1. 3D Imaging of Bacterial Biofilms
Kyle Hillegass, 2016, Chemical Engineering
Prof. Matthew Libera, CEMS
The Issue
Bacterial biofilms are a threat to the safe integration of
biomedical devices into the body. These device
surfaces are designed to enhance adhesion of desirable
tissue cells but do not inhibit bacterial biofilm growth.
Project Scope
Use the Zeiss Auriga Small Dual-Beam Focused Ion
Beam/Scanning Electron Microscope married with
traditional TEM sample preparation methods to
determine the 3-D morphology of an S. Aureus biofilm
grown on a polystyrene petri dish. Such datasets will
serve as a baseline for comparison of S. Aureus biofilms
grown on alternate surfaces.
Procedures and Methods
I&E Summer Scholars
2013
Microtomy
The microtome is used
for high precision slicing
of the sample surface to
allow for even deposition
and milling while using
the SEM/FIB system.
Bacterial Biofilm Culture
1. Initial attachment
(Van der Waals)
2. Irreversible
attachment
(cell adhesion)
3. Maturation I
4. Maturation II
5. Dispersion
TEM Sample Prep
The SEM requires the
sample to be dry, but an
air-dried sample loses its
structure and is too
fragile. Fixing the sample
into an epoxy medium
cures this issue.
FIB-SEM Slice & View
The SEM uses highly
accelerated electrons to
generate a digital image.
The FIB uses highly
accelerated Gallium ions
to mill material on the
order of 10-9 m. The Slice
& View method utilizes
both beams to generate
a set of 2D serial images
that can be rendered into
a 3D image.
Avizo Fire 3D Visualization
Avizo Fire is an advanced
visualization software
that generates 3D images
from a set of 2D serial
images. These 3D images
can be analyzed
qualitatively as well as
quantitatively. Each
image is then segmented
to highlight an area of
interest.
Conclusions
As expected, a thick S. Aureus biofilm grew on the
polystyrene (PS) petri dish thus creating a baseline for
comparison for other surface topographies. Using both
the quantitative and qualitative data from the 3D
model, S. Aureus biofilms can be grown on various
surface topographies under similar conditions to inhibit
growth. Future work would also include the utilization
of cryogenic preservation techniques to shorten the
overall length of the experiment.
References
1 MBW: Role Of Biofilm Matrix In Structural Development. 2013. Photograph. CU Boulder Applied Math Mathematical
Biology GroupWeb. 14 Aug 2013.
<http://mathbio.colorado.edu/mediawiki/index.php/MBW:Role_of_Biofilm_Matrix_in_Structural_Development>.
2 Bozzola, John J. "Conventional Specimen Preparation Techniques for Transmission Electron Microscopy of
Cultured Cells." Trans. Array Electron Microscopy: Methods and Protocols. 2nd. Totowa: Humana Press Inc.,
2007. 1-18. Print.
S. Aureus
1
2
SEM
FIB
PS
Biofilm
Epoxy
Results
Avizo Fire 3D model of S. Aureus biofilm (blue) grown on PS petri dish (yellow)
Epoxy