Surface thermodynamics of bacterial adhesion.

The adhesion of five strains of bacteria, i.e., Staphylococcus aureus (strain 049), Staphylococcus epidermidis (strain 047), Escherichia coli (strains 055 and 2627), and Listeria monocytogenes, to various polymeric surfaces was studied. The design of the experimental protocol was dictated by thermodynamic considerations. From the thermodynamic model for the adhesion of small particles from a suspension onto a solid substratum, it follows that the extent of adhesion is determined by the surface properties of all three phases involved, i.e., the surface tensions of the adhering particles, of the substrate, and of the suspending liquid medium. In essence, adhesion is more extensive to hydrophilic substrata (i.e., substrata of relatively high surface tension) than to hydrophobic substrata, when the surface tension of the bacteria is larger than that of the suspending medium. When the surface tension of the suspending liquid is larger than that of the bacteria, the opposite pattern of behavior prevails. Suspensions of bacteria at a concentration of 10(8) microorganisms per ml were brought into contact with several polymeric surfaces (Teflon, polyethylene, polystyrene, and acetal and sulfonated polystyrene) for 30 min at 20 degrees C. After rinsing, the number of bacteria adhering per unit surface area was determined by image analysis. The surface tension of the suspending medium. Hanks balanced salt solution, was modified through the addition of various amounts of dimethyl sulfoxide. It was found that the number of bacteria adhering per unit surface area correlates well with the thermodynamic predictions and that these data may be used to determine the surface tension of the different bacterial species. The surface tensions of the bacteria obtained in this fashion are in excellent agreement with those obtained by other methods.     

Surface activation of the cell adhesion fragment of fibronectin

One of the earliest events in the adhesion of fibroblasts to a substratum is the recognition by the cells of macromolecular adhesive factors, such as fibronectin. This early event is followed by a complex series of cell alterations leading to adhesion and spreading. To identify cell surface components involved in the initial cell-fibronectin recognition step, we have employed an assay involving latex particles coated with radiolabelled plasma Fibronectin (Fn). In previous studies from this laboratory (Harper & Juliano , J cell biol 87 (1980) 755) [28], we demonstrated that Fn-mediated adhesion of CHO cells is temperature-dependent, cation-dependent and sensitive to cytoskeletal disrupting agents; by contrast, binding of 3H-Fn beads was unaffected by these factors, indicating that this process reflects binding and recognition events at the cell surface which are independent of cytoskeletal and metabolic activity. Biological specificity of 3H-Fn bead-to-cell binding was confirmed by the ability of anti-Fn antisera to completely block the process. To examine surface components which may mediate binding we treated Fn beads with purified glycosaminoglycans (GAGs) or glycolipids prior to incubation with cells. Among the GAGs tested, heparin, heparan sulfate and dermatan sulfate blocked bead binding in a dose-related fashion with heparin being most potent. The gangliosides GT1, and GM1, also inhibited bead binding. However, treatment of cells with neuraminidase had no effect on bead binding while subsequent analysis of treated cells by thin layer chromatography revealed a drastic reduction in the amount of GM3, the predominant CHO cell ganglioside. CHO cells were also incubated with a panel of proteolytic enzymes to study the possible role of cell surface proteins or glycoproteins in Fn bead binding. We found 3H-Fn bead binding to be quite sensitive to pretreatment with thermolysin, pronase, and papain but only moderately sensitive to treatment with trypsin. From our findings we suggest: (1) binding of Fn beads to CHO cells reflects an early step in the adhesion process; (2) glycolipids may block bead binding but are probably not the endogenous binding site for Fn; (3) protease sensitive components (glycoproteins or proteoglycans) may be more likely candidates as cell surface-binding sites for Fn.     

Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion

The ability to control the interaction between proteins and cells with biomaterials is critical for the effective application of materials for a variety of biomedical applications. Herein, the surface modification of the biological dopant dextran sulphate-doped polypyrrole (PPy-DS) with poly(ethylene glycol) to generate a biomaterial interface that is highly resistant to protein and cellular adhesion is described. Thiolated poly(ethylene glycol) (PEG-thiol) was covalently bound to PPy-DS backbone via a thiol-ene reaction. The surface resistance to an extracellular matrix protein fibronectin increased with increasing molecular weight and concentration of PEG-thiol, and was further optimised via increasing the reaction temperature and the pH of the reactant aqueous solution. Optimised surface modification conditions substantially reduced interfacial protein adsorption, with the complete inhibition of adhesion and colonisation by primary mouse myoblasts. PEG-thiol-modified inherently conducting polymers are highly protein resistant multifunctional materials that are promising compounds for a range of biomedical and aquatic applications.     

Cellulose pretreatments of lignocellulosic substrates

Cellulose in inedible plant materials, forestry residues, and municipal wastes must be pretreated to disrupt its physical structure, thereby making its hydrolysis to glucose practical. Developments since 1991 are summarized.     

Surface components of shigellae involved in adhesion and haemagglutination

Strains of Shigella species were studied for their ability to adhere and agglutinate mammalian erythrocytes. Shigella dysenteriae and Sh. flexneri exhibited haemagglutinating (HA) properties when cultured in Casamino Acids-Yeast Extract (CYE) broth in the presence of 1 mmol 1^-1 calcium chloride, but other shigellae did not show this property under the same culture conditions. Repeated subcultivation of Sh. boydii, Sh. sonnei and HA negative strains of Sh. dysenteriae and Sh. flexneri in CYE broth medium induced adhesive and haemagglutinating properties that were inhibited by sodium periodate. HA activities of Shigella spp. were also inhibited by N -acetylneuraminic acid, α₁-glycoprotein and fetuin, but not by protease. Electron microscopy of Sh. dysenteriae 1, Sh. flexneri 2a, Sh. boydii 12 and Sh. sonnei 1 grown in CYE broth showed the presence of an extracellular slime layer that promoted agglutination of erythrocytes. The slime layer extracted from the cell surface of Shigella spp. showed HA properties, whereas lipopolysaccharide (LPS) obtained from the same strains, except Sh. dysenteriae 1, did not agglutinate erythrocytes. This evidence suggests that the cell surface haemagglutinin is a loosely bound slime layer which is expressed in CYE broth medium.     

Surface characterization of two amoebae relative to cell adhesion

This is a comparative study of the surfaces of two Acanthamoeba castellanii strains, one of which clumps during exponential growth. Proteins from the isolated surface membranes of the strains were compared by disc electrophoresis. The number and similarity of the proteins between the two strains depended on the method of solubilization and electrophoresis. Periodic acid Schiff staining indicated that most of the proteins were glycoproteins. The molecular weights of the proteins ranged between 50 000–70 000 with those of the clumping strain being slightly larger. The non-clumping strain's surface membrane exhibited glucosyltransferase activity while the clumping strain did not. Galactosyltransferase activity was present in surface membranes of both strains but the specific activity of the clumping strain was greater. The non-clumping strain agglutinated in the presence of concanavalin A, soybean agglutinin and wheat germ agglutinin while the clumping strain was affected by soybean agglutinin only. Whole cell electrophoresis and enzymic treatments further indicated differences in the surfaces of the two strains. The data is discussed with reference to differences between the two amoebae strains and their possible role in cell-cell adhesion.     

Efficiency of pretreatments for optimal enzymatic saccharification of soybean fiber

The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H₂SO₄), sodium hydroxide (NaOH), and ammonium hydroxide (NH₃OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144 μm_{pp (peak-to-peak)} for 30 and 60 s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H₂SO₄ [1% (w/w), 90 °C, 1.5 h], NaOH [15% (w/w), 65 °C, 17 h], and NH₃OH [15% (w/w), 65 °C, 17 h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH₃OH pretreatment resulted in the highest saccharification yield (63%) after 48 h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aequeous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm^2,† in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis

Surface hydrophobicity, surface electrokinetic potential and the ability to adhere to nitric-acid cleansed glass surfaces has been assessed throughout the growth, in batch culture, of Escherichia coli and Staphylococcus epidermidis . In both instances adhesiveness and surface hydrophobicity decreased in early- to mid-exponential phase. Cell surface charge, on the other hand became more electro-negative for E. coli but electro-neutral for Staph. epidermidis as the cells proceeded to divide. Adhesiveness correlated directly with surface electronegativity and hydrophobicity for Staph. epidermidis but inversely with surface electro-negativity for E. coli.     

Different pretreatments for increasing the anaerobic biodegradability in swine manure

The effect of three methods (mechanical, chemical, and thermal pretreatment) were tested to improve methane production and anaerobic biodegradability of swine wastes. The first experiment was designed to determine the biodegradability enhancement through the separation of liquid and solid matrix by using a 0.25mm pore size screen (mechanical pretreatment). The second approach was the treatment of swine waste by the addition of a flocculant agent and strong chemicals such as acid (HCl) and alkali (NaOH). The third pretreatment studied was thermal application (170 degrees C provided by vapor). The soluble COD was increased by 57% and 32% during the pretreatment period with alkali and thermal application, respectively. In addition, these two pretreatments gave the highest enhancement on methane production with regard to the untreated sample. Meanwhile, the addition of a flocculant improved the methane production of the liquid fraction but not the solid one. On the other hand, mechanical pretreatment did not show any important enhancement. Biodegradability percentage followed the same trend as methane productivity.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces.

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that a cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aqueous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm(2), in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Surface free energies of oral streptococci and their adhesion to solids

Abstract The adhesion of 3 strains of oral streptococci from a buffered suspension onto 3 different solid substrata was studied. Representative strains of streptococci were selected on the basis of their surface free energy ( γ _b), namely Streptococcus mitis L1 ( γ _b= 37 mJ·m⁻²), Streptococcus sanguis CH3 (95 mJ·m⁻²) and Streptococcus mutans NS (117 mJ·m⁻²). Solid substrata were also selected on basis of their surface free energy ( γ _s), and included polytetrafluorethylene ( γ _s= 20 mJ·m⁻²), polymethylmethacrylate (53 mJ·m⁻²) and glass (109 mJ·m⁻²). Bacterial adhesion was measured as the number of bacteria adhering per cm² at equilibrium. Equilibrium was usually obtained within 20 min. S. sanguis CH3, having an intermediate surface free energy did not show a clear preference for any of the 3 solids. S. mitis L1, however, the lowest surface free energy strain, adhered in highest numbers to the low energy solid PTFE, whereas the highest γ _b strain, S. mutans NS, adhered in highest numbers to the highest γ _s solid, glass. Calculation of the interfacial free energy of adhesion ( ΔF _adh) for each bacterial strain showed that this parameter was predictive of bacterial adhesion to solid substrata.     

Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis.

Surface hydrophobicity, surface electrokinetic potential and the ability to adhere to nitric-acid cleansed glass surfaces has been assessed throughout the growth, in batch culture, of Escherichia coli and Staphylococcus epidermidis. In both instances adhesiveness and surface hydrophobicity decreased in early- to mid-exponential phase. Cell surface charge, on the other hand became more electro-negative for E. coli but electro-neutral for Staph. epidermidis as the cells proceeded to divide. Adhesiveness correlated directly with surface electronegativity and hydrophobicity for Staph. epidermidis but inversely with surface electro-negativity for E. coli.     

Application of pretreatments for the isolation of bioactive actinomycetes from marine sediments

Summary Actinomycetes were isolated from marine sediments collected in Sandy Hook Bay, New Jersey. A variety of pretreatments, including heat and phenol, were employed to improve the recovery of these microorganisms. In addition, plating of sediment samples on chitin agar, and filtration through cellulose membrane filters were also utilized. These pretreatments eliminated or severely curtailed the growth of contaminating microorganisms thereby facilitating the isolation of actinomycetes. A total of 120 isolates was obtained, of which 19 displayed significant antimicrobial activity. Most of the activity was directed against gram-positive bacteria, but inhibition of gram-negative species and a yeast were also evident.     

The application of flow cytophotometry in measurements of cell adhesion

A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The application of flow cytophotometry in measurements of cell adhesion

Summary A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These free cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer.A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from maccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%–10%.A comparison was made of this technique with conventional cell counting by light microscopy, in a study of mouse splenocytes and of myoblasts isolated from 12 day old chicken embryos, Suspensions of these cells and of myoblasts were applied to microtitre plate wells precoated with fibronectin or Concanavalin A (Con A). The specific phase of cell contact was blocked by preincubation of the cells in media containing Con A or -methyl-d-mannoside, and the kinetics of cell attachment to Con A coated surfaces were compared. Claims in the literature for a difference between the fibronectin-type and lectin-type curves were not supported by our results. It was found that the curves for the attachment of both cell types to fibronectin or Con A coated surfaces are similar in shape.The results obtained by the FC technique were evaluated statistically and compared with cell counts obtained by microscopy. The definition of adhering cells is different in flow-cytophotometry (i.e. cells which cannot be removed from wells) from that used in microscopic counting (i.e. cells which have lost their round shape). Nevertheless, the results are found to be parallel which allows us to suggest the use of flowcytophotometry as a routine technique for evaluation of cell adhesion.     

Quantitation of selective cell-cell adhesion and its application to assays of species-specific adhesion in cellular slime molds

Species-specific cell-cell adhesion can be demonstrated by analysing the composition of aggregates formed when suspensions of dissociated, differentially labelled cells of two species of slime molds are mixed and gyrated [4]. However, the usefulness of this assay has been limited by the lack of methods for quantifying the extent of specific cell-cell adhesion. In the present report, we introduce two measures of specificity, the purity index and the odds ratio, and show several methods of computing them. Their ability to detect differing amounts of specificity is shown by analysing the composition of aggregates formed by mixing cells from two species under various experimental conditions of differentiation or gyration. Of the two statistical methods considered, the odds ratio seems more useful since it incorporates aggregate size into its formulation and its attendant confidence intervals are easily calculated.