Characterising the structure of photosynthetic biofilms using fluid dynamic gauging

A new configuration of the fluid dynamic gauging technique for measuring soft layers on surfaces was used to monitor the growth of a cyanobacterium, Synechococcus sp. WH 5701, on stainless steel (SS), glass and an indium tinoxide (ITO) on a polyethylene terephthalate (PET) substratum. The biofilm thickness increased steadily over 4weeks and exhibited noticeable changes in microstructure and strength. The biofilms all exhibited a two-layer structure, with a compact layer next to the substratum and a loose layer above. Biofilms on ITO or SS exhibited cohesive failure when removed by fluid shear whereas those on glass exhibited adhesive failure. The technique is able to elucidate various aspects of biofilm behaviour, as illustrated by the action of a biocide (NaOCl) on a mature biofilm.     

Characterising the structure of photosynthetic biofilms using fluid dynamic gauging

A new configuration of the fluid dynamic gauging technique for measuring soft layers on surfaces was used to monitor the growth of a cyanobacterium, Synechococcus sp. WH 5701, on stainless steel (SS), glass and an indium tin oxide (ITO) on a polyethylene terephthalate (PET) substratum. The biofilm thickness increased steadily over 4 weeks and exhibited noticeable changes in microstructure and strength. The biofilms all exhibited a two-layer structure, with a compact layer next to the substratum and a loose layer above. Biofilms on ITO or SS exhibited cohesive failure when removed by fluid shear whereas those on glass exhibited adhesive failure. The technique is able to elucidate various aspects of biofilm behaviour, as illustrated by the action of a biocide (NaOCl) on a mature biofilm.     

Characterisation of the Physical Composition and Microbial Community Structure of Biofilms within a Model Full-Scale Drinking Water Distribution System

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Measurement of the adhesive strength of a gingival cell line to agar

A new technique is described for measuring the adhesive strength of a gingival cell line to an agar substratum by the modification of the original "blister" test for adhesives. A cell monolayer was developed on a Petri dish with a hole in the center of the growing surface, overlayed with agar, and the system pressurized to debond the cells from the agar surface. Pressure changes were measured by a capacitance pressure transducer the output of which was measured by a strip-chart recorder. The modulus (E) of the agar overlay was determined and used in the calculation of the adhesive-bond strength (gammaalpha). The gammaalpha yield for the gingival cell line (cell-agar debond) was 48.8 ergs per cm2, and for the control (no cells) (agar-polystyrene debond) was 30.0 ergs per cm2.     

Phospholipid composition of substrate adhesion sites of normal, virus-transformed, and revertant murine cells.

The phospholipid composition of cell-substratum adhesion sites, obtained after EGTA-mediated detachment of cells from the tissue-culture substratum, was determined for [32P]orthophosphate radiolabeled Balb/c 3T3, SV40-transformed (SVT2), and concanavalin A selected revertant variant cell lines. All of the major phospholipid classes were found in the substrate-attached material, but there was an enrichment for specific phospholipid species in this adhesive material as compared to whole-cell and surface-enriched membranes. The phospholipid composition was remarkable similar for the whole-cell and surface-enriched membrane fractions from the three cell lines. However, pronounced differences in the phospholipid composition of the adhesion sites were observed as a result of viral transformation--SVT2 sites were clearly enriched in phosphatidylethanolamine and depleted in phosphatidylcholine when compared to 3T3 sites. This alteration in adhesion site phospholipids of transformed cells reverted to 3T3-like values in the adhesive material of revertant cells. The composition of adhesive material of newly attaching cells was also examined to differentiate compositional differences between "footpad" adhesion sites and "footprints", adhesive material pinched off from the posterior of cells as they move across the substratum. Pulse and pulse-chase analyses of the [32P]phospholipids revealed some differences in synthesis and turnover rates in the three cell lines; in addition, altered rates of deposition of newly synthesized material into adhesion sites of transformed cells were observed. These data afford further evidence that the cell-substratum adhesion sites are highly specialized areas of the cell surface enriched in components which are intricately involved in the adhesive process. The transformation-dependent changes in adhesion site phospholipids may help to determine the basis for the altered adhesive properties of transformed cells.     

“In vitro” biofilm formation by Penicillum frequentans strains on sandstone,granite, and limestone

The appearance of crusts or patinas on surfaces of stone monuments are indicative signs of weathering. In many cases, microorganisms are mainly responsible for stone decay, giving rise to the formation of characteristic biodeteriorative patinas, called biofilms. In the present work, in vitro biofilm formation on sandstone, limestone, and granite block samples by Penicillium frequentans strains isolated from two Spanish cathedrals is demonstrated. Spore suspensions of P. frequentans strains were inoculated on each block sample of unaltered stone material cited above. Biofilms of 1–2 mm thickness were formed on each of the three rock samples, and analysed by means of Fourier-transform infrared spectroscopy, scanning electron microscopy and X-ray mapping (energy-dispersive X-ray spectrometry, KEVEX system). In the three cases, biofilms were principally composed of: fungal mycelium, mineral particles released from the stony substratum, and newly formed organic salts such as oxalate and citrate. These biofilms enhance and accelerate the deteriorative process of rocks due to the loss of stone material (biopitting and mineral grains captured by mycelium) and to alteration of the mineral crystalline networks (cation release by organic acids). Correspondence to: G. Gómez-Alarcón     

Preliminary results on the effects of salinity and settling conditions on megalopal metamorphosis of fiddler crab Ilyoplax pusilla

Metamorphosis of fiddler crab Ilyoplax pusilla larvaefrom megalopal to first crab stage wasstudied in laboratory experiments under variousconditions of salinity and substratum. The hatchedzoea metamorphose through five stages before reachingmegalopal stage. The megalops were placed in a 20 mlPetri dishes (2–3 megalops/dish), with salinities of10, 20 or 30 . Each salinity level was tested eitherwith or without sandy mud substratum. Thirty to 35megalops were used in each of the six treatments. Theexperiment was carried out at a water temperature of28 ±0.5°C and with daily feeding of the diatomChaetoceros gracilis. Treatment of 20 salinity andsandy mud substratum revealed the highest metamorphicrate (87%), while in contrast no metamorphosis wasobserved at 30 salinity withoutsandy mud substratum. Duration of the metamorphosiswas about 16 days. Numerous malformed juvenile crabswere observed in treatments conductedwithout sandy mud substratum.     

Surface-catalysed disinfection of thick Pseudomonas aeruginosa biofilms

Transition metal catalysts were incorporated into polymers which formed the surface for bacterial attachment and biofilm formation in a constant depth film fermenter (100 μm thickness), flow chamber (about 30 μm thickness) and in batch culture (<30 μm thickness). The catalysts drive the breakdown of persulphates to reactive oxygen species. When Pseudomonas aeruginosa biofilms were exposed to dilute solutions of potassium monopersulphate (20 μg ml⁻¹–1 mg ml⁻¹), significant enhancement of killing was notable for catalyst-containing surfaces over that of controls. The degree of enhancement was greatest for thin films, but was nevertheless significant for the 100 μm thick biofilms. Fluorescence probes and viability staining, in conjunction with laser confocal microscopy, showed that reactive species were generated at the biofilm–substratum interface and killed the biofilm from the inside. Reaction-diffusion limitation now concentrates the active species within the biofilm rather than protecting it, and a diffusion pump is established whereby further treatment agent is drawn to the substratum enabling relatively thick biofilms to be disinfected.     

Cloning,Characterization, and Expression Levels of the <Emphasis Type="Italic">Nectin</Emphasis> Gene from the Tube Feet of the Sea Urchin <Emphasis Type="Italic">Paracentrotus Lividus</Emphasis>

Marine bioadhesives perform in ways that manmade products simply cannot match, especially in wet environments. Despite their technological potential, bioadhesive molecular mechanisms are still largely understudied, and sea urchin adhesion is no exception. These animals inhabit wave-swept shores, relying on specialized adhesive organs, tube feet, composed by an adhesive disc and a motile stem. The disc encloses a duo-gland adhesive system, producing adhesive and deadhesive secretions for strong reversible substratum attachment. The disclosure of sea urchin Paracentrotus lividus tube foot disc proteome led to the identification of a secreted adhesion protein, Nectin, never before reported in adult adhesive organs but, that given its adhesive function in eggs/embryos, was pointed out as a putative substratum adhesive protein in adults. To further understand Nectin involvement in sea urchin adhesion, Nectin cDNA was amplified for the first time from P. lividus adhesive organs, showing that not only the known Nectin mRNA, called Nectin-1 (GenBank AJ578435), is expressed in the adults tube feet but also a new mRNA sequence, called Nectin-2 (GenBank KT351732), differing in 15 missense nucleotide substitutions. Nectin genomic DNA was also obtained for the first time, indicating that both Nectin-1 and Nectin-2 derive from a single gene. In addition, expression analysis showed that both Nectins are overexpressed in tube feet discs, its expression being significantly higher in tube feet discs from sea urchins just after collection from the field relative to sea urchin from aquarium. These data further advocate for Nectin involvement in sea urchin reversible adhesion, suggesting that its expression might be regulated according to the hydrodynamic conditions.     

Glial cell growth in culture: Influence of living cell substrata

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick embryo on the growth of glial cells derived from chick embryonic brain and plated onto the substratum; b) the influence of dissociated cells derived from chick embryonic brain on the growth of established glial cells in culture, and c) the influence of dissociated cells derived from adult rat spinal cord on the growth of established glial cells from newborn rat in culture. The activities of glutamine synthetase (GS) and 2, 3-cyclic nucleotide 3-phosphohydrolase (CNP) were the biochemical probes determined for astrocytes and oligodendrocytes, respectively. We found that glial growth as assessed by both enzyme activities, was enhanced when a nervous tissue derived cell population was plated onto a glial-enriched substratum, whereas glial growth was inhibited when the neuronal-enriched population was the cell substratum.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Enhanced Surface Colonization by Escherichia coli O157:H7 in Biofilms Formed by an Acinetobacter calcoaceticus Isolate from Meat-Processing Environments

A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 × 10⁶ μm³) in a dynamic cocultured biofilm than in a monoculture (3.0 × 10³ μm³), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.Shiga toxin-producing Escherichia coli (STEC) is a food-borne human pathogen responsible for severe gastrointestinal disease (16, 17). Processing, handling, and preparation of food may lead to cross-contamination of food and uncontaminated surface areas of the food chain with pathogens from contaminated surfaces (8). Though most processing plants ensure and maintain good manufacturing practices with elaborated sanitary operations, persisting microorganisms may survive well after cleaning and disinfection procedures (1, 9, 12-14), possibly in the form of biofilms (11). A review of the underlying problems caused by biofilms in the food industry was presented by Carpentier and Cerf (4). Several studies have shown that E. coli, including STEC strains, has the capacity to attach to and form biofilms on various surface materials (5, 18). However, such studies have mainly used monocultures without considering the possible influence of resident organisms from food-processing environments on the surface colonization of E. coli. One recent study showed that resident microflora increased E. coli O157:H7 colonization on solid surfaces under static conditions (10). To our knowledge, no studies have investigated the influence of meat industry resident bacteria on surface colonization by E. coli under dynamic-flow conditions.The aim of this study was to investigate how a biofilm-forming isolate of Acinetobacter calcoaceticus influences surface colonization by E. coli O157:H7. This study focused on the spatial distribution of cells during biofilm formation under static and dynamic growth conditions.Here we used an A. calcoaceticus strain (MF3627) isolated from a clean and disinfected meat-processing environment, as well as Shiga toxin-negative E. coli O157:H7 (ATCC 43888) harboring the plasmid pGFP-uv (Clontech Laboratories, Palo Alto, CA). For static growth conditions, mono- and coculture biofilms were harvested at 25°C in Lab-Tek II chamber slide systems (VWR, Oslo, Norway) consisting of miniature polystyrene medium chambers with a sealed cover glass as the growth surface. For dynamic growth conditions, mono- and coculture biofilms were grown at 25°C in three-channel flow cells with individual channel dimensions of 1 by 4 by 40 mm and a sealed glass coverslip substratum (Knittel Glass, Germany). A 1/10 dilution of Luria-Bertani broth was continuously pumped through the sterile flow cell channels at a rate of 0.5 ml/min. In two of the channels, A. calcoaceticus and E. coli were inoculated individually, while the third channel was reserved for the inoculation of a mixed culture of A. calcoaceticus and E. coli (1:1). The flow cell channels and Lab-Tek chambers were stained with SYTO 61. Horizontal-plane images of the biofilms were acquired using a Leica SP5 AOBS laser scanning confocal microscope (Leica Microsystems, Lysaker, Norway). Three independent biofilm experiments were performed for each biofilm growth condition. Three-dimensional projections were performed with IMARIS software (Bitplane, Zürich, Switzerland). The structural quantification of biofilms (biovolume in cubic micrometers) was performed using the PHLIP Matlab program (http://www.phlip.org/phlip-ml/).Under static growth conditions, E. coli O157:H7 formed a homogeneous flat biofilm yielding biovolumes ranging between 3.3 × 10⁵ and 5.4 × 10⁵ μm³ after 24 and 72 h of biofilm growth. Although the E. coli biovolume revealed no significant differences in monoculture or when cocultured with A. calcoaceticus, microscopic analysis revealed how E. coli cells were gradually covered by a carpet of A. calcoaceticus cells after 72 h of biofilm growth (for visualization, see the supplemental material). A. calcoaceticus monospecies biofilms were heterogeneous, highly structured, and channeled under both static and dynamic conditions (Fig. ​(Fig.11 A), yielding a biovolume of 1.46 × 10⁶ μm³ after 72 h of biofilm growth (Fig. ​(Fig.2).2). E. coli O157:H7 did not form monospecies biofilms under dynamic-flow conditions (Fig. ​(Fig.1A),1A), with biovolume values below 3.5 × 10⁴ μm³ after 72 h (Fig. ​(Fig.2).2). The presence of A. calcoaceticus had a significant impact on E. coli O157:H7 surface colonization with a 400-fold increase in the total biovolume of E. coli O157:H7 from 3.0 × 10³ μm³ to 1.2 × 10⁶ μm³ between 24 and 48 h (Fig. ​(Fig.2),2), as observed from the increase in E. coli O157:H7 biomass between A. calcoaceticus cell clusters (Fig. 1A and B). After 72 h of development, E. coli O157:H7 cell clusters were partially covered by A. calcoaceticus cells. The poor settlement and subsequent poor colonization of E. coli O157:H7 under dynamic-flow conditions could have been attributed to shear forces, which made it difficult for E. coli O157:H7 cells to establish colonies on the substratum. The observed spatial distribution of A. calcoaceticus cells at the liquid-biofilm interface may offer E. coli O157:H7 cells better protection from shear stress and could potentially provide additional protection against disinfectants, as has been observed in other multispecies biofilm studies (2, 21). Whether E. coli cells had increased resistance to antimicrobial agents in our experimental setup as a result of being at the bottom layers of mixed-species biofilms will be the subject of further investigations. Biofilm formation of meat industry surface bacteria can enhance E. coli surface colonization and thereby increase the risk of persistence of and food contamination by potential pathogens. The occurrence of Acinetobacter in food-processing environments is well documented (1, 9, 15), and it has also been isolated from spoiled food products (3, 6, 7). Furthermore, a recent study showed that A. calcoaceticus biofilms are able to interact and coaggregate with other bacteria (19). Cleaning and disinfection procedures used in food industries should thus take into account the risks involved in ignoring the presence of resident flora biofilms.Open in a separate windowFIG. 1.Structural development of A. calcoaceticus and E. coli in mono- and dual-species biofilms under dynamic conditions. (A) Representative biofilms of A. calcoaceticus and pGFP-uv-tagged E. coli O157:H7 grown in flow cells using Luria-Bertani broth as a growth medium at 25°C. The spatial structures in the developing biofilms were studied by laser scanning confocal microscopy. (B) Vertical sections (in the x-z plane) representing the spatial distribution of pGFP-uv-tagged E. coli O157:H7 in the presence of A. calcoaceticus under dynamic-flow conditions after 24, 48, and 72 h of growth. The lower side of each section corresponds to the substratum. Green cells represent pGFP-uv-tagged E. coli O157:H7, red cells represent SYTO 61-stained A. calcoaceticus cells, and yellow cells represent GFP-tagged E. coli O157:H7 marked with SYTO 61.Open in a separate windowFIG. 2.Biovolume of A. calcoaceticus and E. coli O157:H7 biofilm development after 24, 48, and 72 h of growth under dynamic conditions. A. calcoaceticus in monospecies biofilms is represented by the symbol □, A. calcoaceticus in dual-species biofilms is represented by the symbol ▪, E. coli O157:H7 in mono-species biofilms is represented by the symbol Δ, and E. coli O157:H7 in dual-species biofilms is represented by the symbol ▴. Mean values of at least 30 individual images ± the standard errors from three independent experiments are shown.Cleaning and disinfection procedures are employed by the food industry to ensure clean and hygienic surfaces for food production. However, due to the ubiquitous nature of biofilms and their potential to resist antimicrobial treatments (21), new strategies based on preventive actions to reduce the incidence of biofilm formation on food-processing surfaces should be employed (20). In light of the results obtained in this study, combining curative actions with preventive actions based on the use of surface materials with antiadhesive or antifouling surfaces could enhance the hygienic standards of food-processing surfaces.In conclusion, we have shown that under both static and dynamic growth conditions, E. coli cells were found embedded and covered by A. calcoaceticus cells in mixed-species biofilms. Moreover, the presence of an A. calcoaceticus biofilm structure favored E. coli O157:H7 colonization and biofilm formation under dynamic-flow conditions. These results offer new insights into the spatial distribution of pathogenic bacteria and resident flora during cocultured biofilm formation. Conditions allowing active biofilm formation of resident microflora may provide increased opportunities for pathogens to thrive in food-processing environments. The hazardous influences of resident biofilms should therefore not be ignored, since improper cleaning procedures in food-processing environments could potentially increase the risk of food contamination by spoilage and pathogenic bacteria.      

Dynamic surface deformation of silicone elastomers for management of marine biofouling: laboratory and field studies using pneumatic actuation

Many strategies have been developed to improve the fouling release (FR) performance of silicone coatings. However, biofilms inevitably build on these surfaces over time. Previous studies have shown that intentional deformation of silicone elastomers can be employed to detach biofouling species. In this study, inspired by the methods used in soft-robotic systems, controlled deformation of silicone elastomers via pneumatic actuation was employed to detach adherent biofilms. Using programmed surface deformation, it was possible to release > 90% of biofilm from surfaces in both laboratory and field environments. A higher substratum strain was required to remove biofilms accumulated in the field environment as compared with laboratory-grown biofilms. Further, the study indicated that substratum modulus influences the strain needed to de-bond biofilms. Surface deformation-based approaches have potential for use in the management of biofouling in a number of technological areas, including in niche applications where pneumatic actuation of surface deformation is feasible.     

Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces

The properties of barnacle adhesive on silicone surfaces were studied by AFM indentation, imaging, and other tests and compared to the barnacle shear adhesion strength. A multilayered structure of barnacle adhesive plaque is proposed based on layered modulus regions measured by AFM indentation. The fracture of barnacles from PDMS surfaces was found to include both interfacial and cohesive failure of barnacle adhesive plaque, as determined by protein staining of the substratum after forced barnacle release from the substrate. Data for freshly released barnacles showed that there was a strong correlation between the mean Young's modulus of the outermost (softest) adhesive layer (E<0.3 MPa) and the shear strength of adhesion, but no correlation for other higher modulus regions. Linear, quadratic, and Griffith's failure criterion (based on rough estimate of crack length) regressions were used in the fit, and showed significance.     

Microdistribution of benthic invertebrates in a rocky mountain (U.S.A.) stream

A study of the benthic invertebrate community inhabiting a small, foothill trout stream in the Rocky Mountains of Idaho was conducted over a two-year period. Monthly Hess samples and short-term experiments using substratum-filled trays were used to describe the spatial dispersion of the benthos and to examine the response of invertebrate populations to substratum and current. A method was devised for measuring available surface area which involved coating individual stones with latex and measuring the area of the print resulting from inking the impression left on the latex mold.The dispersion of all populations was clumped throughout the year. Alteration of the cross-sectional pattern of current velocity and stream bed composition changed the pattern of distribution but not the extent of clumping. Collections made in areas of depositing and eroding substrata revealed a more diverse fauna in the latter. Most groups of organisms found in the riffle were scarcer in the pools or absent from them. The pool fauna contained no important additions over those found in the riffles.After a year's study of invertebrate populations in an otherwise undisturbed riffle, the substratum was altered and the flow made more uniform; an increase in the abundance of most of the benthic invertebrates followed. No single factor was responsible for the increase, but the change in substratum size and degree of compaction accounted for most of the change. Interpretation of the results was aided by findings from experiments using substratum-filled trays.Two series of stream experiments using the trays were conducted: one to test the relative importance of current and substratum and the other to test the effect of particle size on the distribution of the benthic fauna. In the first series, placement of trays of stones in a pool resulted in an increase in numbers of some but not all of the invertebrates over numbers usually occurring in the pool. Trays filled with stones and placed in a riffle supported fewer animals than found on the adjacent stream bed but more than in the pool. Variations are attributed to differences in current velocity and amounts of imported organic and inorganic debris. Three different relationships of population numbers to current velocity were found for different members of the community (direct, indirect, and parabolic) over the range of 10 to 60 cm/sec. The second series of experiments consisted of two sets of trays filled with stones of medium or large pebbles, respectively. Nine taxa, as well as all of the combined taxa, showed a preference for trays of small stones over the natural stream bed. A few taxa were noticeably more abundant on the small substratum than on the large but most of the fauna showed only slight increases in numbers or remained constant on the two substrata. Only three taxa showed a direct relation of numbers to total surface area presented by the stones.Number and kinds of organisms found in trays filled with a uniform size of substratum did not correspond to those taken in Hess samples from the natural stream bed. This has important implications in terms of currently recommended pollution monitoring techniques. However, it is suggested that if the substratum composition of the trays more nearly matched that of the stream, the correspondence would be much better. The results of the present study also throw considerable doubt on the adequacy of generalizations derived from earlier studies of responses to substratum size and suggest several reasons for reevaluating current ideas regarding the influence of substratum on invertebrate distribution.     

Surface elastic modulus of barnacle adhesive and release characteristics from silicone surfaces

The properties of barnacle adhesive on silicone surfaces were studied by AFM indentation, imaging, and other tests and compared to the barnacle shear adhesion strength. A multilayered structure of barnacle adhesive plaque is proposed based on layered modulus regions measured by AFM indentation. The fracture of barnacles from PDMS surfaces was found to include both interfacial and cohesive failure of barnacle adhesive plaque, as determined by protein staining of the substratum after forced barnacle release from the substrate. Data for freshly released barnacles showed that there was a strong correlation between the mean Young's modulus of the outermost (softest) adhesive layer (E< 0.3 MPa) and the shear strength of adhesion, but no correlation for other higher modulus regions. Linear, quadratic, and Griffith's failure criterion (based on rough estimate of crack length) regressions were used in the fit, and showed significance.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure,composition, and adhesive characteristics

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell‐substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular‐like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3‐linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS‐PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.