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1.
The experimental study has assessed a novel membrane bioreactor for mammalian cell culture. In the absence of a gas phase, the key features of cell damage associated with laminar and turbulent flow have been identified. The bioreactor employs a dimpled membrane in order to enhance transverse mixing in a narrow channel, but a fall in viable cell density has been observed at Reynolds numbers above Re = 83. In the laminar flow regime wall shear is the critical mechanism and an accurate calculation of shear rate in a complex channel has been achieved using the Reynolds analogy. Flow generating a wall shear rate in excess of 3000 s(-1) has been shown to cause damage. Power dissipation measurements have been used to distinguish between laminar and turbulent flow and also to predict Kolmogorov eddy lengths. An additional turbulent bulk stress damage mechanism at higher Reynolds numbers (Re > 250) results in a very rapid fall in viable cell density.  相似文献   

2.
Streptococcus mutans plays an important role in the formation of dental plaque. To study biofilm growth on hydroxyapatite (HA) in vitro, a flow system based on a Modified Robbins Device (MRD) and a method for the quantification of the biomass using fluorescent staining with SYTO(R) 9 were developed. The combined approach was used to assess the inhibitory effect of plant extracts on biofilm formation in concentrations below their minimal inhibitory concentrations.  相似文献   

3.
Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.  相似文献   

4.
In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.  相似文献   

5.
A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (PVC) disks immobilized in a modified Robbins device using a seed and feed model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained onin situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.  相似文献   

6.
Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.  相似文献   

7.
The aim of this study was to compare biofilm accumulation and heterotrophic bacterial diversity on three pipe materials-cast iron, medium density polyethylene (MDPE), and unplasticised polyvinyl chloride (uPVC) - using a laboratory model system run over a short period (21 d) and a longer period (7 months). Newly Modified Robbins Devices (nMRD) were run in parallel, each containing 25 discs of one material with cold tap water flowing through the devices at 3 ml min(-1) (Reynolds Number 9.05) for 21 d. The numbers of bacteria on each material increased exponentially between 0 and 11 d when the biofilm viable count remained constant. The mean doubling times of the heterotrophic population on the materials during the exponential phase was 13.2 h for cast iron and 15.6 h for MDPE and uPVC. The same experiment was repeated under different environmental conditions with a lower temperature, higher free chlorine and lower number of organisms ml(-1) of incoming water. The exponential phase lengthened to 16 d but the steady state count remained the same. The mean viable count after 21 d and after 7 months was on average 97% higher on cast iron than on the other materials. Very few different colony types were isolated from each material with the largest number (nine) recovered from cast iron. The numbers of planktonic bacteria in the effluent water leaving each of the nMRDs directly correlated with the numbers in the biofilm phase on each of the materials. In addition the distribution and thickness of the biofilms on the MDPE and uPVC were observed using confocal scanning laser microscopy. In conclusion, MDPE and uPVC support the lowest numbers of bacteria in a steady state biofilm in the short term (21 d) and over a longer term (7 months). The diversity of heterotrophic bacteria was greatest on cast iron.  相似文献   

8.
AIMS: Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. METHODS AND RESULTS: The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. CONCLUSIONS: We were unable to demonstrate that stagnant conditions promoted Legionella colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.  相似文献   

9.
Moderate and severe arterial stenoses can produce highly disturbed flow regions with transitional and or turbulent flow characteristics. Neither laminar flow modeling nor standard two-equation models such as the kappa-epsilon turbulence ones are suitable for this kind of blood flow. In order to analyze the transitional or turbulent flow distal to an arterial stenosis, authors of this study have used the Wilcox low-Re turbulence model. Flow simulations were carried out on stenoses with 50, 75 and 86% reductions in cross-sectional area over a range of physiologically relevant Reynolds numbers. The results obtained with this low-Re turbulence model were compared with experimental measurements and with the results obtained by the standard kappa-epsilon model in terms of velocity profile, vortex length, wall shear stress, wall static pressure, and turbulence intensity. The comparisons show that results predicted by the low-Re model are in good agreement with the experimental measurements. This model accurately predicts the critical Reynolds number at which blood flow becomes transitional or turbulent distal an arterial stenosis. Most interestingly, over the Re range of laminar flow, the vortex length calculated with the low-Re model also closely matches the vortex length predicted by laminar flow modeling. In conclusion, the study strongly suggests that the proposed model is suitable for blood flow studies in certain areas of the arterial tree where both laminar and transitional/turbulent flows coexist.  相似文献   

10.
This study was designed to evaluate the effects of the inorganic electrolytes present in human tear film on the viability and colonization of bacteria to hydrogels. Pseudomonas aeruginosa and Staphylococcus aureus were used in these experiments. A D-value test was performed to investigate any bacteriostatic effect by measuring the reduction of viable test microorganisms over time when exposed to the inorganic electrolyte solution. No D-value was calculable for S. aureus in electrolyte solution whereas a D-value of 8.1 h was obtained for P. aeruginosa in electrolyte solution. The D-value data indicate that staphylococci have a greater survivability potential in a hypertonic environment than do pseudomonads. Bacterial adhesion to high water, ionic hydrogels was studied using the Modified Robbins Device (MRD). The data for P. aeruginosa recovered from the lenses showed an 82% decrease in bacterial counts in electrolyte solution as compared to bacteria incubated in control solution. In contrast there were slight increases in S. aureus counts recovered from the lenses. Journal of Industrial Microbiology & Biotechnology (2000) 25, 17–19. Received 12 August 1999/ Accepted in revised form 05 January 2000  相似文献   

11.
Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 108 CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 102 CFU/cm2) than in a batch system (reaching 107 CFU/cm2), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.  相似文献   

12.
A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·108 cells·cm-2 and the latter 8.2·107 cells·cm-2.Non-common abbreviation NSS Nine salt solution  相似文献   

13.
Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.  相似文献   

14.
Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h?1 in the turbulent flow cell and 1.0 μm h?1 in the laminar flow cell.  相似文献   

15.
Microbial biofilms were formed on coupons with defined coatings in once-through laminar flow fields of controlled bulk-phase composition and shear. Dilute media were utilized to select for biofilm growth. The formation, succession, and stability of the biofilms were monitored with non-destructive on-line methods (fluorescence, bioluminescence, attenuated total reflectance Fourier transform infrared spectrometry [ATR-FTIR] and electrochemical impedance spectroscopy) and by high resolution destructive analysts (viable and direct counts and phospholipid fatty acid signature methods) at the termination of the experiments. Biofilms of reproducible composition can be formed and the order of inoculation of multi-component biofilms affects their composition at harvest. The corrosion rates of mild steel depended on the biofilm composition but not the attached biomass. Examination of biofilms with the scanning vibrating electrode in a microscope field showed effects of heterogeneity in biofilm structure which promoted localized anodic activity. Pseudomonas stains were engineered to contain the lux gene cassette as a "reporter"; and the formation of the exopolymer alginate was shown not to promote attachment of the strain or secondary colonization by Vibrio. Examination of mutants forming different alginate structures showed differential attachment and biofilm structure. Studies of mutants of lipopolysaccharide structure showed differential attachment to substrata. Specific antifouling and fouling-release coatings showed a wide range of attachment and release properties as well as sublethal toxicity.  相似文献   

16.
A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.  相似文献   

17.
Comparative evaluation of biofilm disinfectant efficacy tests   总被引:1,自引:0,他引:1  
Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.  相似文献   

18.
The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.  相似文献   

19.
The combined use of antibiotics with low levels of electrical current has been reported to be more effective in controlling biofilms (the bioelectric effect) than antibiotics alone. An electrical colonisation cell was designed to study the effect of antibiotics on biofilms formed on a dialysis membrane away from the electrode surface. To avoid the electrochemical generation of toxic products,Pseudomonas aeruginosa biofilms were formed in minimal salts medium that excluded chloride-containing compounds. Under these conditions, electrical currents of up to 20 mA cm–2 did not prevent biofilm formation or have any detrimental effect on an established biofilm. Tobramycin alone at concentrations of 10 g ml–1 did not affect the biofilm, but were significantly enhanced by 9 mA cm–2. The effect of tobramycin concentrations of 25 g ml–1 were enhanced by a 15 mA cm–2 electrical current. In both cases higher levels of electrical current, up to 20 mA cm–2, did not further enhance the effect of the antibiotic. The possible mechanisms of action of the bioelectric effect have been reported to involve electrophoresis, iontophoresis and electroporesis, thus overcoming the biofilm biomass and cell wall barriers. Our results suggest that other factors may also be important, such as the metabolic activity and growth rate of the bacteria. Such factors may be critical in maximising antibiotic efficacy.  相似文献   

20.
In the abdominal segment of the human aorta under a patient's average resting conditions, pulsatile blood flow exhibits complex laminar patterns with secondary flows induced by adjacent branches and irregular vessel geometries. The flow dynamics becomes more complex when there is a pathological condition that causes changes in the normal structural composition of the vessel wall, for example, in the presence of an aneurysm. This work examines the hemodynamics of pulsatile blood flow in hypothetical three-dimensional models of abdominal aortic aneurysms (AAAs). Numerical predictions of blood flow patterns and hemodynamic stresses in AAAs are performed in single-aneurysm, asymmetric, rigid wall models using the finite element method. We characterize pulsatile flow dynamics in AAAs for average resting conditions by means of identifying regions of disturbed flow and quantifying the disturbance by evaluating flow-induced stresses at the aneurysm wall, specifically wall pressure and wall shear stress. Physiologically realistic abdominal aortic blood flow is simulated under pulsatile conditions for the range of time-average Reynolds numbers 50 < or = Rem < or = 300, corresponding to a range of peak Reynolds numbers 262.5 < or = Repeak < or = 1575. The vortex dynamics induced by pulsatile flow in AAAs is depicted by a sequence of four different flow phases in one period of the cardiac pulse. Peak wall shear stress and peak wall pressure are reported as a function of the time-average Reynolds number and aneurysm asymmetry. The effect of asymmetry in hypothetically shaped AAAs is to increase the maximum wall shear stress at peak flow and to induce the appearance of secondary flows in late diastole.  相似文献   

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