Development of a Portable Fluorescence Bacterial Detector

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Development of a Portable Fluorescence Bacterial
Detector

• Texas AM- Commerce

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People

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

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Why is monitoring important to people both on
earth and in space?
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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.

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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

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Necessity of Monitoring

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

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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
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Detection Methods

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

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

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What is Our Method How Does it Work
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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)

Test photo from microscope. Note artifacts are
not bacteria, nor should cloudy areas exist.
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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)

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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.
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Sample Preparation
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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

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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
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The Detector previous work
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The Detector
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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
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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
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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
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Software

• Stepper motor controller program
• Nikon D80 camera software
• IMAGEJ
• Counting Macro
• Major Problem Solved Computer Science Graduate
  Student Joining Team Next Semester

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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
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The Detector

• Current Work
• Integrate camera trigger and stepper controller
• Increase UV light intensity
• Increase structural integrity refinement of
  device

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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.
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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.

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Camera Trigger

• Trigger activated via stepper motor controller

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Camera Trigger

• Force limited by solenoid controller board so as
  not to damage trigger
• Operated off 19.2VDC battery activated by 5VDC
  TTL signal

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Strengthening of Device Structure

• Must be rigid otherwise focus changes are
  possible. Focal length isvery small.
• L brackets added.

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Strengthening of Device Structure

• Motor shim added to assist in maintaining
  coplanar focus.
• Critical to function and ability of get clear,
  uniformly focused pictures.

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Future Work
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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.

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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)
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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.

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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.

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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
• 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 cuvette. Enzyme Microb.
  Technol., vol. 13, June, 1991.
• Heid, C. A., J. Stevens, K. J. Livak, and P. M.
  Williams. Real time quantitative PCR. Genome Res.
  6986-994. 1996.
• Lyons, Sharon, et al. Quantitative real-time PCR
  for Porphyromonas gingivalis and total bacteria.
  Journal of Clinical Microbiology, June, Vol. 38,
  p.2362-2365. 2000.
• Cools, I. et al. Solid phase cytometry as a tool
  to detect viable but non-culturable cells of
  Campylobacter jejuni. Journal of Microbiological
  Methods. Vol. 63. Issue 2. p. 107-114. 2005.
• Bach, HJ. et al. Enumeration of total bacteria
  and bacteria with genes for proteolytic activity
  in pure cultures and in environmental samples by
  quantitative PCR mediated amplification. Journal
  of Microbial Methods. 49235-245. 2002.
• Li, C.S. et al. Fluorochrome and flow cytometry
  to monitor microorganisms in treated hospital
  water. J Environ Sci Health A Tox Hazad Subst
  Environ Eng. Feb42(2)195-203. 2007.
• Davey, H.M., Kell, D. B. Flow cytometry and cell
  sorting of heterogeneous microbial populations
  the importance of single-cell analyses.
  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.

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Questions
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