In situ and laboratory studies of bacterial survival using a microporous membrane sandwich.

A new device and procedure for the study of bacterial survival in an aquatic environment are described. The device uses two appressed presterilized microporous membranes to expose a bacterial cell suspension to the environment at a cell concentration that closely resembles those levels found in natural aquatic ecosystems. The device has been used under laboratory controlled conditions and in situ to study and compare bacterial survival times. In laboratory studies, Escherichia coli and Streptococcus faecalis survived the longest at 12 degrees C, pH 5, and in the presence of iron or calcium ions and cysteine. Cells in mid-stationary growth phase survived longer than those in mid- or late-logarithmic phase, whereas those maintained for a year or more as stock cultures survived for shorter period of time than did recent environmental isolates. In situ studies indicate that 5% of the starting number of E. coli and S. faecalis cells may survive longer than 96 h at 16 degrees C in potable lake water, whereas survival times in polluted lake water were approximately 12 h.     

In situ and laboratory studies on the behaviour and survival of Pacific salmon (genus Oncorhynchus)

Juvenile Pacific salmon display a marked surface water orientation during downstream migration, estuarine and nearshore coastal rearing phases. Many estuaries in British Columbia are vertically stratified with a shallow, well-defined halocline which can restrict the dispersion of wastes discharged into less saline surface waters and impose constraints upon aquatic organisms. In situ experiments in an estuary receiving a surface discharge of treated pulp mill wastes, revealed conditions which were lethal to underyearling salmon at, and below the halocline (4.0–6.5 m depth). Behavioural bioassays determined that juvenile chinook salmon were biased towards the water surface and avoided waters at depth. Dissolved oxygen was the variable which affected this distribution most significantly. Surface waters receiving effluent from another pulp mill were lethal to juvenile salmon within 350 m, and a significant vertical avoidance response occurred within 350–950 m of the outfalls. The behavioural response was significantly correlated with in situ temperature, pH and colour (effluent).As a complement to field experiments we developed a 4500 l water column simulator (WCS) to examine salmon behaviour in the laboratory. We investigated the surface water orientation behaviour of juvenile salmon in relation to variations in salinity and dissolved oxygen. Under simulated vertically stratified estuarine conditions, the fish moved freely between overlying fresh water and salt water. Induction of hypoxic conditions in fresh water elicited a downward distribution shift towards the halocline and oxygenated, but more saline, waters. Avoidance reactions (50% level) occurred consistently up to 7–8 mg · l^–1 dissolved oxygen. Salmon continued to examine the hypoxic freshwater zone despite sub-optimal conditions.     

In situ and laboratory assessment of heart rate in a Mediterranean limpet using a noninvasive technique

Heart rate of the Mediterranean limpet Patella caerulea L. was investigated on the natural shore and in the laboratory by using a technique based on infrared phototransducers. Field recording occurred in the Gulf of Trieste (northern Adriatic) during March and June 1997. A consistent dependence of heart rate on temperature was observed in limpets both when submerged and when exposed to air in the two periods, but thermal acclimation was evident. During spontaneous activity at high tide, heart rate increased 1.5-1.7 times the values observed during resting in water at corresponding temperatures. The dependence of heart rate on temperature (10 degrees, 16 degrees, and 22 degrees C) and size (wet weight <1.25 and >1.30 g) in submerged limpets from different populations (northern Adriatic and Tyrrhenian) was tested in the laboratory by adopting a factorial design. The results showed a marked effect of temperature, body weight, and their interaction, independent from the site of origin. Smaller limpets showed a linear increase of heart rate in the whole range of temperature tests, while in the larger ones the increase between 10 degrees and 16 degrees C was greater than between 16 degrees and 22 degrees C. Heart rate decreased with increasing body size at control (16 degrees C) and high (22 degrees C) temperature, while at lower temperature (10 degrees C) no effect of body size was evident. When removed from their home scar, limpets increased heart rate to about 1.5 times the reference value. Finally, correlation of oxygen consumption with heart rate of submerged limpets maintained at a different temperature (10 degrees -22 degrees C) was statistically significant.     

In situ plant water balance studies using a portable NMR spectrometer

A portable ¹H NMR spectrometer has been applied to whole plantsin situ, i.e. in climate rooms and in a greenhouse, to studyplant water relations under these conditions. The spectrometerconsists of a 30 kg permanent magnet system of 0.235 T, modifiedBruker Minispec electronics, and a standard XT pc for spectrometercontrol. Unattended, the system automatically measures the xylemsap stream and tissue water content in a well-defined sectionof the plant stem. Because of the small size of the magnet thesemeasurements can be made at different positions on the plantstem or on different plants in succession. Magnetic field driftdue to the varying climates that occur in greenhouses was correctedby field locking. Results are presented for a single plant ina climate room, demonstrating a method for the study of plantwater hydraulics. In addition, a single plant in a greenhousecrop was measured, and the NMR results were compared with thewater uptake and transpiration rates under (natural) variationof light intensity and relative humidity, demonstrating thereliability of the portable NMR under realistic greenhouse conditions.Finally, the application for the measurement of the root wateruptake efficiency is demonstrated for a number of grafted cucumberplants under constant climatic conditions in a phytotron. Key words: Portable NMR system, plant water balance sensor, greenhouse applications, flow, water content     

In situ electrosynthetic bacterial growth using electricity generated by a deep-sea hydrothermal vent

Electroautotrophic microorganisms have attracted great attention since they exhibit a new type of primary production. Here, in situ electrochemical cultivation was conducted using the naturally occurring electromotive forces at a deep-sea hydrothermal vent. The voltage and current generation originating from the resulting microbial activity was observed for 12 days of deployment, with fluctuation in response to tidal cycles. A novel bacterium belonging to the genus Thiomicrorhabdus dominated the microbial community specifically enriched on the cathode. Metagenomic analysis provided the draft genome of the bacterium and the gene repertoire indicated that the bacterium has the potential for thio-autotrophic growth, which is a typical physiological feature of the members of the genus, while the bacterium had a unique gene cluster encoding multi-heme cytochrome c proteins responsible for extracellular electron transfer. Herein, we propose this bacterium as a new species, specifically enriched during electricity generation, as ‘Candidatus Thiomicrorhabdus electrophagus’. This finding suggests the natural occurrence of electrosynthetic microbial populations using the geoelectricity in deep-sea hydrothermal environments.Subject terms: Soil microbiology, Biogeochemistry     

Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains

Evaluating chondrocytes in situ to document the effectiveness of cartilage preservation techniques has proven exceedingly difficult. This study was conducted to determine the effectiveness of WST-1 on porcine chondrocytes in situ after cooling to −10°C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10 mm in diameter) were harvested from sexually mature pigs within 24 h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at −10°C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at −10°C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 μm slices. Representative images were digitized and green and red pixel numbers determined the percent recovery of intact cells. Mitochondrial activity (WST-1) was determined using 20 slices of 70 μm thickness per sample to obtain reliable readings using a spectrophotometer at 450 nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144 h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7 days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48 h. MIS was most accurate when “absolute” cell recovery was compared to original controls, taking into account cell density. In conclusion, WST-1 can track metabolic activity of chondrocytes in situ over time but “absolute” cell recovery determined by MISs after 48 h incubation may be the most accurate determination of the number of live chondrocytes in situ.     

An in situ method for determining bacterial survival on food preparation surfaces using a redox dye

A simple method is described for the direct enumeration of viable bacteria dried on test surfaces. Inoculated surfaces were overlayed with agar and after incubation nitroblue tetrazolium solution (pale yellow) was used to stain colonies (purple) at the agar-test surface interface. Stained colonies could be readily detected and counted even against the opaque background of ceramic tile or stainless steel or when present within opaque films of milk or serum. Recovery of bacteria by this method was approximately fivefold greater than using a conventional swabbing procedure. The method was used to demonstrate the marked effect of the composition of the suspension fluid, in which bacteria were dried, and the length of surface exposure upon bacterial survival.     

In situ 19F NMR studies of an E. coli membrane protein

In this report, (19)F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl-phenylalanine ((19) F-tfmF). Site-specific (19)F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that (19)F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while (19)F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.     

In situ molecular level studies on membrane related peptides and proteins in real time using sum frequency generation vibrational spectroscopy

Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study the molecular structures of surfaces and interfaces in different chemical environments. This review summarizes recent SFG studies on hybrid bilayer membranes and substrate-supported lipid monolayers and bilayers, the interaction between peptides/proteins and lipid monolayers/bilayers, and bilayer perturbation induced by peptides/proteins. To demonstrate the ability of SFG to determine the orientations of various secondary structures, studies on the interactions between different peptides/proteins (melittin, G proteins, alamethicin, and tachyplesin I) and lipid bilayers are discussed. Molecular level details revealed by SFG in these studies show that SFG can provide a unique understanding on the interactions between a lipid monolayer/bilayer and peptides/proteins in real time, in situ and without any exogenous labeling.     

A simple method for conducting laboratory treatability studies to assess potential for In situ bioremediation

Summary A simple, sealed flask system for conducting laboratory treatability studies to assess potential for in situ bioremediation was developed, evaluated, and compared to treatability studies which were open to the atmosphere. The treatability studies were conducted in 40 ml volatile organic analysis (VOA) vials fitted with Teflon septum caps. Contaminant loss was significantly less in the sealed-flasks relative to systems open to the atmosphere.     

In situ measurement of the electrical potential across the lysosomal membrane using FRET

The progressive acidification of the endocytic pathway is generated by H(+) pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the proton-motive force (pmf), diminishing the pH gradient proportionally. Validation of this notion requires direct measurement of the electrical potential that develops across the endosomal/lysosomal membrane. To date, the measurement of lysosomal membrane potential (ψ(φ) ) in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that individual organelles cannot be discerned when using potentiometric fluorescent dyes. Here, we describe a noninvasive procedure to estimate ψ(φ) in intact cells, based on fluorescence resonance energy transfer (FRET). At steady state, ψ(φ) averaged 19 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A (CcA). ψ(φ) was considerably increased by alkalinization of the lysosome lumen by NH(4) Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of ψ(φ) to pmf is relatively small. Our method should enable systematic studies of endosomal/lysosomal potential.     

In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry

Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of model and cell membranes.     

Performance of a tubular microporous membrane filter used for in situ sampling of mammalian cell culture medium

Summary The transmembrane permeation rate of the medium and the membrane transmission of low- and high-molecular medium components were evaluated for a microfiltration sampling system as a function of the operation time.     

In situ studies with membrane diffusion chambers of antibiotic resistance transfer in Escherichia coli.

Coliform bacteria were isolated from raw sewage and sewage effluent-receiving waters and tested for their antibiotic susceptibility patterns and their ability to transfer antibiotic resistance to Escherichia coli K-12 C600. An environmental isolate of E. coli (MA527) capable of transferring antibiotic resistance to C600 was mated, both in vitro and in situ, with an antibiotic-sensitive E. coli environmental isolate (MA728). In situ matings were conducted in modified membrane diffusion chambers, in the degritter tank at the Grant Street (Melbourne, Fla.) sewage treatment facility, and in the sewage effluent-receiving waters in Melbourne, Fla. The transfer frequencies in situ were 3.2 x 10(-5) to 1.0 x 10(-6), compared with 1.6 x 10(-4) to 4.4 x 10(-5) observed in vitro. Transfer was shown to occur in raw sewage but was not detected in the effluent-receiving waters. The presence of a 60-megadalton plasmid species in both donor and transconjugants, but not in the recipients, provided physical evidence for the transfer of antibiotic resistance in situ.     

In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

In situ identification of bacterial species in marine microfouling films by using an immunofluorescence technique

An immunofluorescence technique was developed for the in situ identification of specific bacteria in marine microfouling films. Microorganisms adherent to glass plates after 30 days of immersion in a synthetic seawater system were cultured and classified by biochemical tests, flagellar arrangement, and the API 20E system. All isolates were gram-negative aerobic or facultative motile rods, predominantly Pseudomonas spp. Rabbit antisera to the five dominant organisms including Achromobacter spp., Comamonas terrigena, P. putrefaciens, a yellow-pigmented Pseudomonas sp., and Vibrio alginolyticus were prepared. These antisera were shown to be species specific in indirect immunofluorescence assays against a battery of 26 marine isolates from 14 bacterial species, with the exception of antisera to the Pseudomonas spp, which cross-reacted with each other but not with test bacteria of other genera. These immunofluorescent reagents enabled the in situ identification of all five bacterial species in microfouling films. Low-surface-energy test plates had smaller numbers of adherent bacteria in microfouling films than medium-surface-energy test plates, suggesting that the degree of microfouling may be influenced by the surface energy. In addition, the reagents could identify up to 39% of the attached bacteria in microfouling films spontaneously formed on steel plates in flow cells deployed in different areas of the Atlantic Ocean. The microbial composition of the ocean-formed films varied with the geographical area of their formation. The present results indicate that immunofluorescence techniques may provide a rapid and reliable means to identify, in situ, specific bacteria in marine microfouling films.     

In situ protein folding and activation in bacterial inclusion bodies

Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.     

In situ behavioural studies on echinoderm aggregations

Summary 1. On the west coast of Ireland, a number of echinoderm species have been found to exhibit extreme aggregation. Population densities for some of these animals are amongst the highest on record.2. Detailed studies have been carried out on aggregations of the holothurianPseudocucumis mixta Östergren. These provided information on habitat preference, mode of burrowing, method of feeding and the animal's sensitivity.3. In situ observation established the existence of a diurnal feeding rhythm which may be primarily controlled by light.4. The manner of defecation and the nature of the faeces would appear to rule out self-fouling by the aggregation.

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In-situ-Verhaltensstudien an Aggregationen von Echinodermen. Teil I.Pseudocucumis mixta

Kurzfassung An der Westküste Irlands wurden extrem dichte Aggregationen einiger Echinodermen-Arten beobachtet. Die für die Populationsdichten einiger dieser Stachelhäuter ermittelten Werte gehören zu den höchsten, die bisher bekannt geworden sind. Die Untersuchungen konzentrieren sich auf die Lebensweise der HolothuriePseudocucumis mixta Östergren unter besonderer Berücksichtigung der Habitatpräferenz, der Art des Einbohrens in den Untergrund, der Nahrungsweise, der Defäkation und des Sinneslebens. In-situ-Studien zeigten, daß ein offensichtlich vom Tag-Nacht-Wechsel gesteuerter Freßrhythmus vorliegt.