Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

Behavior of bacterial stream populations within the hydrodynamic boundary layers of surface microenvironments

Phase and computer-enhanced microscopy were used to observe the surface microenvironment of continuous-flow slide cultures during microbial colonization and to document the diversity of bacterial colonization maneuvers among natural stream populations. Surface colonization involved 4 discrete types of cell movement, which were designated as packing, spreading, shedding, and rolling maneuvers. Each maneuver appeared to be associated with a specific species population within the community. The packing maneuver resulted in the formation of a monolayer of contiguous cells, while spreading maneuvers resulted in a monolayer of adjacent cells. During the shedding maneuver, cells attached perpendicular to the surface and the daughter cells were released. The rate of growth of new daughter cells gradually decreased as the attached mother cell aged. During the rolling maneuver, cells were loosely attached and continuously somersaulted across the surface as they grew and divided. Only those populations with a packing maneuver conformed fully to the assumptions of kinetics used previously to calculate growth and attachment rates from cell number and distribution. Consequently, these kinetics are not applicable to stream communities unless fluorescent antisera are used to study specific species populations within natural communities. Virtually all of the cells that attached to the surface were viable and underwent cell division. The abundance of unicells on surfaces incubated in situ was thus primarily the consequence of bacterial colonization behavior (shedding and spreading maneuvers) rather than the adhesion of dead or moribund cells.     

The surface growth and activity ofNitrobacter

The effect of surface attachment on oxidation of nitrite to nitrate byNitrobacter was studied in batch culture, on glass coverslips, and in continuous culture on glass beads and anion exchange resin beads in an air-lift column fermenter. In batch culture, the surfaces stimulated specific growth rate, while in continuous culture, activity of attached cells was less than that of freely suspended cells. Nitrate productivity, free cell productivity, and attached cell concentration increased exponentially at the same specific rate, termed the colonization rate, and nitrate productivity was found to be a convenient estimate of biomass concentration. Permanent attachment was mediated by production of slime material. Surface growth resulted in multiple steady states and the ability to respond quickly to changes in dilution rate. The air-lift column fermenter system provided a convenient system for the study of growth and activity of attached cells and was most suitable when using ion exchange resins as a substratum for attachment.     

Ultrastructure of the in situ adherence of Mobiluncus to vaginal epithelial cells

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacteria cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.     

Chemostat and in‐situ colonization kinetics of thermothrix thiopara on calcite and pyrite surfaces

Growth and attachment rates of Thermothrix thiopara on calcite and pyrite were quantitated in a thiosulfate‐limited chemostat and in the thermal spring where the organism is found in nature. Surface growth rates were quantitated by using the surface colonization and exponential growth equations. These two models were compared as means of determining surface growth rates. In the chemostat, T. thiopara cells colonizing calcite and pyrite surfaces grew at approximately one‐third the rate of suspended cells. However, T. thiopara attached to pyrite faster than to calcite. In the thermal spring, growth and attachment rates were equal on calcite and pyrite. It was concluded that the exponential growth equation overestimates in‐situ surface growth rates and that T. thiopara grows more slowly when colonizing mineral surfaces than when growing in suspension. Lower growth rates on surfaces may be due to a reduced cell surface area for nutrient uptake or an increased specific maintenance rate.     

Evaluation of a proposed surface colonization equation usingThermothrix thiopara as a model organism

The colonization equation shown below was evaluated usingThermothrix thiopara as a model organism. $$N = (A/\mu )e^{\mu t} - A/\mu $$ where: N=number of cells on surface (cells field^?1); A = attachment rate (cells field^?1 h^?1); M=specific growth rate (h^?1); t=incubation period (h). Previous studies of microbial surface colonization consider attachment and growth independently. However, the proposed colonization equation integrates the effects of simultaneous attachment and growth. Using this equation, the specific growth rate ofT. thiopara was found to be 0.38±0.3 h^?1 during in situ colonization. Estimates ofμ were independent of incubation period after 4 h (2 generations). Shorter incubations were inadequate to produce sufficient microcolonies for accurate determination of specific growth rate. Empirical data for the time course of colonization fell within the 95% confidence interval of predicted values. The attachment rate, although assumed to be constant, was found to continuously increase with time. This increase may have been an artifact due to the continuous deposition of travertine on the surface, or may indicate the need for a function to replace A in the colonization equation. Using the exponential growth equation, the progeny of cells that attach during incubation are considered to be progeny of cells that attach initially. This erroneously inflated the growth rate by 55%.     

A mathematical model for the growth of bacterial microcolonies on marine sediment

Counts of bacterial microcolonies attached to deep-sea sediment particles showed 4-, 8-, 16-, and 32-celled microcolonies to be very rare. This was investigated with a mathematical model in which microcolonies grew from single cells at a constant growth rate (), detached from particles at constant rate (), and reattached as single cells. Terms for attachment of foreign bacteria (a) and death of single cells (d) were also included. The best method of fitting the model to the microcolony counts was a weighted least-squares approach by which(0.83 hour^–1) was estimated to be about 20 times greater than(0.038 hour^–1). This showed that the bacteria were very mobile between sediment particles and this mobility was explained in terms of attachment by reversible sorption. The implications of the results for the frequency of dividing cell method for estimating growth rates of sediment bacteria are discussed. The ratio of and was found to be very robust both in terms of the errors associated with the microcolony counts and the range of microcolony sizes used to obtain the solution.     

A computer simulation of surface microcolony formation during microbial colonization

Several models of microbial surface colonization have been devised to quantitate growth and attachment rates on surfaces. One of these, the surface growth rate equation, is based on the assumption that the number of microcolonies of a given size (C_i) reaches a constant value (C_max) that is equal to the attachment rate (A) divided by the specific growth rate (Μ). In this study, a computer simulation was used to determine the time required to reach C_max. It was shown that C_i approaches C_max asymptotically. The time required is dependent solely upon the growth rate and size of microcolonies. The number of one-celled microcolonies reaches 95% of C_max after 4.3 generations. At low growth rates, a relatively long incubation period is required. Alternate methods that shorten the incubation time are considered.     

Detachment ofPseudomonas fluorescens from biofilms on glass surfaces in response to nutrient stress

The effects of glucose and nitrogen depletion on the colonization of glass Petri plates byPseudomonas fluorescens were studied in batch culture. Colonization of the surfaces was initiated before colonization of the bulk phase, and biofilm formation was observed. This resulted in an apparent lag in the batch growth curve for the cell suspension. The lag phase was an artifact caused by the partitioning of cells between the bulk and solid phase of the culture and was not due to a reduction in the growth rate of unattached cells. The specific growth rate of the unattached cells (0.331 hour^–1) was almost twice that determined for the total population (0.171 hour^–1). Consequently the growth rate of biofilm-forming bacteria cannot be determined in batch culture unless the growth of both attached and unattached cells is monitored, and batch growth curves may contain artifacts due to the formation and dispersion of biofilms. The depletion of either glucose or nitrogen led to the active detachment of cells from the biofilm. An increase in the hydrophobicity of unattached cells was noted on depletion of carbon. This increase was the result of emigration of cells from the surface into the bulk phase.Paper contribution number 128, Centre de Rechcrches Alimentaires de St Hyacinthe.     

LapF,the second largest Pseudomonas putida protein,contributes to plant root colonization and determines biofilm architecture

We have investigated the role of LapF, one of the two largest proteins encoded in the genome of Pseudomonas putida KT2440, in bacterial colonization of solid surfaces. LapF is 6310 amino acids long, and is localized on the cell surface. The C‐terminal region of the protein is essential for its secretion, which presumably requires the ABC transporter encoded by an operon (lapHIJ) adjacent to the lapF gene. Although the initial attachment stages are not different between the wild type and a lapF mutant, microcolony formation and subsequent development of a mature biofilm is impaired in the mutant. This is consistent with the expression pattern of lapF; activation of its promoter takes place at late stages of growth and is regulated by the alternative sigma factor RpoS. A lapF mutant is also affected in individual and competitive plant root colonization. In these assays, mixed microcolonies formed by cells of both the wild‐type and the mutant strains could be observed but microcolonies of the mutant alone were not found. These data and the localization of the protein at discrete spots in areas of contact between cells in biofilms suggest that LapF determines the establishment of cell–cell interactions during sessile growth.     

Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot− Pseudomonas fluorescens

Computer-enhanced microscopy (CEM) was used to monitor bacteria colonizing the inner surfaces of a 1×3 mm glass flow cell. Image analysis provided a rapid and reliable means of measuring microcolony count, microcolony area, and cell motility. The kinetics of motile and nonmotilePseudomonas fluorescens surface colonization were compared at flow velocities above (120m sec^–1) and below (8m sec^–1) the strain's maximum motility rate (85m sec^–1). A direct attachment assay confirmed that flagellated cells undergo initial attachment more rapidly than nonflagellated cells at high and low flow. During continuous-flow slide culture, neither the rate of growth nor the timing of recolonization (cell redistribution within surface microenvironments) were influenced by flow rate or motility. However, the amount of reattachment of recolonizing cells was both flow and motility dependent. At 8m sec^–1 flow, motility increased reattachment sixfold, whereas at 120m sec^–1 flow, motility increased reattachment fourfold. The spatial distribution of recolonizing cells was also influenced by motility. Motile cells dispersed over surfaces more uniformly (mean distance to the nearest neighbor was 47.0m) than nonmotile cells (mean distance was 14.2m) allowing uniform biofilm development through more effective redistribution of cells over the surface during recolonization. In addition, motile cell backgrowth (where cells colonize against laminar flow) occurred four times more rapidly than nonmotile cell backgrowth at low flow (where rate of motility exceeded flow), and twice as rapidly at high flow (where flow exceeded the rate of motility). The observed backgrowth of Mot⁺ cells against high flow could only have occurred as the result of motile attachment behavior. These results confirm the importance of motility as a behavioral mechanism in colonization and provides an explanation for enhanced colonization by motile cells in environments lacking concentration gradients necessary for chemotactic behavior.     

Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl xanthate

Ion exchange resins and glass microscope slides were used to investigate factors affecting attachment of nitrifying bacteria to solid surfaces and the effect of attachment on inhibition ofNitrobacter by potassium ethyl xanthate. The ammonium oxidizerNitrosomonas attached preferentially to cation exchange resins while the nitrite oxidizerNitrobacter colonized anion exchange resins more extensively. Colonization was always associated with growth, and the site of substrate (NH₄ ⁺ or NO₂ ^–) adsorption was the major factor in attachment and colonization. The specific growth rate of cells colonizing either ion exchange resin beads or glass surfaces was greater than that of freely suspended cells, butNitrobacter populations colonizing glass surfaces were more sensitive to the inhibitor potassium ethyl xanthate. The findings indicate that surface growth alone does not protect soil nitrifying bacteria from inhibition by potassium ethyl xanthate and explain different patterns of inhibition for ammonium and nitrite oxidizers in the soil.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Studies on relations of bacteria with skin surface of Carassius auratus L. and Poeciloia reticulata

Inocula taken from body surfaces of goldfish and guppy indicated the presence of a variety of aquatic bacteria. However, bacteria were not seen when skin samples were studied by means of histology and scanning electron microscopy. Skin from the body flank was essentially a barren field of Malpighian cell surfaces, interrupted occasionally by goblet cell secretions. Under experimental conditions, Aeromonas hydrophila (Gram-negative, motile rod) attached in large numbers to the dermal surface of artificially made wounds in goldfish, but none was found on adjacent Malpighian cells. Freshly collected skin secretions from goldfish did not influence survival or growth of A. hydrophila in vitro . The study concludes that, under normal conditions, cuticle overlying the Malpighian cells prevents firm attachment and colonization of the skin by aquatic bacteria. It is argued that bacteria isolated by microbiological studies were simply in suspension near the body surface.     

Utilization of surface localized substrate by non-adhesive marine bacteria

Thirty-four marine bacteria were isolated from the eluate of seawater passed through a column of glass beads coated with stearic acid. Irreversible attachment of these isolates to stearic acid-coated glass surfaces ranged from 7.6–100% of the total attached population, with 7 isolates exhibiting less than 10% irreversible adhesion. All 14 isolates tested were able to utilize surface bound¹⁴C-stearic acid, even though some showed mostly reversible adhesion to the surface. More detailed studies were made comparing the reversibly adheringVibrio MH3 with the irreversibly adheringPseudomonas NCMB2021. MH3 cells were readily removed from the surface by a gentle shear force, and a significant degree of¹⁴C-labeling of MH3 cells, but not of NCMB2021 cells, in the bulk phase was observed. The ecological significance of nutrient scavenging at solid surfaces by reversibly attached bacteria is considered.     

Ammonium and attachment of <Emphasis Type="Italic">Rhodopirellula baltica</Emphasis>

A dimorphic life cycle has been described for the planctomycete Rhodopirellula baltica SH1^T, with juvenile motile, free-swimming cells and adult sessile, attached-living cells. However, attachment as a response to environmental factors was not investigated. We studied the response of R. baltica to nitrogen limitation. In batch cultures, ammonium limitation coincided with a dominance of free-swimming cells and a low number of aggregates. Flow cytometry revealed a quantitative shift with increasing ammonium availability, from single cells towards attached cells in large aggregates. During growth of R. baltica on glucose and ammonium in chemostats, an ammonium addition caused a macroscopic change of the growth behaviour, from homogeneous growth in the liquid phase to a biofilm on the borosilicate glass wall of the chemostat vessel. Thus, an ammonium limitation—a carbon to nitrogen supply ratio of 30:1—sustained free-living growth without aggregate formation. A sudden increase in ammonium supply induced sessile growth of R. baltica. These observations reveal a response of Rhodopirellula baltica cells to ammonium: they abandon the free-swimming life, attach to particles and form biofilms.     

An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal

The use of hexamethyldisilazane (HMDS) as a drying agent was investigated in the specimen preparation for scanning electron microscopy (SEM) imaging of bacterial surface colonization on sub-bituminous coal. The ability of microbes to biofragment, ferment and generate methane from coal has sparked interest in the initial attachment and colonization of coal surfaces. HMDS represents an attractive alternative to critical point drying (CPD) in the imaging of cells on coal, negating the need for expensive equipment. Coal is easily fragmented into sub-micron particles, which can be problematic in critical point drying procedures. In this study, both individual and aggregated cells appeared well shaped with minimal occurrence of flattened cells, signifying the suitability of HMDS in cell attachment studies on sub-bituminous coal. In the absence of glucose, microcolonies of short and long cells showed similar positive results using this method. EPS shrinkage found in microcolonies was inevitable, though this enabled observation of points of attachment between cells and with coal, which would be less effective if the EPS was intact. Overall the use of HMDS drying is preferred over the more commonly used CPD method as it is safer, cheaper and more practical.     

Characteristics of attachment and growth of Thiobacillus caldus on sulphide minerals: a chemotactic response to sulphur minerals?

To further our understanding of the ecological role of sulphur-oxidizing microorganisms in the generation of acid mine drainage (AMD), growth and attachment of the chemoautotrophic sulphur-oxidizing bacterium, Thiobacillus caldus , on the sulphide minerals pyrite, marcasite and arsenopyrite was studied. Growth curves were estimated based on total cells detected in the system (in suspension and attached to mineral surfaces). In general, higher cell numbers were detected on surfaces than in suspension. Fluorescent in situ hybridizations to cells on surfaces at mid-log growth confirmed that cells on surfaces were metabolically active. Total cell (both surface and solution phase) generation times on pyrite and marcasite (both FeS₂) were calculated to be ≈ 7 and 6 h respectively. When grown on pyrite (not marcasite), the number of T. caldus cells in the solution phase decreased, while the total number of cells (both surface and solution) increased. Additionally, marcasite supported about three times more total cells (≈ 3 × 10⁹) than pyrite (≈ 8 × 10⁸). This may be attributed to the dissolution rate of marcasite, which is twice that of pyrite. Epifluorescent and scanning electron microscopy (SEM) were used to analyse the cell orientation on surfaces. Results of Fourier transform analysis of fluorescent images confirmed that attachment to all three sulphides occurred in an oriented manner. Results from high-resolution SEM imaging showed that cell orientation coincides with dissolution pit edges and secondary sulphur minerals that develop during dissolution. Preferential colonization of surfaces relative to solution and oriented cell attachment on these sulphide surfaces suggest that T. caldus may chemotactically select the optimal site for chemoautotrophic growth on sulphur (i.e. the mineral surface).     

Derivation of a growth rate equation describing microbial surface colonization

A surface growth rate equation is derived which describes simultaneous growth and attachment during microbial surface colonization. The equation simplifies determination of attachment and growth rate, and does not require a computer program for solution. This rate equation gives the specific growth rate (Μ) as a function of the number of cells on the surface (N), the incubation period (t), and the number of colonies (C_i) containing either one cell, two cells, four cells, etc, as shown below. $$\mu = \frac{{\ln (\frac{N}{{C_i }} + 1)}}{t}$$ The attachment rate (A) is given by the following relationship: $$A = \mu C_i $$ The proposed colonization kinetics are compared with exponential growth kinetics using 3-dimensional computer plots. Colonization kinetics diverged most from exponential kinetics when the growth rate was low or the attachment rate was high. Using these kinetics, it is possible to isolate the effects of growth and attachment on microbial surface colonization.