Download Development of a Portable Fluorescence Bacterial Detector

Development of a Portable
Fluorescence Bacterial Detector
Texas A&M- Commerce
People
• Team Members
– David Andrew Jacob
– Will Negrete
– Jeff E. Landry
– Holly Pryor
• Faculty Advisor
– Dr. Frank Miskevich
Why is monitoring
important
to people both
on earth and in space?
Introduction
• Microorganisms can be found almost
anywhere on earth.
• There are more microorganisms living in and
on a human than the sum of the cells that
make up that human.
• Some are dangerous to humans, others are
benign.
Introduction
• 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
• Bacteria Causes
– Allergy
– Food Spoilage /
Poisoning
– Material Degradation
– Infectious Disease
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Tuberculosis
Dysentery
Pneumonia
Cholera
Plague
Tetanus
Monitoring Critical in Space
• 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
Fungus Growing on
Wall of ISS
Detection Methods
• Culture Dependent
– Plate Counting
– Cytosensor (ΔpH)
• Culture Independent
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Turbidimetry
ATP Bioluminesence
Quantitative PCR
Solid Phase Cytometry
Flow Cytometry*
*Used to validate results.
What is Our Method
& How Does it
Work
Our Method
Bacterial Fluorescent Units
Test photo from microscope.
Note: artifacts are not bacteria, nor
should “cloudy” areas exist.
• 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
• 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
• 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.
Sample Preparation
Sample Preparation
• 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
The Detector
&
previous work
The Detector
Detector Overview
1. Digital Camera
2. Infinitube
3. UV LED
4. Bandpass filter
5. Microscope
objective lens
6. Stepper motor
7. Laptop
8. 19.2 VDC Power
supply
9. Motor driver
10. Laptop Interface
11. Dichroic mirror
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
Note Addition on Solenoid and
controller board;
Triggered from PSMC
Software
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Stepper motor controller program
Nikon D80 camera software
IMAGEJ
Counting Macro
Major Problem Solved: Computer Science
Graduate Student Joining Team Next Semester
IMAGEJ
• 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
The Detector
Current Work:
•Integrate camera trigger and stepper
controller
•Increase UV light intensity
•Increase structural integrity &
refinement of device
Increase UV Intensity
Light generated by UV LED(s).
Reflected off dichroic lens towards
sample or generated by “ring of LEDs”
near sample.
Ring of LEDs added to increase light
intensity. Single LED source from
microscope tube proved to be
inadequate.
Both sources are going to be used in
future.
Activated on same circuit as original
LED.
Increase UV Intensity
• Five UV LEDs in series
for ~19.2V draw from
battery.
• LEDs will be focused so
that their beams
converge on the same
point within the focal
plane of the camera.
Camera Trigger
•Trigger activated
via stepper motor
controller
Camera Trigger
•Force limited by solenoid
controller board so as not
to damage trigger
•Operated off 19.2VDC
battery activated by 5VDC
TTL signal
Strengthening of Device Structure
• Must be rigid
otherwise focus
changes are
possible. Focal
length isvery small.
• “L” brackets added.
Strengthening of Device Structure
•Motor shim
added to assist in
maintaining
coplanar focus.
•Critical to
function and
ability of get
clear, uniformly
focused pictures.
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
Graduate computer science student
Recruited to assist with integration of
Software components into
single, user-friendly package.
White Blood Cell Counts
• Erythrocytes (Red Blood Cells) are anucleated.
• White blood cells have nuclear material.
Left: Electron micrograph of
RBC
Above: stained in purple, WBC
(neutrophil)
White Blood Cell Counts
•Our dye (Hoechst 33258)
stains only DNA.
•Therefore, we can select
preferentially for WBC and
utilize the same process to
estimate number of WBCs
present in a given volume on
blood.
White Blood Cell Counts
• Method of operation very similar.
• Given a specific volume of blood our detector
can generate WBCs per volume data.
• White blood cell counts good marker for
immune function and disease states.
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, Texas A&M- Commerce.
Bruce, Rebekah. Microbial Surveillance During Long-Duration Spaceflight. Bioastronautics Technology Forum. URL:
http://advtech.jsc.nasa.gov/btf05.htm 2005
Rasband, Wayne. Introduction to ImageJ. ImageJ website. 2008. http://rsb.info.nih.gov/ij/docs/intro.html
Obuchowska, Agnes. Quantitation of bacteria through adsorption of intracellular biomolecules on carbon paste and screen-printed carbon electrodes
and volammetry of redox-active probes. Ana Bioanal Chem. 2008.
Ortmanis, A., Patterson W.I., Neufeld, R.J. Evaluation of a new turbidimeter design incorporating a microprocessor-controlled variable pathlength
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Microbiological Reviews. Dec. p.641-696. 1996.
Alsharif, Rana. Godfrey, William. Bacterial Detection and Live/Dead Discrimination by Flow Cytometry. BD Biosciences, San Jose, CA, 2002.
La Duc, MT, Nicholson, WL, Kern, R, Venkateswaran, K Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility.
Environmental Microbiology. 2003.
Questions