Development of a Portable Fluorescence Bacterial Detector

1
Development of a Portable Fluorescence Bacterial
Detector

• Texas AM- Commerce

2
People

• Team Members
• David Andrew Jacob
• Will Negrete
• Jeff E. Landry
• Holly Pryor
• Faculty Advisor
• Dr. Frank Miskevich

3
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

4
Necessity of Monitoring

• Bacteria Causes
• Allergy
• Food Spoilage / Poisoning
• Material Degradation
• Infectious Disease
• Tuberculosis
• Dysentery
• Pneumonia
• Cholera
• Plague
• Tetanus

5
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

6
Detection Methods

• Culture Dependent
• Plate Counting
• Cytosensor (?pH)

• Culture Independent
• Turbidimetry
• ATP Bioluminesence
• Quantitative PCR
• Solid Phase Cytometry
• Flow Cytometry
• Used to validate results.

7
Our Method
Bacterial Fluorescent Units

• 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)

8
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)

9
Proof of Concept

• Work done by Joseph Harvey, M.S.
• BFU results generated from our method correlates
  (P0.8051) to flow cytometer results

Flow Cytometer results pictured above. Shows
both dead and alive bacteria.
10
The Detector
11
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
12
Light Path
light generated by UV LED Reflected off dichroic
lens towards sample emission from sample passes
through dichroic lens toward camera
13
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
14
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
15
Software

• Stepper motor controller program
• Nikon D80 camera software
• IMAGEJ
• Counting Macro

16
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
17
Sample Preparation
18
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

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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

20
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
21
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

22
Future Work
23
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

24
Future Work

• Develop antibody based, species specific
  fluorescent tags to give organism level
  identification capabilities
• Would require that multiple light frequencies and
  dyes be used

25
Future Work

• 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

26
Future Work

• 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

27
Questions
??
28
References

• 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 AM- 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/in
  tro.html
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• Alsharif, Rana. Godfrey, William. Bacterial
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• La Duc, MT, Nicholson, WL, Kern, R,
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