1. Development of a Portable Fluorescence Bacterial Detector Texas A&M- Commerce

2. People  Team Members David Andrew JacobDavid Andrew Jacob Will NegreteWill Negrete Jeff E. LandryJeff E. Landry Holly PryorHolly Pryor  Faculty Advisor Dr. Frank MiskevichDr. 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 AllergyAllergy Food Spoilage / PoisoningFood Spoilage / Poisoning Material DegradationMaterial Degradation Infectious DiseaseInfectious 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 CountingPlate Counting Cytosensor (ΔpH)Cytosensor (ΔpH)  Culture Independent Turbidimetry ATP Bioluminesence Quantitative PCR Solid Phase Cytometry Flow Cytometry* * Used to validate results.

7. 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

8. 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

9. 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.

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.  Escherichia coli suspensions used to test device Gram-negative rod, Non-sporulatingGram-negative rod, Non-sporulating 2 μm long X 0.5 μm in diameter2 μm long X 0.5 μm in diameter Cell volume = ~0.6 - 0.7 μm 3Cell volume = ~0.6 - 0.7 μm 3 Very common floraVery common flora in human GI tract in human GI tract

19. 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. 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 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 Scale down detector size and weight to allow for greater portability Custom cut lens to reduce length and focal distance Custom cut lens to reduce length and focal distance Replace camera with high quality, small CCD Replace camera with high quality, small CCD Integrate laptop and detector into one functional unit 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 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 A&M- Commerce.  Bruce, Rebekah. Microbial Surveillance During Long-Duration Spaceflight. Bioastronautics Technology Forum. URL: http://advtech.jsc.nasa.gov/btf05.htm 2005 http://advtech.jsc.nasa.gov/btf05.htm 2005 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 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, 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. 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 Eng. Feb;42(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.