Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations.

A simplified procedure is presented for releasing and concentrating indigenous microbial cells from soil for viewing by transmission electron microscopy as thin sections or replicas of frozen-etched preparations. This procedure is compared with two others reported earlier, and their relative merits are discussed as concerns the choice of procedure for the cellular information desired from the soil. Freeze-etching showed that the cell types and size distributions for cells which have been released and concentrated from soil are in general agreement with those for cells in a crude soil slurry in which no attempt to release and concentrate cells was made. Microcolonies were present both in the crude slurry and in the discard soil debris centrifugation pellets from the cell release and concentration procedures. In contrast to the historic assumptions, these microcolonies, as well as some individual cells embedded in soil debris could not be broken up and (or) dislodged so that they would be washed from the soil. The relative numbers of these cells remaining with the soil debris, however, could not be quantitated in the present study.     

Method to concentrate protein solutions based on dialysis-freezing-centrifugation: enzyme applications

One of the recurrent methodological problems in preparative biochemical work is the concentration of dilute protein solutions, including culture supernatants resulting from biotechnological processes. A procedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach includes a new device that facilitates the sample freezing and the subsequent solute elution from the frozen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fructan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression system. The optimal centrifugation speed, time, and temperature were 6150 g, 20 min, and 4 °C, respectively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT, whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure allowed concentrating proteins efficiently without affecting their enzymatic activity.     

Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences.

Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities. By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids. Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new "double" polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences. This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.     

Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences.

Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities. By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids. Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new "double" polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences. This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.     

An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction

Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Evaluation of methods for extraction of bacteria from soil

Abstract Several methods for dispersion of soil were tested for possible use in procedures for extraction of bacteria. Physical cell damage on cells and efficiency in extraction of indigenous cells from soil, were investigated. Cell damage by the dispersion methods was investigated by measuring the physical cell integrity and viability of pure cultures of Escherichia coli and Bacillus subtilis , as well as soil bacteria extracted from soil, when dispersed in slurries of γ-sterilized soil. Separation of bacteria and soil particles on the basis of buoyant density was conducted with the nonionic density gradient medium Nycodenz. When slurries of γ-sterilized soil with added pure cultured cells were centrifuged (10000 × g ) over cushions of Nycodenz (1.3 g ml⁻¹), practically all the added cells were recovered in a layer on top of the cushion. This proves that a reversible attachment and cosedimentation is not an important phenomenon in this procedure. The efficiency of the different dispersion methods for the extraction of indigenous soil bacteria, was assessed after separation of dislodged and attached soil bacteria. This separation was done either on the basis of sedimentation rate by low speed centrifugation, or buoyant density by Nycodenz density gradient centrifugation. The physical dispersion by ultrasonic treatment and chemical dispersion by the use of a chelating agent together with a detergent, were inferior to physical dispersion either by Waring blender (for large volumes) or a rotating rubber pestle treatment (for smaller volumes). The physical dispersion did not appear to be destructive to the cells tested.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton.

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.     

Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20–30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 m³) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 m³) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.     

Patterns in the immunoenzyme analysis of bacterial cells

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.     

Modulation by centrifugation of cell susceptibility to chlamydial infection.

Enhancement of chlamydial infection of cell monolayers by centrifugation was shown to depend on induced cell surface changes. Evidence for this came from analysis of two forms of organism attachment which take place during centrifugation. In 'productive binding', organisms attached to cells and then entered and infected them. In 'unproductive binding', organisms became attached to cells but were not ingested. These organisms could be stripped from the cells by treatment with trypsin and could then infect fresh monolayers. Measurement of attachment kinetics during centrifugation showed that cells passed through three different susceptibility states. Only productive binding occurred in the first 20 min; cells then entered a refractory state during which no attachment took place At about 45 min, attachment recommenced but this allowed only unproductive binding. Induced movement of cell surface structures may enhance infection by promoting specific or non-specific interactions. Failure of ingestion may result from insufficient cell 'receptors' for circumferential binding of the whole chlamydial surface so that engulfment cannot take place.     

Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Community level physiological profile of soil bacteria unaffected by extraction method.

Extraction and purification of bacteria from soil by the Nycodenz gradient centrifugation procedure described by Bakken and Lindahl (1995; Recovery of bacterial cells from soil. In: van Elsas, J.D., Trevors, J.T. (Eds.), Nucleic Acids in the Environment: Methods and Applications. Springer Verlag, Berlin, pp. 9-27) were compared to soil slurry extractions. Bacterial communities from four different soils were described by the bacterial abundance, CTC-reducing capacity, culturability and the community level physiological profiles (CLPP) in BIOLOG GN plates. A significant loss of both total and culturable number of bacteria g(-1) soil dry weight were found after extraction and purification of cells. The origin of soil influenced the yield of cells and a difference between the four soils and an interaction between the soils and extraction procedure were found. The culturability and the CLPP were different between the four soils but were unaffected by the extraction procedure. The bacterial community obtained after extraction and purification thus represented the same fraction of the indigenous bacterial community.     

Simple method to demonstrate radiation-inducible radiation resistance in microbial cells.

A simple method for detection of radiation-inducible radiation resistance was developed by irradiating aliquots (0.01 ml) of cell suspension on agar plates. Part of each experimental plate was subjected to an induction treatment, and subsequent radiation resistance was compared with that of untreated cells on the same plate. The UV radiation resistance of a Micrococcus sp. was increased approximately 1.6 times by an induction treatment. This simple procedure of irradiating cells in a "fixed" position on agar avoided washing, centrifugation, and cell enumeration required in traditional methods.     

Separation and Purification of Bacteria from Soil

Bacteria were released and separated from soil by a simple blending-centrifugation procedure. The percent yield of bacterial cells (microscopic counts) in the supernatants varied over a wide range depending on the soil type. The superantants contained large amounts of noncellular organic material and clay particles. Further purification of the bacterial cells was obtained by centrifugation in density gradients, whereby the clay particles and part of the organic materials sedimented. A large proportion of the bacteria also sedimented through the density gradient, showing that they had a buoyant density above 1.2 g/ml. Attachment to clay minerals and humic material may account for this apparently high buoyant density. The percent yield of cells was negatively correlated with the clay content of the soils, whereas the purity was positively correlated with it. The cell size distribution and the relative frequency of colony-forming cells were similar in the soil homogenate, the supernatants after blending-centrifugation, and the purified bacterial fraction. In purified bacterial fraction from a clay loam, the microscopically measured biomass could account for 20 to 25% of the total C and 30 to 40% of the total N as cellular C and N. The amount of cellular C and N may be higher, however, owing to an underestimation of the cell diameter during fluorescence. A part of the contamination could be ascribed to extracellular structures as well as partly decayed cells, which were not revealed by fluorescence microscopy.     

Ultrastructure of double minutes from a human tumor cell line

Double minutes (dm) have been isolated from human tumor cells by zonal centrifugation and by differential pelleting of chromosome suspsension. These methods allowed collection of dm in sufficient quantity and purity for visualization with electron microscopy. Ultrastructurally, the chromatin fibers in dm resemble thrance fragments was found. When the two isolation protocols were compared, differential pelleting was shown to increase purity twofold to 85% dm by mass. The differential pelleting procedure enables easy collection of dm in sufficient quantity and purity for chemical analysis.     

Simple method to demonstrate radiation-inducible radiation resistance in microbial cells

A simple method for detection of radiation-inducible radiation resistance was developed by irradiating aliquots (0.01 ml) of cell suspension on agar plates. Part of each experimental plate was subjected to an induction treatment, and subsequent radiation resistance was compared with that of untreated cells on the same plate. The UV radiation resistance of a Micrococcus sp. was increased approximately 1.6 times by an induction treatment. This simple procedure of irradiating cells in a "fixed" position on agar avoided washing, centrifugation, and cell enumeration required in traditional methods.