to lysed cells permitted analysis of extruded contents -including cell protoplast (protoplasm without associated cell wall material) and chromatin fibrils. The microprobe detection of DNA-associated cations was most effective with freshly extracted chromatin and showed the presence of bound K, Ca and transition metals

Explaining the causes of cell death in cyanobacteria: what role for asymmetric division?

Cyanobacteria contribute a significant fraction of global primary production and are therefore of great ecological significance. An individual cyanobacteria cell has four potential fates: to divide, perhaps after a dormant period, to be eaten, to undergo viral lysis, or to undergo cell death. In some studies, cyanobacteria cell death has been classified as programmed cell death, borrowing a concept more widely known in metazoan cells, and there are various biochemical parallels to support such a categorisation. However, at the same time there is a growing awareness of asymmetric division as a fundamental process in bacterial division which can result in non-equal daughter cells with differing fitness. Thanks to recent theoretical and experimental advances it is now possible to explore cyanobacteria cell death in the light of asymmetric division and to test hypotheses on the ultimate causes of cyanobacterial cell death. Assessing the degree of protein damage within individual cells during population growth is a sensible initial research target as is the application of techniques which allow the tracking of cell lineages. The existence of asymmetric division in cyanobacteria is likely given its suggested ubiquity across the bacterial domain of life. It will be technically difficult to test the interaction of asymmetric division with environmental variability, and how that leads to individual cell death via differing susceptibilities to environmental stress. However, testing such ideas could confirm asymmetric division as the ultimate cause of cell death in cyanobacteria and thereby allow a better understanding of the patterns of cell death in natural populations

Aerosols in atmospheric chemistry and biogeochemical cycles of nutrients

Atmospheric aerosols have complex and variable compositions and properties. While scientific interest is centered on the health and climatic effects of atmospheric aerosols, insufficient attention is given to their involvement in multiphase chemistry that alters their contribution as carriers of nutrients in ecosystems. However, there is experimental proof that the nutrient equilibria of both land and marine ecosystems have been disturbed during the Anthropocene period. This review study first summarizes our current understanding of aerosol chemical processing in the atmosphere as relevant to biogeochemical cycles. Then it binds together results of recent modeling studies based on laboratory and field experiments, focusing on the organic and dust components of aerosols that account for multiphase chemistry, aerosol ageing in the atmosphere, nutrient (N, P, Fe) emissions, atmospheric transport, transformation and deposition. The human-driven contribution to atmospheric deposition of these nutrients, derived by global simulations using past and future anthropogenic emissions of pollutants, is put into perspective with regard to potential changes in nutrient limitations and biodiversity. Atmospheric deposition of nutrients has been suggested to result in human-induced ecosystem limitations with regard to specific nutrients. Such modifications favor the development of certain species against others and affect the overall functioning of ecosystems. Organic forms of nutrients are found to contribute to the atmospheric deposition of the nutrients N, P and Fe by 20%–40%, 35%–45% and 7%–18%, respectively. These have the potential to be key components of the biogeochemical cycles since there is initial proof of their bioavailability to ecosystems. Bioaerosols have been found to make a significant contribution to atmospheric sources of N and P, indicating potentially significant interactions between terrestrial and marine ecosystems. These results deserve further experimental and modeling studies to reduce uncertainties and understand the feedbacks induced by atmospheric deposition of nutrients to ecosystems

Combatting cyanobacteria with hydrogen peroxide: a laboratory study on the consequences for phytoplankton community and diversity

Experiments with different phytoplankton densities in lake samples showed that a high biomass increases the rate of hydrogen peroxide (HP) degradation and decreases the effectiveness of HP in the selective suppression of dominant cyanobacteria. However, selective application of HP requires usage of low doses only, accordingly this defines the limits for use in lake mitigation. To acquire insight into the impact of HP on other phytoplankton species, we have followed the succession of three phytoplankton groups in lake samples that were treated with different concentrations of HP using a taxa-specific fluorescence emission test. This fast assay reports relatively well on coarse changes in the phytoplankton community; the measured data and the counts from microscopical analysis of the phytoplankton matched quite well. The test was used to pursue HP application in a Planktothrix agardhii-dominated lake sample and displayed a promising shift in the phytoplankton community in only a few weeks. From a low-diversity community, a change to a status with a significantly higher diversity and increased abundance of eukaryotic phytoplankton species was established. Experiments in which treated samples were re-inoculated with original P. agardhii-rich lake water demonstrated prolonged suppression of cyanobacteria, and displayed a remarkable stability of the newly developed post-HP treatment state of the phytoplankton community

Genotype × genotype interactions between the toxic cyanobacterium Microcystis and its grazer, the waterflea Daphnia

Toxic algal blooms are an important problem worldwide. The literature on toxic cyanobacteria blooms in inland waters reports widely divergent results on whether zooplankton can control cyanobacteria blooms or cyanobacteria suppress zooplankton by their toxins. Here we test whether this may be due to genotype × genotype interactions, in which interactions between the large-bodied and efficient grazer Daphnia and the widespread cyanobacterium Microcystis are not only dependent on Microcystis strain or Daphnia genotype but are specific to genotype × genotype combinations. We show that genotype × genotype interactions are important in explaining mortality in short-time exposures of Daphnia to Microcystis. These genotype × genotype interactions may result in local coadaptation and a geographic mosaic of coevolution. Genotype × genotype interactions can explain why the literature on zooplankton–cyanobacteria interactions is seemingly inconsistent, and provide hope that zooplankton can contribute to the suppression of cyanobacteria blooms in restoration projects