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Development of an Evaporative Deposition Method to Probe the Strength of Bacteria-Surface Interactions

Objective

Spontaneous ordering of materials is of importance for numerous technological applications and has captured the interest of scientists for decades. A well-known technique to deposit materials on surfaces is through evaporative deposition from a drying drop. In addition to the obvious importance in controllably arranging particles on surfaces for engineering applications there is potential to use this technique to probe fundamental particle-particle and particle-surface interactions. This could open a new avenue for characterizing the colloidal interactions of environmentally important materials such as bacteria.<P> Our goal is to develop this method for use in probing bacteria-bacteria and bacteria-surface interactions. We recently investigated the evaporative deposition of bacteria from aqueous drops on mica. We show that we can systematically vary the deposition patterns from multi-rings to honeycomb patterns by the simple process of changing the surface wettability through timed exposure to the laboratory atmosphere.<P> Further studies with negatively charged polystyrene microspheres have shown that microsphere deposits also change with mica exposure time in a very similar way. For sufficiently long mica exposure times the mica-microsphere interaction becomes attractive. This leads to the immobilization of some of the microspheres. These stationary microspheres disrupt the long range connectivity of the deposited network and lead to patchy deposition patterns. This suggests it is possible to differentiate particle deposition patterns into those that are affected by attractive particle-surface interactions (short range connectivity in patchy films) from those that are determined by repulsive particle-surface interactions (long range connectivity in cellular films). This suggests that one could rank the relative particle-surface attraction strength of different particles or bacteria by determining the mica exposure time that produces a transition from patterns with long range connectivity to those with short range connectivity.<P> We propose to investigate this by depositing two different types of microspheres of known composition as a function of mica exposure time and salt composition. The results of this work will be used to design experiments to determine the relative bacteria-surface attraction of bacteria. This is potentially useful for understanding differences in adhesiveness of different types of bacteria as well as differences in adhesiveness of a single type of bacteria subjected to genetic modifications or environmental conditions. <P>Objectives: <OL> <LI> Characterize deposition patterns formed using commercially obtained sulfate terminated polystyrene microspheres (relatively hydrophobic) of known surface charge as a function of mica exposure time and increasing salt concentration. <LI> Contrast deposition patterns formed using carboxy-terminated microspheres (hydrophilic) and sulfate terminated microspheres (hydrophobic) as a function of mica exposure time. <LI> Develop a phase diagram for deposition patterns as a function of mica exposure time, and microsphere hydrophilicity, concentration and salt composition.

More information

Non-Technical Summary: With the advent of DNA sequencing and proteomics much progress has been made in unraveling the details of the pathways of biomacromolecule production in bacterial cells. Much less work has been done in understanding the physical properties of bacterial cells which are related to bacterial interactions in the environment and survivability. Bacterial adhesion is, of course, the first step in biofilm formation, which is associated with numerous agricultural, ecological, medical, and industrial problems. In addition, bacterial adhesion is the key event that retards movement of bacteria in soils. A quantitative understanding of bacterial movement in soil is critical to several areas important in agriculture. One such area is the prediction of dissemination of pathogenic bacteria in the environment, e.g., from biosolids-amended crops, or root infecting plant pathogens. A second area is the movement of bacteria in response to environmental perturbation, e.g., application of pesticides or other cropping procedures that disturb the natural soil environment. Adhesion is mediated by the bacterial membrane, the composition of which varies with nutrient status, attachment state and environmental stressors. For instance, in response to drying, cell surface lipids become more rigid and proteins undergo conformational changes. As a further complication, in the environment and for that matter even in refrigerated storage in a laboratory, bacterial cells change genetically over time. We know very little about how genetic changes and environmental stresses affect the interaction of bacteria with each other and with environmental surfaces. One of the difficulties with this problem is that there are no straightforward methods of characterizing the bacteria-surface interaction without perturbing the bacteria through immobilization strategies. Flow-cell methods, in which biofilm biomass is monitored, are too complex to gain unambiguous information about the initial adhesion events that lead to the biofilm development. Recently we have studied the deposition of bacteria from drying drops (evaporative deposition) with the goal of producing ordered bacterial films for use in investigations of bacterial surface properties. During these studies we found that bacteria can be deposited in a variety of patterns on a surface and this appears to be part of a process which can be exploited to characterize bacteria-surface interactions. In this work we aim to systematically develop this method using synthetic well characterized microspheres which are negatively charged and similar in size to bacteria. <P> Approach: We will address these objectives experimentally using evaporative deposition of microspheres on mica. Ruby mica (S & J Trading Inc. NY) will be used as the substrate for these experiments because mica is commonly used in surface studies and is easily prepared into molecularly smooth surfaces that are clean when initially cleaved. Thick pieces of mica will be cut into samples typically 2.5 to 3.0 cm wide and about 3 to 7 cm long. The mica will be cleaved and the samples placed on the working surface of the laminar flow hood until drop deposition experiments are performed. The laminar flow hood (air flow speed 0.44 m/s) will be used to minimize dust contamination. The time the mica is exposed to the atmosphere (in the laminar flow hood) prior to drop deposition will be controlled and will range from less than a minute up to several months. Drops of microsphere suspension (4 microliters) will be deposited on the mica samples using a P100 Gilson pipetter fitted with a polypropylene tip. In order to keep the suspension well-mixed, the suspension will be vortexed before each drop or set of drops is deposited. In order to analyze the particle residue patterns the dried residues will be imaged using an Olympus IMT-2 inverted optical microscope with a Hamamatsu ORCA-100 CCD camera. Where appropriate, AFM images of dried residues will be collected in tapping mode on a Veeco Dimension 3100 Nanoscope IV (Santa Barbara CA) with NSC15 Silicon Nitride cantilevers (Mikromasch). Sulfate-terminated polystyrene microspheres 1 micrometer in diameter (hydrophobic) and carboxy-terminated microspheres 1 micrometer in diameter will be obtained from Molecular Probes. The carboxylate microspheres are also polystyrene microspheres however they are coated with a hydrophilic polymer with pendant carboxylic acid groups making them hydrophilic.

Investigators
Curry, Joan
Institution
University of Arizona
Start date
2010
End date
2014
Project number
ARZT-136020-H-21-163
Accession number
222765