Transcript Development of a Portable Fluorescence Bacterial Detector

Development of a Portable
Fluorescence Bacterial
Detector
Texas A&M- Commerce
People
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Team Members
• David Andrew Jacob
• Will Negrete
• Jeff E. Landry
• Holly Pryor
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Faculty Advisor
• Dr. Frank Miskevich
Introduction
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Bacteria are a major
contributor to human
disease
Fast generation time
(exponential growth)
Can spread quickly in
compact populations
as seen in space
stations and space
craft
Necessity of Monitoring
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Bacteria Causes
• Allergy
• Food Spoilage /
Poisoning
• Material Degradation
• Infectious Disease
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Tuberculosis
Dysentery
Pneumonia
Cholera
Plague
Tetanus
Monitoring Critical in Space
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Air and Water Recycled
Limited Personal Hygiene
Infectious Disease
spreads quickly in close
living quarters
Difficult to isolate sick
individual from crew
Despite our best efforts
microbes still inhabit the
space station
Detection Methods
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Culture Dependent
• Plate Counting
• Cytosensor (ΔpH)
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Culture Independent
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Turbidimetry
ATP Bioluminesence
Quantitative PCR
Solid Phase
Cytometry
• Flow Cytometry*
*Used to validate results.
Our Method
Bacterial Fluorescent Units
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Culture Independent
Bacteria marked
with a non-toxic,
fluorescent DNA
binding dye
(Hoechst 33258)
Each fluorescing
bacteria is counted
to give X bacterial
fluorescent units
(BFUs)
Our Method
Bacterial Fluorescent Units
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Counts both dead and alive bacteria
Does not require prior knowledge of
organism to be cultured to quantify
Estimated that only 1% of present bacteria
grow in culture dependent bacteria (La
Duc, 2003)
Proof of Concept
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Work done by Joseph
Harvey, M.S.
BFU results generated
from our method
correlates (P=0.8051)
to flow cytometer
results
Flow Cytometer results
pictured above. Shows
both dead and alive
bacteria.
The Detector
Detector Overview
1.
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Digital Camera
Infinitube
UV LED
Bandpass filter
Microscope
objective
lens
6. Stepper motor
7. Laptop
8. 19.2 VDC
Power
supply
9. Motor driver
10. Laptop
Interface
11. Dichroic
mirror
Light Path
light generated by UV LED
Reflected off dichroic lens
towards sample
emission from sample passes
through dichroic lens toward
camera
Filters
Dichroic lens reflects 350nm light
and allows 450nm sample emission
to pass through
450nm bandpass filter selects for
light very close to the 450nm
spectrum
“cleans up” picture seen by camera
by reducing noise
Integration of Parts
Stepper motor and UV LED
activation coordinated by
programmable step motor
controller
Relay Used to allow 5 VDC
TTL activation of UV LED
Single USB hook up to
laptop controller
Software
Stepper motor controller program
 Nikon D80 camera software
 IMAGEJ
 Counting Macro
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IMAGEJ
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Free software by National Institute of
Health (NIH)
•Raw Images sharpened
•Delineates boundaries positive
for bacteria and background
•Counting macro used to count
bacteria
•Clusters of bacteria counted
based on area and individual
number of bacteria estimated
bacterial image
selected areas
Sample Preparation
Sample Preparation
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Escherichia coli suspensions used to
test device
• Gram-negative rod, Non-sporulating
• 2 μm long X 0.5 μm in diameter
• Cell volume = ~0.6 - 0.7 μm3
• Very common flora
in human GI tract
Sample Preparation
•Hoechst 33258 is added to liquid bacteria
sample at 1 micro liter per milliliter sample
•Liquid sample is then drawn up into syringe
•Sample is pass through 0.2 micron filter
•Filter is put into sample holder and
photographed
Sample Holder
Polycarbonate Filter Sandwiched
between parts B and C (Above &
Right)
Parts A and D attached to stepper
motor. Allows parts B & C to be held
in front of the camera assembly
Post-Development Testing
Filters will be experimented with to
get best picture quality and least
noise
 Counting Macro will be “tweaked”
such that results match that of the
flow cytometer
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Future Work
Future Work
Integrate all software (camera controller,
motor / LED controller, IMAGEJ and
counting macro) into one easy to use
package that can be loaded onto the
detectors memory stick and allow USB
“Plug & Play” compatibility
Future Work
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Develop antibody based, species specific
fluorescent tags to give organism level
identification capabilities
Would require that multiple light frequencies and
dyes be used
Future Work
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Scale down detector size and weight to
allow for greater portability
• Custom cut lens to reduce length and focal
distance
• Replace camera with high quality, small CCD
• Integrate laptop and detector into one
functional unit
Future Work
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Research the possibility of using a liquid filled
column to pass the bacteria sample in front a
camera to eliminate the need of the black
polycarbonate filters and decrease required
handling and preparation of the sample
Questions
References
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Harvey, Joseph E. "The development and implementation of a portable fluorescence bacterial detector." Thesis.
Miskevich, Frank, and Matthew Elam. Life at the Edge: Biology Beyond the Earth. Biology / Industrial Engineering,
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