Leslie M. Shor

Mobility of Protozoa through Narrow Channels

ABSTRACT Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individuals unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 μm s−1 (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 μm s−1. Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.

Dynamic Dosing Assay Relating Real-Time Respiration Responses of Staphylococcus aureus Biofilms to Changing Microchemical Conditions

Bacterial biofilms are a metabolically heterogeneous community of bacteria distributed in an extracellular matrix comprised primarily of hydrated polysaccharides. Effective inhibitory concentrations measured under planktonic conditions are not applicable to biofilms, and inhibition concentrations measured for biofilms vary widely. Here, we introduce a novel microfluidic approach for screening respiration inhibition of bacteria in a biofilm array morphology. The device geometry and operating conditions allow antimicrobial concentration and flux to vary systematically and predictably with space and time. One experiment can screen biofilm respiratory responses to many different antimicrobial concentrations and dosing rates in parallel. To validate the assay, onset of respiration inhibition following NaN₃ exposure is determined optically using an O₂-sensing thin film. Onset of respiration inhibition obeys a clear and reproducible pattern based on time for diffusive transport of the respiration inhibitor to each biofilm in the array. This approach can be used for high-throughput screening of antimicrobial effectiveness as a function of microbial characteristics, antimicrobial properties, or antimicrobial dosing rates. The approach may also be useful in better understanding acquired antimicrobial resistance or for screening antimicrobial combinations.

Protozoan migration in bent microfluidic channels.

ABSTRACT Microfluidic devices permit direct observation of microbial behavior in defined microstructured settings. Here, the swimming speed and dispersal of individual marine ciliates in straight and bent microfluidic channels were quantified. The dispersal rate and swimming speed increased with channel width, decreased with protozoan size, and was significantly impacted by the channel turning angle.

Tape underlayment rotary-node (TURN) valves for simple on-chip microfluidic flow control

We describe a simple and reliable fabrication method for producing multiple, manually activated microfluidic control valves in polydimethylsiloxane (PDMS) devices. These screwdriver-actuated valves reside directly on the microfluidic chip and can provide both simple on/off operation as well as graded control of fluid flow. The fabrication procedure can be easily implemented in any soft lithography lab and requires only two specialized tools—a hot-glue gun and a machined brass mold. To facilitate use in multi-valve fluidic systems, the mold is designed to produce a linear tape that contains a series of plastic rotary nodes with small stainless steel machine screws that form individual valves which can be easily separated for applications when only single valves are required. The tape and its valves are placed on the surface of a partially cured thin PDMS microchannel device while the PDMS is still on the soft-lithographic master, with the master providing alignment marks for the tape. The tape is permanently affixed to the microchannel device by pouring an over-layer of PDMS, to form a full-thickness device with the tape as an enclosed underlayment. The advantages of these Tape Underlayment Rotary-Node (TURN) valves include parallel fabrication of multiple valves, low risk of damaging a microfluidic device during valve installation, high torque, elimination of stripped threads, the capabilities of TURN hydraulic actuators, and facile customization of TURN molds. We have utilized these valves to control microfluidic flow, to control the onset of molecular diffusion, and to manipulate channel connectivity. Practical applications of TURN valves include control of loading and chemokine release in chemotaxis assay devices, flow in microfluidic bioreactors, and channel connectivity in microfluidic devices intended to study competition and predator/prey relationships among microbes.

Window on a microworld: simple microfluidic systems for studying microbial transport in porous media.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 microm. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Direct Tracking of Particles and Quantification of Margination in Blood Flow

Margination refers to the migration of particles toward blood vessel walls during blood flow. Understanding the mechanisms that lead to margination will aid in tailoring the attributes of drug-carrying particles for effective drug delivery. Most previous studies evaluated the margination propensity of these particles via an adhesion mechanism, i.e., by measuring the number of particles that adhered to the channel wall. Although particle adhesion and margination are related, adhesion also depends on other factors. In this study, we quantified the margination propensity of particles of varying diameters (0.53, 0.84, and 2.11 μm) and apparent wall shear rates (30, 61, and 121 s-1) by directly tracking fluorescent particles flowing through a microfluidic channel. The margination parameter, M, is defined as the total number of particles found within the cell-free layers normalized by the total number of particles that passed through the channel. In this study, an M-value of 0.2 indicated no margination, which was observed for all particle sizes in water. In the case of blood, larger particles were found to have higher M-values and thus marginated more effectively than smaller particles. The corresponding M-values at the device outlet were 0.203, 0.223, and 0.285 for 0.53-, 0.84-, and 2.11-μm particles, respectively. At the inlet, the M-values for all particle sizes in blood were <0.2, suggesting that non-fully-developed flow and constriction may lead to demargination. For particle velocities transverse to the flow direction (vy), all particle sizes showed a larger standard deviation of vy as well as a higher effective diffusivity when the particles were suspended in blood relative to water. These higher values are attributed to collisions between the blood cells and particles, further supporting recent simulation results. In terms of flow rates, for a given particle size, the higher the flow rate, the higher the M-value.

Protist-Facilitated Particle Transport Using Emulated Soil Micromodels

Microbial processes in the subsurface can be visualized directly using micromodels to emulate pore-scale geometries. Here, emulated soil micromodels were used to measure transport of fluorescent beads in the presence and absence of the soil ciliate Colpoda sp. under quiescent conditions. Beads alone or beads with protists were delivered to the input wells of replicate micromodels that contained three 20 mm(2) channels emulating a sandy loam microstructure. Bead abundance in microstructured channels was measured by direct counts of tiled confocal micrographs. For channels with protists, average bead abundances were approximately 320, 560, 710, 830, and 790 mm(-2) after 1, 2, 3, 5, and 10 days, respectively, versus 0, 0, 0.3, 7.8, and 45 mm(-2) without protists. Spatial and temporal patterns of bead abundance indicate that protist-facilitated transport is not a diffusive-type process but rather a function of more complex protist behaviors, including particle uptake and egestion and motility in a microstructured habitat. Protist-facilitated transport may enhance particle mixing in the soil subsurface and could someday be used for targeted delivery of nanoparticles, encapsulated chemicals, or bacteria for remediation and agriculture applications.

Microfluidic passive samplers for in situ collection of live aquatic protists

This paper describes the development of microfluidic passive samplers for the collection of live protists from natural aquatic habitats. Microfluidic passive samplers provide several potential benefits over existing sampling methods. For example, they offer greater versatility, higher throughput, and do not require the disruption of specimens through the use of fixatives, stains, or by extraction. In lab testing, a marine ciliate Cyclidium sp. was concentrated from 600 cells per mL in a laboratory microcosm to above 2 × 108 cells per mL within individual microfluidic observation galleries. In field experiments, live protists and other microorganisms were collected from surface water and sediment in a northeastern Connecticut stream. Protists were accumulated to 1 × 107 cells per mL in individual observation galleries. Concentrating and isolating protists enables high-resolution, long-term observation of live, unstained protists. The compact arrangement of observation galleries facilitates high-throughput analysis. Sampler versions were created that differed in the degree of channel branching, the spatial density of galleries, and the size and shape of gallery entrance constrictions. Lab and field testing illustrated tradeoffs in performance among sampler variations in terms of the fraction of occupied chambers, overall on-chip biomass density, and in the types of protists and in the range of sizes of protists collected. Recommendations are provided to facilitate the adoption of microfluidic passive samplers for environmental characterization, research, and educational purposes.

Pore-scale water dynamics during drying and the impacts of structure and surface wettability

Plants and microbes secrete mucilage into soil during dry conditions, which can alter soil structure and increase contact angle. Structured soils exhibit a broad pore size distribution with many small and many large pores, and strong capillary forces in narrow pores can retain moisture in soil aggregates. Meanwhile, contact angle determines the water repellency of soils, which can result in suppressed evaporation rates. Although they are often studied independently, both structure and contact angle influence water movement, distribution, and retention in soils. Here, drying experiments were conducted using soil micromodels patterned to emulate different aggregation states of a sandy loam soil. Micromodels were treated to exhibit contact angles representative of those in bulk soil (8.4° ± 1.9°) and the rhizosphere (65° ± 9.2°). Drying was simulated using a lattice Boltzmann single component, multi-phase model. In our experiments, micromodels with higher contact angle surfaces took four times longer to completely dry versus micromodels with lower contact angle surfaces. Microstructure influenced drying rate as a function of saturation and controlled the spatial distribution of moisture within micromodels. Lattice Boltzmann simulations accurately predicted pore scale moisture retention patterns within micromodels with different structures and contact angles.

Bioavailability of PAHs to Bacteria in Estuarine Sediment

Because of the toxicity and persistence of polycyclic aromatic hydrocarbons (PAHs) in the biosphere and their hydrophobicity and association with particles, there is a need to understand the bioavailability and fate of PAHs in soil and sediment environments. Factors affecting bioavailability include the physical/ chh@ical nature of the soil/sediment particles and the intrinsic biological properties of the organisms affected. In our studies, we examined (1) the bioavailability and biodegradation of endogenous field-aged PAHs from two sites in the greater New York/New Jersey harbor estuary system, Piles Creek (PC) and Newtown Creek (NC), separated into high-density and low-density fractions, and (2) the effect of aging on PAH bioavailability in low and high organic matter sediment material. A Mycobacterium sp. strain PC01 isolated on PAHs from Piles Creek was used as an indicator of bioavaila bility in the series of experiments. In both PC and NC sediment, most of the PAHs were preferentially associated with the lowdensity fraction which represented a very small fraction of the total sediment mass. Yet in the low density fraction, the rate of degradation was slower and the percent PAHs degraded was less than in high density or whole sediment. These observations suggest that the low density fraction is the factor limiting or controlling PAH bioavailability in whole sediment. In order to examine aging of PAHs in sediment, 14C-labeled phenanthrene was aged in PC sediment for over 384 days. The rate and extent of degradation after 139 days in both the low and high organic sediment did not differ substantially from that observed on day 0. Samples taken after 251 and 384 days, however, show that the rate and extent of degradation was significantly reduced in the high organic sediment, while reduced only to a minor extent in the low organic sediment. The impact of aging time, thus, appears to be more important for high organic sediment.