Elizabeth Rudolph

Bacteria size determination by elastic light scattering

Light extinction and angular scattering measurements were performed on three species of bacteria with different sizes and shapes ( Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis). The Gaussian ray approximation of anomalous diffraction theory was used to determine the average bacteria size from transmission measurements. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering. Particle shape and size distribution is then obtained from the rescale spectra. Particle characteristics (size and/or shape) retrieved from both methods are in good agreement with size and shape measured under scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.

BACTERIA-CLAY INTERACTION: STRUCTURAL CHANGES IN SMECTITE INDUCED DURING BIOFILM FORMATION

Bacteria play an important role in determining the properties and behavior of clay minerals in natural environments and such interactions have great potential for creating stable biofilms and carbon storage sites in soils, but our knowledge of these interactions are far from complete. The purpose of this study was to understand better the effects of bacteria-generated biofilms on clay interlayer expansion. Mixtures of a colloidal, 2-water hectorite clay and Pseudomonas syringae in a minimal media suspension evolve into a polysaccharide-rich biofilm aggregate in time-series experiments lasting up to 1 week. X-ray diffraction analysis reveals that upon aggregation, the clay undergoes an initial interlayer contraction. Short-duration experiments, up to 72 h, result in a decrease in the d001 value from 1.50 to 1.26 nm. The initial interlayer contraction is followed in long-duration (up to 1 week) experiments by an expansion of the d001 value of 1.84 nm. The expansion is probably a result of large, biofilm-produced, polymeric molecules being emplaced in the interlayer site. The resultant organo-clay could provide a possible storage medium for carbon in a microbial colony setting.

In situ determination of refractive index and size of Bacillus spores by light transmission

Light-extinction measurements in the wavelength range of 400 to 1000 nm are performed in situ on Bacillus subtilis endospores during heat-shock-induced activation. Simultaneous information on particle size and refractive indices during activation is calculated from the transmission spectra by use of the Gaussian ray approximation of anomalous diffraction theory. During activation the refractive index of the core decreases from 1.51 to 1.39, and the size increases from 0.38 to 0.6 microm.

Effects of smectite clay on biofilm formation by microorganisms

We have investigated the role of smectite clay particles in biofilm formation by several different species of bacteria ( Pseudomonas syringae, Escherichia coli , Staphylococcus aureus and Bacillus subtilis ). We observed that the presence of clay particles enhances the formation of biofilms and, after 24 h, the bacterial populations in the clay mixtures were greater than the respective populations in media without clay. Smectite-bearing clay slurries uniformly develop bacteria–clay aggregates with a substantial biofilm component within 24 h, while the exclusively bacterial suspensions do not develop any observable biofilm component. The biofilm–clay aggregates vary in size from tens of micrometers to several millimeters. Biofilm formation was evaluated by phase contrast microscopy and fluorescence staining. Biofilm promotion by smectite clays may indicate the importance of transport of bacteria by aerosol dust particles.

Broadband light scattering measurements of the time evolution of the fractal dimension of smectite clay aggregates

The characterization of the initial stages of complex aggregate flocculation is a central problem of colloidal science and environmental studies. A fast, white light scattering technique for monitoring aggregation kinetics is used to provide a unique probe of the changes that transpire during flocculation. The time evolution of an accreting smectite clay aggregate is monitored on the basis of changes in the fractal dimension (Df) initiated by the addition of an electrolyte (MgCl2). The evaluation of the Df change reveals for the first time three distinct stages in aggregate formation that characterize smectite flocculation: a rapid, initial jump; followed by a slow decrease; and a final gradual increase, eventually reaching a plateau. The three stages in the time evolution of Df correlate with (1) collapse of loosely packed face-to-edge structures and the formation of compact face-to-face composite structures; (2) the merging of the face-to-face composites; and (3) aggregation of the composites. Stage 3 eventually reaches a quasi-equilibrium condition. The quasi-equilibrium value of Df varies from 1.86 in 1 mM MgCl2—corresponding to diffusion-limited colloidal aggregation—to 2.20 in 57 mM MgCl2—corresponding to reaction-limited colloidal aggregation. These unexpected results originate from the formation of intermediate composite particles during aggregation and the increase in the compactness of the intermediate composite particles with electrolyte concentration. The new white light optical technique demonstrated here is non-invasive and has significant implications in the investigation of the growth dynamics of colloidal systems.

Bacteria-clay interactions investigated by light scattering and phase contrast microscopy

Light scattering experiments and phase contrast microscopy are used to evaluate the aggregate-forming characteristics of simple clay-bacteria mixtures. Colloidal suspensions of negatively charged Pseudomonas syringae (Ps) and Mg2+-, Li+ - or Ca2+ -exchanged smectite (and non-exchanged smectite) are flocculated in neutral (pH 7) aqueous media. Aggregate formation is monitored using changes in optical transmission. Clustering is observed in all the clay-bacteria preparations. The Li+-substituted clay aggregates average 50-300 microns in diameter, in contrast to the Ca2+- substituted clay that produces aggregates of 10-50 microns in diameter. Light scattering measurements indicate that aggregates begin forming 3 hours after mixing and that the (larger sized) aggregates exhibit less scattering than a mixture with an equivalent concentration of unattached Ps and clay particles.

In situ identification of bacteria size by light scattering

Optical transmission and light scattering measurements were performed on three species of bacteria with different sizes and shapes (Pseudomonas aeruginosa. Staphylococcus aureus and Bacillus subtilis). The average bacteria size was determined from transmission measurements by using the Gaussian Ray Approximation of Anomalous Diffraction Theory. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering in order to determine particle shape and size distribution. Particle size and shape as determined by both methods are in good agreement with size and shape measured by scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.

Refractive index changes during germination of bacillus subtilis spores

Light transmission measurements in the wavelength range of 400 to 1000 nm were performed on Bacillus subtilis spores at periodic time intervals after heat-shock induced activation. The Gaussian ray approximation, using a concentric sphere model for the spore coat and spore core, was used to calculate the scattering cross-section of the spores. Analysis of transmission spectra determined that the refractive index of the spore core was 1.515. In the three hours following heat shock, the core refractive index decreased to 1.39, and subsequently remained constant. During the measurements, the spore radius increased from 0.38 microns to 0.6 microns. The results were confirmed by phase contrast microscopy.

Changes in refractive index and size of Bacillus subtilis spores during activation, measured by light transmission

Refractive index and size changes in Bacillus subtilis spores were measured during heat shock activation by light scattering. The refractive index decreased from 1.51 to 1.38 while the size increased from 0.37 to 0.6 microns.

Fluorescence detection of proteins released by Bacillus subtilis spores during heat shock germination

Fluorescence and absorption spectroscopy were performed on Bacillus subtilis spores which were heat treated at temperatures ranging from 20°C to 90°C. The tryptophan (trp) emission from the spore suspensions treated at temperatures greater than 60°C was shifted towards longer wavelength as compared to the spores which were heat treated at lower temperatures. These spectral changes were the result of proteins released by the spores into the suspension. Trp residues in the emitted proteins are in a more polar environment and therefore exhibit a larger Stokes shift. Fluorescence and absorption measurements show that the concentration of proteins in the supernatant was greater for spores treated at higher temperatures. Electrophoresis gel analysis showed the presence of a 47 KDa protein in the supernatant.