Development of a Portable Development of a Portable Fluorescence Bacterial DetectorFluorescence Bacterial Detector

Texas A&M- CommerceTexas A&M- Commerce

People• Team Members– David Andrew Jacob– Will Negrete– Jeff E. Landry– Holly Pryor

• Faculty Advisor– Dr. Frank Miskevich

Why is monitoring Why is monitoring important important

to people bothto people both

on earth and in space?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

• 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 onWall of ISS

Detection Methods

• Culture Dependent– Plate Counting– Cytosensor (ΔpH)

• Culture Independent– Turbidimetry– ATP Bioluminesence– Quantitative PCR– Solid Phase Cytometry – Flow Cytometry*

*Used to validate results.

What is Our Method & How Does it

Work

Our Method

• 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)

Bacterial Fluorescent Units

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

Our Method

• 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)

Bacterial Fluorescent Units

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 The Detector & &

previous workprevious work

The Detector

Detector Overview1. Digital Camera2. Infinitube3. UV LED4. Bandpass filter5. Microscope

objective lens6. Stepper motor7. Laptop8. 19.2 VDC Power

supply9. Motor driver10. Laptop Interface11. Dichroic mirror

FiltersDichroic 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 PartsStepper 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

• 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 DetectorThe DetectorCurrent Work:Current Work:

•Integrate camera trigger and Integrate camera trigger and stepper controllerstepper controller

•Increase UV light intensityIncrease UV light intensity•Increase structural integrity & Increase structural integrity &

refinement of devicerefinement of device

Increase UV IntensityLight 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 WorkFuture Work

Future WorkFuture Work

Integrate all software (camera controller, motor / LED Integrate all software (camera controller, motor / LED controller, IMAGEJ and counting macro) into one easy to controller, IMAGEJ and counting macro) into one easy to use package that can be loaded onto the detectors use package that can be loaded onto the detectors memory stick and allow USB “Plug & Play” compatibilitymemory stick and allow USB “Plug & Play” compatibility

Graduate computer science student Graduate computer science student Recruited to assist with integration of Recruited to assist with integration of Software components intoSoftware components intosingle, user-friendly package.single, user-friendly package.

White Blood Cell Counts

• Erythrocytes (Red Blood Cells) are anucleated.• White blood cells have nuclear material.

Left: Electron micrograph of RBCAbove: stained in purple, WBC (neutrophil)

•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 CountsWhite Blood Cell Counts

• Method of operation very similar.Method of operation very similar.• Given a specific volume of blood our detector Given a specific volume of blood our detector

can generate WBCs per volume data.can generate WBCs per volume data.• White blood cell counts good marker for White blood cell counts good marker for

immune function and disease states.immune function and disease states.

White Blood Cell CountsWhite Blood Cell Counts

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 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

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. 6:986-994. 1996.• Lyons, Sharon, et al. Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria. Journal of Clinical Microbiology, June, Vol. 38,

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quantitative PCR mediated amplification. Journal of Microbial Methods. 49:235-245. 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

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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.

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