Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2ⁿ cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.     

Growth kinetics ofPseudomonas fluorescens microcolonies within the hydrodynamic boundary layers of surface microenvironments

Computer-enhanced microscopy (CEM) was used to study the growth kinetics of bacterial microcolonies attached to the wall of a continuous-flow slide culture. Image processing increased effective microscope resolution and quantitated colony growth at 10 min intervals. Three growth parameters were used to determine growth rate: the time required for cell fission, the specific rate of increase in cell number, and the specific rate of increase in cell area. Growth rate was initially constant regardless of colony size, as assumed previously in deriving colonization kinetics. However, at low substrate concentrations growth rate varied depending on laminar flow velocity. Growth was flow-dependent at a glucose concentration of 100 mg/liter and flow-independent at a concentration of 1 g/liter. This indicated that the surface microenvironment became substrate-depleted in the absence of sufficient laminar flow velocities and that glucose rather than oxygen was rate limiting.     

Simplified Automated Image Analysis for Detection and Phenotyping of Mycobacterium tuberculosis on Porous Supports by Monitoring Growing Microcolonies

Background

Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings.

Methods

Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies “computer vision” and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures.

Significance

Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.     

Label-free identification of bacterial microcolonies via elastic scattering

Label‐free microcolony identification via elastic light scattering was investigated for three different genera: Salmonella enterica serovar Montevideo, Listeria monocytogenes F4244, and Escherichia coli DH5α. Microcolonies were defined as bacterial colonies in their late‐lag phase to early‐exponential phase with the diameter range of 100–200 µm. To link biophysical characteristics with corresponding scattering patterns, a phase contrast microscope and a confocal displacement meter were used to measure the colony diameter and its 3D height profile. The results indicated that the growth characteristics of microcolonies were encoded in their morphologies which correlated to the characteristic diffraction patterns. Proposed methodology was able to classify three genera based on comprehensive phenotypic map which incorporated growth speed, ring count, and colony diameter. While the proposed method illustrated the possibility of discriminating microcolonies in their early growth stage, more thorough biophysical understanding is needed to expand the technology to other species. Biotechnol. Bioeng. 2011; 108:637–644. © 2010 Wiley Periodicals, Inc.     

The dual nature of lymphocyte interaction with allogenic stem cells

The interaction of lymphocytes from mouse lymph nodes with allogeneic stem cells was studied using exogenous colony formation inhibition test. Dual nature of the interaction was revealed: great amounts of lymphocytes inhibited, while small amounts stimulated colony formation. This dependence holds true for macro- and microcolonies as well as for erythrocyte and granuloid microcolonies in the bone marrow during fixation on day 8 and 11 after cell mixture transplantation.     

The effect of sodium chloride concentration and pH on the growth of Salmonella typhimurium colonies on solid medium

The growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium) was followed. Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of [NaCl] (0.5–3.5% (w/v)) and pH (7.0–5.0) on colony growth at 30°C was observed; colonies were point inoculated from serial dilutions. Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony. Overall, both increasing the [NaCl] and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth. Initial specific growth rate (μ) ranged from 0.73 to 0.87 h⁻¹. Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation. A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.     

Colony growth of Streptomyces coelicolor A3(2) under conditions of continuous nutrient supply

Colony growth of Streptomyces coelicolor A3(2) was studied on a cellophane membrane beneath which was passed a continuous supply of liquid medium. Colony development and differentiation occurred normally but hyphal extension rates and colony radial growth rates were reduced and branch formation was increased in comparison with colonies grown on the same medium solidified with agar. These changes are thought to result from continuous removal of staling compounds which would otherwise suppress branching at the colony margin. Glucose concentrations in the range of 0–1 g · l⁻¹ had little effect on radial growth and branching except at a concentration of 1 g glucose · l⁻¹, at which branching at the colony margin was suppressed. This concentration of glucose did not permit continued growth on solid medium.     

Chemical and Physical Factors Influencing Growth of Phytophthora parasitica var. nicotianae on Vegetable Oils

Factors influencing the growth of Phytophthora parasition var. nicotiona on vegetable oil liquid media were studied. This fungus grew poorly on oleic acid, triolein, and tristearin. Cholesterol and α-tocopherol reversed the toxicity of oleic acid. Growth was often stimulated by addition of mineral oil to a glucose medium, and sterols dissolved in mineral oil frequently stimulated grwoth. Shaking produced a 5-fold or more increase in growth on either glucose or cottonseed oil media. Cholesterol (20 μ/ml further increased growth on glucose meidum in shake culture by at least 100%. Growth was usually better at pH 6.7 than at 5.4 or 7.7. Growth in shake culture on a glucose medium supplemented with cholesterol and α-tocopherol approached that occuring on vegetable oil media.     

A critical stage in the formation of acid mine drainage: Colonization of pyrite by Acidithiobacillus ferrooxidans under pH-neutral conditions

A dominant Acidithiobacillus ferrooxidans ssp. was isolated from the supergene copper deposit in Morenci, Arizona, USA. Washed bacterial suspensions (10⁸ MPN per treatment), in pH‐neutral buffer, were inoculated onto pyrite cubes for 24 h. Heterogeneous bacterial absorption onto the pyrite removed approximately 90% of the viable bacteria from the inoculum. At T = 0, the bacteria were observed primarily in regions enriched in phosphorus. Over 30 days, the bacterial population on the pyrite cubes increased from 1.3 × 10⁷ to 2.9 × 10⁸ bacteria cm^?2. During this growth stage, low levels of thiobacilli (228 ± 167 MPN mL^?1) were also recovered from the fluid phase; however, this population decreased to zero within 30 days. Growth on pyrite occurred as micrometre‐scale planar microcolonies, a biofilm, coating the mineral surfaces. These microcolonies possessed viable thiobacilli, even after 4 months at ‘circumneutral pH’. Imaging the pyrite cubes using SEM‐EDS and scanning force microscopy demonstrated that the thiobacilli grew as iron oxy‐hydroxide‐cemented cells, leading to the formation of mineralized microcolonies. Removing the iron oxy‐hydroxides with oxalic acid did not dislodge the bacteria, demonstrating that the secondary minerals were not responsible for ‘gluing’ the bacteria to the pyrite surface. Removing organic material, i.e. the cells, by an oxygen plasma treatment revealed the presence of corrosion pits the size and shape of bacteria. Because of the inherent geochemical constraints on pyrite oxidation at neutral pH, the colonization of pyrite under circumneutral pH conditions must be facilitated by the development of an acidic nanoenvironment between the bacteria and the pyrite mineral surface.     

Method for Assessment of Viability and Morphological Changes of Bacteria in the Early Stage of Colony Formation on a Simulated Natural Environment

A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.     

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.     

Influence of Structural Properties and Kinetic Constraints on Bacillus cereus Growth

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter⁻¹. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.     

Localization of Active Microorganisms in Cheese by Autoradiography

By using an autoradiographic technique, one can follow, during ripening of a cheese, the distribution, size, and metabolic activity of microcolonies. Fragments of cheese were labeled with [³H]leucine, fixed, and mounted in epoxy resin. After exposure and development, sections were examined by optical microscopy. In Camembert cheese, bacterial microcolonies synthesized protein rapidly during the beginning of the ripening process. At the end of the ripening process, active bacterial clusters were scarcer and of two types: (i) large microcolonies with reduced labeling, and (ii) microcolonies having the same size as those observed at the beginning of the ripening process, but with slight or no labeling.     

Protists with different feeding modes change biofilm morphology

The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders), Spumella , and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder Dexiostoma campylum did not alter biofilm volume and porosity but stimulated the formation of larger microcolonies compared with ungrazed biofilms. In contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the biofilm surface, leading to smaller microcolonies and lower maximal and basal layer thickness compared with ungrazed biofilms. Microcolony formation was not stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped bacteria from the outer surface leading to mushroom-shaped microcolonies. In the presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times lower compared with ungrazed biofilms. However, the biofilm porosity and the ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times higher, respectively. Thus, exchange of nutrients and gases between the biofilm and its surrounding fluid should also be improved in deeper biofilm layers, hence accelerating microbial growth.     

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

Regulation of Bacterial Growth Rates by Dissolved Organic Carbon and Temperature in the Equatorial Pacific Ocean

Abstract The effect of dissolved organic matter (DOM) and temperature on bacterial production was examined in the equatorial Pacific Ocean. Addition of glucose, glucose plus ammonium, or free amino acids stimulated bacterial production ([³H]thymidine incorporation), whereas changes in bacterial abundance were either negligible or much less than changes in bacterial production. The average bacterial growth rate also greatly increased following DOM additions, whereas in contrast, addition of ammonium alone never affected production, bacterial abundance, or growth rates. Since the large glucose effect was not observed in previous studies of cold oceanic waters, several experiments were conducted to examine DOM-temperature interactions. These experiments suggest that bacteria respond more quickly and to a greater extent to DOM additions at higher temperatures, which may explain apparently conflicting results from previous studies. We also examined how temperate affects the kinetic parameters of sugar uptake. Maximum uptake rates (V_max) of glucose and mannose increased with temperature (Q₁₀= 2.4), although the half-saturation constant (K_m) was unaffected; K_m+ S was roughly equal to glucose concentrations (S) measured by a high pressure liquid chromographic technique. Bacterial production and growth rates appear to be limited by DOM in the equatorial Pacific, and thus bacterial production follows primary production over large spatial and temporal scales in this oceanic regime, as has been observed in other aquatic systems. Although temperature may not limit bacterial growth rates in the equatorial Pacific and similar warm waters, it could still affect how bacteria respond to changes in DOM supply and help set steady-state DOM concentrations. Received: 26 July 1995; Revised: 19 January 1996     

Wheat root colonization by Azospirillum brasilense strains with different motility

Migration of associative bacteria Azospirillum brasilense in semisolid media is performed mainly by swarming (Swa⁺ phenotype), which depends on the flagellar functioning and intercellular contacts. Non-swarming mutants of A. brasilense Sp245 lacking a polar flagellum migrate in semisolid media with microcolony formation using a unrevealed mechanism (Gri⁺ phenotype). The study of wheat root colonization dynamics demonstrated that A. brasilense Sp245 Gri⁺ mutants exhibited lower capacity for wheat root adsorption. However, after “anchoring” has occurred, both A. brasilense Sp245 and its Swa-Gri⁺ mutants colonized the growing roots with virtually the same efficiency. All strains under study formed microcolonies on the surface of roots, stimulated root branching, and exhibited changes in the composition of protein antigens exposed on the bacterial cell surface. Indirect evidence was obtained for enhanced production of genus-specific protein antigens in the process of A. brasilense Sp245 adaptation to growth on plant roots.     

Numbers,diversity, and morphological characteristics of aerobic,chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina

The aerobic, chemoheterotrophic bacteria indigenous to deep aquifers and other subsurface sediments (depths to 265 m) at a site in South Carolina were characterized by direct microscopy, enumeration of viable cells, analysis of colony morphologies on plates, and analysis of cell morphologies of isolated strains. Substantial numbers of viable bacteria (10^5‐10⁸/g) were present in all transmissive, aquifer sediments, and their numbers did not decrease with depth. Fewer bacteria (<10³/g) were detected in nontransmissive, confining layers. The highest viable counts were obtained on dilute media, but 10–50% of the bacteria in most aquifer sediments also grew rapidly on concentrated, nutrient‐rich media (indicating a high degree of metabolic flexibility). Most of the bacteria were mesophilic; relatively few psychrophiles or thermophiles were detected (<10³/g; in many cases, none). The bacterial flora was diverse (11–62 distinct colony types on enumeration plates of most aquifer sediments). Diversity did not decrease with depth, but the composition of the microflora (based on colony analysis) varied extensively from one geological formation to another. Almost 95% of the platable colonies that grew on enumeration plates contained nonstreptomycete bacteria, more than 80% of which were gram‐negative rods. Light microscopy of films released from aquifer sediments by flotation revealed the presence of dividing cells and microcolonies, thus implying that the in situ deep aquifer microflora was more metabolically active than that seen previously in shallow aquifers.     

Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

Motility in liquid and semisolid media of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> associative rhizobacteria differing in exopolysaccharide yield and properties

Paenibacillus polymyxa rhizobacteria associate with a wide range of plants and promote plant growth and development. These bacteria produce exopolysaccharides, which are very important for P. polymyxa adaptation to changing environmental conditions. In this study, five P. polymyxa strains differing in exopolysaccharide yield and properties (CCM 1465, CCM 1460, CCM 1459^T, 88A, and 92) were investigated for motility in a liquid and a semisolid nutrient medium with either glucose or sucrose as the carbon source. In the liquid medium, all strains except CCM 1460 were motile and swam by peritrichous flagella. After being stab inoculated into the semisolid (0.4% agar) medium, all strains except CCM 1460 switched to collective swarming and formed concentric macrocolonies with different diameters, depending on the strain and the carbon source used. On the semisolid medium containing the vital dye Congo red, P. polymyxa adsorbed the dye, forming stained colonies. No changes in colony morphology were observed. At 37.5 μg ml^?1 of Congo red, swarming was strongly suppressed: the swarming ring diameters of strains 92, CCM 1459^T, 88A, and CCM 1465 ranged from 12.4 to 17% (glucose) and from 9.5 to 16.0% (sucrose) of the colony diameters obtained from bacterial growth without the dye. The results suggest that the speed of collective migration of P. polymyxa on agarized media may be affected, among other factors, by the yield and physicochemical properties of the bacterial exopolysaccharides. Further, the results suggest that Congo red influences the collective migration speed of P. polymyxa by forming a complex with the bacteria’s carbohydrate polymers – an event that alters bacterial surface structure and affects intercellular interactions.