Electrophoretic Mobility of Mycobacterium avium Complex Organisms

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured. The EPMs of 15 clinical isolates ranged from −1.9 to −5.0 μm cm V⁻¹ s⁻¹, and the EPMs of 15 environmental isolates ranged from −1.9 to −4.6 μm cm V⁻¹ s⁻¹ at pH 7.     

Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

Leaf structure and translocation in sugar beet

Anatomical and ultrastructural details of a translocating 10-cm leaf of sugar beet (Beta vulgaris L. var. Klein Wanzleben) were correlated with translocation rate data. The minor veins were found to be 13 times as extensive as the major veins and measure 70 cm/cm² leaf lamina. Measurements disclosed that a 33-μ length of minor vein services 29 mesophyll cells with the result that translocate moves an average of 73 μ or 2.2 cell diameters during transport from mesophyll cells to a minor vein. High-resolution, freeze-dry autoradiography revealed that assimilates accumulate in organelle-rich cells of the minor vein phloem. Correlation of phloem volume and loading rate for minor veins yielded an uptake rate of 735 μmoles of sucrose per g fresh weight of phloem. The arrangement and structural features of minor veins appeared to be consistent with the concept that vein loading precedes translocation.     

Zooplankton Growth,Respiration and Grazing on the Australian Margins of the Tropical Indian and Pacific Oceans

The specific activity of aminoacyl-tRNA synthetases (spAARS), an index of growth rate, and of the electron transport system (spETS), an index of respiration, was measured in three size fractions (73–150 μm, >150 μm and >350 μm) of zooplankton during five cruises to tropical coastal waters of the Kimberley coast (North West Australia) and four cruises to waters of the Great Barrier Reef (GBR; North East Australia). The N-specific biomass of plankton was 3–4-fold higher in the Kimberley than on the GBR in all 3 size classes: Kimberley 1.27, 3.63, 1.94 mg m^-3; GBR 0.36, 0.88 and 0.58 mg m^-3 in the 73–150 μm, >150 μm and >350 μm size classes, respectively. Similarly, spAARS activity in the Kimberley was greater than that of the GBR: 88.4, 132.2, and 147.6 nmol PPi hr^-1 mg protein ^-1 in the Kimberley compared with 71.7, 82.0 and 83.8 nmol PPi hr^-1 mg protein ^-1 in the GBR, for the 73–150 μm, >150 μm and >350 μm size classes, respectively. Specific ETS activity showed similar differences in scale between the two coasts: 184.6, 148.8 and 92.2 μL O₂ hr^-1 mg protein^-1 in the Kimberley, against 86.5, 88.3 and 71.3 μL O₂ hr^-1 mg protein^-1 in the GBR. On the basis of these measurements, we calculated that >150 μm zooplankton grazing accounted for 7% of primary production in the Kimberley and 8% in GBR waters. Area-specific respiration by >73 μm zooplankton was 7-fold higher in the Kimberley than on the GBR and production by >150 μm zooplankton was of the order of 278 mg C m^-2 d^-1 in the Kimberley and 42 mg C m^-2 d^-1 on the GBR. We hypothesize that the much stronger physical forcing on the North West shelf is the principal driver of higher rates in the west than in the east of the continent.     

Water-to-Air Transfer and Enrichment of Bacteria in Drops from Bursting Bubbles

An electrostatic induction technique was used to determine both drop size distribution and concentration of bacteria in the film drops produced by bubbles bursting at the surface of a suspension of Serratia marcescens. Film drops are produced from the collapse of the thin film of water that just before bursting separates the air in the bubble from the atmosphere. Bubbles of 1.7-mm diameter produced from 10 to 20 film drops which ranged from <2 μm to over 30 μm in diameter. Half the drops were <10 μm. For bubbles rising a distance of less than 2 cm through the bacterial suspension, bacterial enrichment factors in the drops were between 10 and 20. Electrostatic methods can be used to determine the enrichment of bacteria in film drops as a function of bubble size and distance of rise through the bacterial suspension.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-μm nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 μmol m⁻² s⁻¹). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 μmol m⁻² s⁻¹, but in the presence of saturating red light, fluence rates as low as 25 μmol m⁻² s⁻¹ induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.     

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness,Nuclear Volume,Contractility, and Cell Stiffness

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.     

Size Distribution of Air Bubbles Entering the Brain during Cardiac Surgery

Background

Thousands of air bubbles enter the cerebral circulation during cardiac surgery, but whether high numbers of bubbles explain post-operative cognitive decline is currently controversial. This study estimates the size distribution of air bubbles and volume of air entering the cerebral arteries intra-operatively based on analysis of transcranial Doppler ultrasound data.

Methods

Transcranial Doppler ultrasound recordings from ten patients undergoing heart surgery were analysed for the presence of embolic signals. The backscattered intensity of each embolic signal was modelled based on ultrasound scattering theory to provide an estimate of bubble diameter. The impact of showers of bubbles on cerebral blood-flow was then investigated using patient-specific Monte-Carlo simulations to model the accumulation and clearance of bubbles within a model vasculature.

Results

Analysis of Doppler ultrasound recordings revealed a minimum of 371 and maximum of 6476 bubbles entering the middle cerebral artery territories during surgery. This was estimated to correspond to a total volume of air ranging between 0.003 and 0.12 mL. Based on analysis of a total of 18667 embolic signals, the median diameter of bubbles entering the cerebral arteries was 33 μm (IQR: 18 to 69 μm). Although bubble diameters ranged from ~5 μm to 3.5 mm, the majority (85%) were less than 100 μm. Numerous small bubbles detected during cardiopulmonary bypass were estimated by Monte-Carlo simulation to be benign. However, during weaning from bypass, showers containing large macro-bubbles were observed, which were estimated to transiently affect up to 2.2% of arterioles.

Conclusions

Detailed analysis of Doppler ultrasound data can be used to provide an estimate of bubble diameter, total volume of air, and the likely impact of embolic showers on cerebral blood flow. Although bubbles are alarmingly numerous during surgery, our simulations suggest that the majority of bubbles are too small to be harmful.     

Bacterioplankton in Antarctic Ocean Waters During Late Austral Winter: Abundance, Frequency of Dividing Cells, and Estimates of Production

Bacterioplankton productivity in Antarctic waters of the eastern South Pacific Ocean and Drake Passage was estimated by direct counts and frequency of dividing cells (FDC). Total bacterioplankton assemblages were enumerated by epifluorescent microscopy. The experimentally determined relationship between in situ FDC and the potential instantaneous growth rate constant (μ) is best described by the regression equation ln μ = 0.081 FDC − 3.73. In the eastern South Pacific Ocean, bacterioplankton abundance (2 × 10⁵ to 3.5 × 10⁵ cells per ml) and FDC (11%) were highest at the Polar Front (Antarctic Convergence). North of the Subantarctic Front, abundance and FDC were between 1 × 10⁵ to 2 × 10⁵ cells per ml and 3 to 5%, respectively, and were vertically homogeneous to a depth of 600 m. In Drake Passage, abundance (10 × 10⁵ cells per ml) and FDC (16%) were highest in waters south of the Polar Front and near the sea ice. Subantarctic waters in Drake Passage contained 4 × 10⁵ cells per ml with 4 to 5% FDC. Instantaneous growth rate constants ranged between 0.029 and 0.088 h⁻¹. Using estimates of potential μ and measured standing stocks, we estimated productivity to range from 0.62 μg of C per liter · day in the eastern South Pacific Ocean to 17.1 μg of C per liter · day in the Drake Passage near the sea ice.     

Root Cortical Cell Spherical Bodies Associated with an Induced Resistance Reaction in Monoxenic Cultures of Meloidogyne incognita

The root-knot nematode Meloidogyne incognita was monoxenically cultured on excised roots of soybean cv. Pickett and tomato cv. Rutgers in agar media containing either 0 to 1,600 μg/ml ammonium nitrate or 0 to 100 μg/ml urea. Observations with scanning and transmission electron microscopy indicated that an elevated concentration of ammonium nitrate or urea inhibited giant cell formation and suppressed nematode development in the infected soybean roots. In the tomato roots, concentrations of ammonium nitrate above 400 μg/ml or urea above 25 μg/ml inhibited giant cell formation and nematode development. Coincident with the nitrogen concentrations that suppressed giant cell formation was the appearance of electron-dense spherical bodies in the cortical parenchyma cells of both the soybean and tomato roots. These bodies, which were 1-4 μm in diameter, appeared to form in the cytoplasm and migrate to the cell vacuole.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Measurement of the Size Distribution of Zymogen Granules from Rat Pancreas

Zymogen granules are obtained in pure form and processed for electron microscopy. Thin sections are photographed and diameters measured with a Zeiss particle size analyzer. Since sectioning cuts any given particle in random way, these diameters are not the true diameters of the particles. The true size distribution is obtained by comparing the observed diameter distribution with a generated diameter distribution. The generated distribution is constructed from an assumed parent distribution (of true diameters) by the Monte-Carlo technique. “Goodness of fit” is judged by the value of “chi-squared” resulting from the comparison. Appropriate adjustments of the parameters of the true distribution are made on the basis of minimizing chi-square. A result of this process is that the zymogen granules follow a normal distribution: mean = 0.984 ±0.005 μm, SD = 0.190 ±0.005 μm. A second preparation of granules was made and diameters were measured directly with a scanning electron microscope. The distribution was again found to be normal, thus supporting the first result.     

Modeling and Mapping of Atmospheric Mercury Deposition in Adirondack Park,New York

The Adirondacks of New York State, USA is a region that is sensitive to atmospheric mercury (Hg) deposition. In this study, we estimated atmospheric Hg deposition to the Adirondacks using a new scheme that combined numerical modeling and limited experimental data. The majority of the land cover in the Adirondacks is forested with 47% of the total area deciduous, 20% coniferous and 10% mixed. We used litterfall plus throughfall deposition as the total atmospheric Hg deposition to coniferous and deciduous forests during the leaf-on period, and wet Hg deposition plus modeled atmospheric dry Hg deposition as the total Hg deposition to the deciduous forest during the leaf-off period and for the non-forested areas year-around. To estimate atmospheric dry Hg deposition we used the Big Leaf model. The average atmospheric Hg deposition to the Adirondacks was estimated as 17.4 g m yr with a range of −3.7–46.0 g m yr. Atmospheric Hg dry deposition (370 kg yr) was found to be more important than wet deposition (210 kg yr) to the entire Adirondacks (2.4 million ha). The spatial pattern showed a large variation in atmospheric Hg deposition with scattered areas in the eastern Adirondacks having total Hg deposition greater than 30 μg m⁻² yr⁻¹, while the southwestern and the northern areas received Hg deposition ranging from 25–30 μg m⁻² yr⁻¹.     

Microflora of soil as viewed by transmission electron microscopy

Several procedures were evaluated for separating and concentrating indigenous microorganisms from soil without the occurrence of growth. Electron microscopy of nontangential, thin sections through these cells revealed that all of the cells examined were less than 0.9 μm in diameter, and up to 72% were „dwarf” cells less than 0.3 μm in diameter. Some were small enough that they should not be resolved with the light microscope. Approximately 27% had a fine structure bearing some resemblance to that of a bacterial cyst or microcyst, but this value may be low because cells having their outer layers partially stripped off were not included in the count. Approximately 25% showed a distinct periplasmic space, which often contained stainable material. Other fine structure features are presented together with frequencies of occurrence for the populations examined.     

High Motility Reduces Grazing Mortality of Planktonic Bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 μm s⁻¹ as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 μm s⁻¹) and a highly motile strain (>45 μm s⁻¹) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 μm³. Grazing mortality was lowest for cells of >0.5 μm³ and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (≤0.1 μm³, >50 μm s⁻¹) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using ¹³C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (K_m approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h⁻¹ g⁻¹ cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. ³¹P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h⁻¹ g⁻¹ cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h⁻¹ g⁻¹ cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.     

Automatic Determination of Bacterioplankton Biomass by Image Analysis

Image analysis was applied to epifluorescense microscopy of acridine orange-stained plankton samples. A program was developed for discrimination and binary segmentation of digitized video images, taken by an ultrasensitive video camera mounted on the microscope. Cell volumes were estimated from area and perimeter of the objects in the binary image. The program was tested on fluorescent latex beads of known diameters. Biovolumes measured by image analysis were compared with directly determined carbon biomasses in batch cultures of estuarine and freshwater bacterioplankton. This calibration revealed an empirical conversion factor from biovolume to biomass of 0.35 pg of C μm⁻³ (± 0.03 95% confidence limit). The deviation of this value from the normally used conversion factors of 0.086 to 0.121 pg of C μm⁻³ is discussed. The described system was capable of measuring 250 cells within 10 min, providing estimates of cell number, mean cell volume, and biovolume with a precision of 5%.