Detection of Microorganisms in the Environment and the Preliminary Appraisal of Their Physiological State by X-ray Microanalysis

The paper deals with the X-ray microanalysis of the elemental composition of bacteriomorphic particles in 170000-year-old Antarctic permafrost sediments and in indoor dust. A comparative analysis of the phosphorus, sulfur, calcium, and potassium contents and the Ca/K and P/S ratios in these particles and in reference microbial cells occurring in different physiological states showed that the absence of P and/or S peaks in the X-ray spectrum of an object may indicate that it is abiotic. Resting microbial forms can be revealed on the basis of the following characteristic features: an increased content of Ca, a high Ca/K ratio, and a low P/S ratio. Model experiments with nonviable bacterial and yeast micromummies with alterations in the structural and barrier functions of the cytoplasmic membrane showed that micromummies can be recognized by a superhigh content of a marker element (e.g., P, K, or Si), accumulated due to facilitated diffusion along a deliberately created concentration gradient. Such an analysis of the permafrost sediment and dust made it possible to suggest the presence of mummified cells in these objects. The possibility of using X-ray microanalysis for the detection of microbial cells in natural habitats in order to enhance the efficiency of ecological monitoring of the environment is discussed.     

Comparative Study of the Elemental Composition of Vegetative and Resting Microbial Cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the content of elements S, P, Ca, and K and Ca/K and P/S ratios. Viable resting forms (cystlike refractive cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of elements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable micromummies, even those morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for the quick analysis of the physiological state of microorganisms in natural environments is discussed.     

Comparative study of the elemental composition of vegetative and dormant microbial cells

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the contents of bioelements S, P, Ca, and K and the Ca/K and P/S ratios. Viable resting forms (cystlike refractory cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of bioelements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable forms, even morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for quick analysis of the physiological state of microorganisms in natural environments is discussed.     

Lipid and elemental composition as indicators of the physiological state of sporangiospores in Mucor hiemalis cultures of different ages

The number of sporangiospores used as inocula may significantly affect the development of the morphogenetic programs in mucoraceous fungi, including manifestations of dimorphism. In order to assess the physiological state of sporangiospores differing in their viability and germination patterns, lipid composition of sporangiospores from 6-, 14-, and 20-day cultures of Mucor hiemalis F-1431 was determined and the element ratios in these spores (Ca/K and P/S) were measured using X-ray microanalysis. While the lipid composition of the “old” (20 days) and “young” (6 days) spores was generally similar, older spores contained no cerebrosides, had half the level of polar lipids and γ-linolenic acid, and also contained 6 times more monoacylglycerols and 2.5 times more phosphatidic acid. The ratio of esterified to free sterols (ES/FS) characterizing the sterol storage pool was lowest in the spores from 20-day cultures. X-ray microanalysis revealed the highest P/S ratio in 6-day spores and lowest ratio in 14-day ones. Mature 14-day spores had the highest Ca/K ratio, ten times exceeding that for the 20-day spores. Higher values of P/S and Ca/K ratios in young (ripening) spores than in old 20-day spores indicate their higher metabolic activity and correlate with their higher viability and mycelial type of germination. Together with the lipid characteristics, the Ca/K and P/S ratios are the parameters which may be used to develop the criteria for assessment of the physiological state of the cells, including viable fungal sporangiospores. This complex may be also used to assess the capacity of mucoraceous fungi for dimorphism, including the species (M. hiemalis) for which this capacity has not been demonstrated previously.     

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora)

Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.     

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora)

Summary Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: application in PCR

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

Correlation between potassium and phosphorus content and their nonuniform distribution in Acanthamoeba castellanii

Summary Biologically important elements: K, Na, Mg, Ca, Cl, P, and S were analyzed in Acanthamoeba castellanii. A higher potassium content, as compared with other cations, was detected. Total content of the cation-forming elements: K, Na, Mg, and Ca was ca. 360 mmoles/kg dry weight of the cells. Phosphorus content was estimated as 492 mmoles/kg dry weight. Content of chlorine, a basic cellular anion, was 173 mmoles/kg dry weight. The low level of chlorine appears not the be sufficient to balance all the cations in Acanthamoeba.Distribution of potassium in Acanthamoeba cells was nonuniform and similar to that of phosphorus as shown by X-ray microanalysis technique. Quantitative correlation between phosphorus and potassium as well as the similar distribution of these elements suggests that in Acanthamoeba phosphorus is an essential anion which, being nonuniformly distributed in the cell, determines also a nonuniform distribution of potassium.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Elemental composition of extremely alkaliphilic anaerobic bacteria

The contents of several chemical elements were assessed in the haloalkaliphilic acetogenic bacterium Natroniella acetigena and the alkaliphilic sulfate-reducing bacterium Desulfonatronum lacustre using X-ray microanalysis, stereoscanning microscopy, and mass spectrometry. The organisms were found to differ significantly in their relative contents of S, K, P, and Cl. The P/S ratio in cells of the alkaliphilic bacteria studied grown on mineral media at different pH was pH-dependent. With a pH increase from 9 to 10, potassium extrusion from cells was observed, suggesting that secondary K+/H+ antiport activity accounts for the homeostasis of cytosolic pH. Deenergization of bacterial cells in the presence of inhibitors and ionophores results in specific changes in the P/S ratio, which may be considered an indicator of the cell energetic state. In Natroniella acetigena, the content of intracellular Cl was directly proportional to the NaCl concentration in the medium. Some metals were shown to be necessary for the N. acetigena viability; the requirement for Ni and Co was absolute. Although little demand for Mg was characteristic of the bacteria studied, their growth was stimulated by an increase in Mg concentration, and the cell resistance to lysis was enhanced.     

ELEMENTAL COMPOSITION,CORRELATIONS, AND RATIOS WITHIN A POPULATION OF STAURASTRUM PLANCTONICUM (ZYGNEMATALES): AN X-RAY MICROANALYTICAL STUDY1

Individual cells of Staurastrum planctonicum (Teil.) within a mixed freshwater phytoplankton sample were analyzed by scanning electron microscope X-ray microanalysis to determine their elemental composition. X-ray emission spectra routinely showed clear peaks of P, S, and Cl, plus monovalent (Na, K, and divalent (Mg, Ca) cations. Si and Cu were also present in lower quantities. Concentrations of individual elements (expressed as mmol.kg⁻¹ dry weight) varied widely among cells, with values over the sample population approximating to a normal distribution. Although intracellular anion and cation levels varied considerably, significant correlations occurred between concentrations of monovalent and divalent cations (mean ratio 1.4), major diffusible anions and cations (mean ratio 1,2), and total levels of electropositive and electronegative elements (mean ratio 1.2). The monovalent cations of K and Na occurred at a mean ratio of 1.2 and were not significantly correlated. Concentrations of individual elements (except Si) showed clear positive correlations within the analyses, with 12 highly significant (99% probability) correlations out of 36 possible combinations. Principal factor analysis showed that elemental correlations were determined by two major factors, with two resulting groups of elements—(Na, S, Cl, Ca, Cu) and (Mg, P, K). Statistical relationships between elements followed a clear correlation pattern, which retained its characteristics even when elemental concentrations were expressed per unit P rather than per unit dry weight. Elemental concentrations were closely similar in matching, but not nonmatching, smicells. The statistical pattern of elemental associations noted in Staurastrum parallels that seen in X-ray micro-analytical studies of other algae but differs in detail. This pattern of statistical associations has biological implications in terms of cell compartmentation, characterization of different cell types, and cell interactions with their environment.     

Elemental Composition and Water Content of Neuron and Glial Cells in the Central Nervous System of the North American Medicinal Leech (Macrobdella decora)

Elemental (Na, P, S, Cl, K, Ca, Mg) composition and water content of neurons and glial cells of the leech (Macrobdella decora) were determined by x-ray microanalysis of frozen hydrated and dried section techniques. Results are reported as elemental mass fractions (mass/mass) and water content as percent mass. Specific cell compartments and cell types had distinct elemental patterns and water content which suggests that chemical composition of specific cell types is unique and may represent an expression of cell differentiation analogous to morphological specialization. Water content of cells was also cell specific and ranged from 55% (neurons) to 90% (vacuolated zone of glial cells). K and Na were present in concentrations greater than predicted by ion-selective microelectrode measurements, indicating that not all the K and Na were simultaneously accessible to such electrodes.     

Morphometry and elemental analysis of rat exocrine pancreas following administration of trypsin inhibitor

The morphological responses of the exocrine pancreas of the adult male rat to soybean trypsin inhibitor (STI) were studied by ultrastructural morphometry and electron probe X-ray microanalysis. STI administered orally in drinking water for 14 days resulted in a 72% increase in the wet weight of the pancreas. This enlargement was due, largely, to an increase in acinar cell mass. Volume increases in the acinar cell mass and extra-acinar cell compartment were 72 and 30%, respectively. The estimated total number of acinar cells in the mean exocrine pancreas was 500 million in the control and 630 million in the experimental group, representing an increase of 27%. Acinar cell volume was 1,790 microns 3 for the control and 2,457 microns 3 for the STI group. The pronounced morphometric changes of the organelles in the STI group were: the mean nucleolar volume increased by 56%; the volume of zymogen granular mass per cell increased by 93%; the volume of the Golgi complex and the condensing vacuoles per cell increased by 52 and 100%, respectively, whereas the membrane area of the Golgi complex and the condensing vacuoles increased by 98 and 47%, respectively. Spectral analysis of seven elements (Na, Mg, P, S, Cl, K and Ca) showed significant changes for nuclei, zymogen granules and mitochondria following STI: nuclei showed Na, P, K increased; zymogen granules showed Na, P, S, K increased, Cl decreased; mitochondrial particles showed Mg, P, Cl, Ca increased, and the mitochondrial matrix showed S decreased. The persistent uptake of STI probably resulted in a continual release of a trophic hormone acting on pancreatic tissue components, consequently causing hyperplasia and hypertrophy of the exocrine pancreas to accommodate a heightened demand for synthesis of exportable proteins.     

Changes in elemental concentrations in LNCaP cells are associated with a protective effect of neuropeptides on etoposide-induced apoptosis

One of the mechanisms that has been put forward for the development of the androgen-resistant status is neuroendocrine differentiation. Neuroendocrine cells secrete neuropeptides that may represent one of the possible molecular bases by which hormone-dependent prostate cancer cells could escape treatment. LNCaP prostate cancer cells were treated with either etoposide or neuropeptides. Morphological changes related to apoptosis and cell viability were assessed. Changes in intracellular ion content were quantitatively analyzed by electron probe X-ray microanalysis. Etoposide treatment consistently induces a decrease in K and an increase in Na, which are inhibited by bombesin or calcitonin. The Na/K ratio increased markedly after exposure to etoposide, and both bombesin and calcitonin blocked this increase. Etoposide also caused changes in the intracellular P and S concentrations that to a large extent could be blocked by neuropeptides. These results support the hypothesis that neuropeptides confer anti-apoptotic capabilities onto non-neuroendocrine cells in close proximity to neuroendocrine cells.     

Mineral partitioning in the phloem during autumn senescence of beech leaves

Summary During the period of leaf senescence in fall, the minerals Mg, Ca, K, P, Cl, S, and Si were compared for occurrence and density in tissue compartments of leaf blade, petiole, and subtending stem of beech (Fagus sylvatica L.). Measurements were made by energy-dispersive X-ray microanalysis. The plant material was collected on 2,9, 16 and 23 October, and showed green, greenyellow, yellow, and red-brown autumn leaf coloration. Mg, K, and P were retrieved from the leaf blade prior to shedding, and deposited mainly in cortex and pith tissues of the stem. S and Ca remained in the leaf, and Si and Cl appeared to accumulate in the leaf prior to shedding. During the four stages of leaf senescence, the phloem compartments of the petiole showed considerable changes in mineral content. In addition, leaf senescence in several cases was accompanied by ion shifting from symplastic to apoplastic compartments and vice versa.     

Ion Distribution Across the Cortex of Soybean Nodules: Possible Involvement in Control of Oxygen Diffusion

X-ray microanalysis was used to study ion distribution in nodulesof soybean [Glycine max (L.)] cv. Clarke formed with two strainsof Bradyrhizobium japonicum ; RCR3407 and RCR3442. Previousstudies have shown that the oxygen diffusion barrier in nodulesof the RCR3442 symbiosis has slower responses to acetylene andincreased external oxygen than those of the RCR3407 symbiosisand this variation is correlated with differences in glycoproteincontent. X-ray maps and cross-cortical cell counts of nodulesformed by either strain show very similar zonal distributionsof Mg, K, S and Ca across the cortex. Levels of K appear tobe similar but levels of Mg, S and Ca seem to be lower in nodulesof the RCR3442 symbiosis. These results suggest that the contentand distribution of Mg, S and Ca reflect an involvement in theoperation of the cortical diffusion barrier.Copyright 1994,1999 Academic Press Glycine max, X-ray microanalysis, nodules, oxygen diffusion, ion distribution, Mg, Ca, K, S     

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds

Summary Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K⁺ in alkaloids transport is discussed.