Projects

The composition of microbial communities within and around plant species is dependent on dynamic physical and chemical signaling events that occur within the local environment and at the root surface.  Visualization and quantification of these events in natural systems is challenging. However, emerging technologies that combine advances in nanostructure fabrication, microfluidics and imaging provide a means of recreating these events within model systems.  These systems mimic aspects of their natural counterparts while providing tractable experimental platforms in which both individual cellular responses and population dynamics can be recorded and analyzed.

Model systems, amenable to imaging, that allow dynamic modulation of local physicochemical cues in a controllable manner have been developed to recreate the interactions between microbes and their hosts.  A nanostructured microfluidic platform has been created in order to examine the chemotactic responses of isolates to specific plant-associated signals.  This platform is created using a combination of electron beam lithography and anisotropic silicon etching techniques.  It can be easily replicated via silicone molding and facilitates the physical tracking of hundreds of microbes within a quasi-two-dimensional space that confines microbes within the focal volume of a conventional phase contrast microscope without significantly impeding natural motility.

In this study, confocal laser scanning microscopy was used to examine the spatial and temporal dynamics of microbial colonization of Populus roots using microbial isolates expressing GFP. Standard molecular methods were used to introduce GFP into microbial isolates. Atomic force microscopy was also used to characterize the morphology, surface characteristics, and dynamics of biofilm formation of selected microbes isolated from the Populus rhizosphere. Gamma-proteobacteria isolate YR343 was observed to attach to Poplar roots after approximately 5 hours co-culture. The cells were observed to grow and form colonies on the surface of the root. Microbial isolate GM30 was examined during biofilm formation using atomic force microscopy. Expression of pili over the time course of biofilm formation and distinct morphotypes were revealed by the AFM image analysis. These colonization studies provide direct evidence that microbes collected from the rhizosphere directly associate with Populus roots. The dynamics of colonization were observed in real time using GFP-expressing microbes.

Karyotype analysis and classification of buckwheat chromosomes was performed with out chemical banding or staining using atomic force microscopy. F. esculentum and F. tartaricum chromosomes were isolated from the root tissues using enzymatic maceration technique and spread over a glass substrate. Air dried chromosomes had
a surface with ridges and the height of common and tartary buckwheat were approximately 350 and 150 nm.
Volumes of metaphase sets of buckwheat chromosomes were calculated using 3D atomic force microscopy
measurements. Chromosomes were morphologically characterized by the size, volume, arm lengths and ratios.
The calculated volumes of the F. esculentum and F. tartaricum chromosomes were in the ranges of 1.08 to 2.09 µm3 and 0.49 to 0.78 µm3 respectively. The parameters such as the relative arm length, centromere position and the chromosome volumes measured using AFM provides accurate karyomorphological classification by avoiding the subjective inconsistencies in banding patterns of conventional methods. The karyotype evolutionary trend indicates that F. esculentum is an ancient species compared to F. tartaricum. This is the first report of karyotyping of buckwheat using AFM.

Morphological and structural features of buckwheat starch granules and nanocrystals were examined using atomic force microscopy. Partially digested starch granules revealed a clear pattern of growth rings with the central core showing lamellar structure. Atomic force microscopy and dynamic light scattering experiments revealed that the buckwheat starch granules were round or polygonal in shape and were in the range of 3 to 12 µm in diameter. Aqueous suspensions of starch using acid hydrolysis produced starch nanocrystals. The starch nanocrystals were in the shape of rods with lengths ranging from 120 to 200 nm, and diameters ranging from 2 to 6 nm respectively. New understanding of buckwheat starch components morphology and the granule concentric growth rings has been achieved through our study. Biocompatibility nature of buckwheat starch nanocrystals and their structural properties makes them a promising green nanocomposite material.

A carbon dioxide sensor was developed using polyaniline boronic acid conducting polymer as the electrically conductive region of the sensor and was demonstrated for use in detecting incipient or ongoing spoilage in stored grain. The developed sensor measured gaseous CO2 levels in the range of 380–2400 ppm of CO2 concentration levels. The sensor was evaluated for the influence of temperature (at - 25 °C to simulate storage and for the operating temperature range of +10 °C to +55 °C) as well as relative humidity (from 20 to 70%). The variation in the resistance with humidity was curvilinear and repeatable, and had a less pronounced effect on the sensor’s performance compared to temperature. The sensor was able to respond to changes in CO2 concentration at various humidity and temperature levels. The response of the PABA film to CO2 concentration was not affected by the presence of alcohols and ketones at 1% of vapour pressure, proving that the developed sensor is not cross-sensitive to these compounds which may be present in spoiling grain. The sensor packaging components were selected and built in such a way as to avoid contamination of the sensing material and the substrate by undesirable components including grain dust and chaff. The developed conducting polymer carbon dioxide sensor exhibited effective response, recovery time, sensitivity, selectivity, stability and response slope when exposed to various carbon dioxide levels inside simulated grain bulk conditions.

Link to paper

Page 6 of 9

Contact Us

Bionanotechnology Laboratory
Suresh Neethirajan

School of Engineering
University of Guelph
Guelph, Ontario
Canada N1G 2W1

Office:
Room 3513 - Richards Building
50 Stone Road East

Lab: THRN 2133 BioNano Lab

Phone: (519) 824-4120 Ext 53922
Fax: (519) 836-0227

E-mail: sneethir@uoguelph.ca

 
© 2016 Bionano Lab - University of Guelph. All Rights Reserved.