BACTERIAL SPECIES SPECTROSCOPIC CHARACTERIZATION BY FTIR, NORMAL RAMAN AND SURFACE ENHANCED RAMAN SCATTERING

By Tatiana Luna Pineda A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE In Chemistry UNIVERSITY OF PUERTO RICO MAYAGEZ CAMPUS 2006
Approved by: ________________________________ Samuel P. Hernndez Rivera, Ph.D. President, Graduate Committee ________________________________ Carlos Rios Velazquez, Ph.D. Member, Graduate Committee ________________________________ Julio G. Briano Peralta, Ph.D. Member, Graduate Committee ________________________________ Nairmen Mina Camilde, Ph.D. Member, Graduate Committee ________________________________ Guillermo Colon Burgos, Ph.D. Representative of Graduate Studies ________________________________ Francis Patrn, Ph.D. Chairperson of the Chemistry Department __________________ Date __________________ Date __________________ Date __________________ Date __________________ Date __________________ Date

ABSTRACT Bioterrorism and its high potential for mass destruction has been subject of increasing international concern. Only modest microbiological skills are needed to produce and effectively use biological weapons. Production costs are low and aerosol dispersal equipment from commercial sources can be adapted for biological weapon dissemination. These prevailing conditions have motivated the increased interest in the application of several physicochemical analytical techniques for the rapid detection and identification of microorganisms. Fourier Transform Infrared (FTIR), Raman spectroscopy and Surface Enhanced Raman Scattering (SERS) require minimum sample and allow for fast identification of microorganisms. The use of these techniques for characterization of the spectroscopic signatures of these agents and their simulants has recently gained considerable attention because these techniques can be easily adapted for standoff detection from considerable distances. The techniques also show high sensitivity, selectivity, and offer near real time detection duty cycles. This research focuses in laying the grounds for the simultaneous spectroscopic detection and differentiation of Staphylococcus spp., Pseudomonas spp., Bacillus spp., and Enterobacteriaceae spp., together with discrimination of their species. In order to achieve the proponed objective, protocols to handle, cultivate and analyze the bacterial species have been developed. Spectroscopic similarities and marked differences have been found for FTIR and Spontaneous or Normal Raman spectra, and SERS spectra using silver nanoparticles assisted spectroscopy. The use of principal component analysis (PCA), discriminate factor analysis (DFA) and a hierarchical cluster analysis (HCA) were used to evaluate the efficiency of identifying potential threat bacterial from their spectra collected on single bacteria. The first DFA and HCA for the Raman, SERS and FTIR spectra of the bacteria showed low discrimination between the diverse bacterial species. However, the results obtained from a second DFA and HCA demonstrate the high discrimination capability of the techniques. The spectroscopic study could be extended to examine the spores produced by selected strains since these are more prone to be used as Biological Warfare Agents (BWA) due to their increased mobility and possibility of airborne transport.

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RESUMEN El bioterrorismo y su alto potencial de destruccin masiva, ha sido un tema que ha aumentado la preocupacin de la comunidad internacional. Las habilidades microbiolgicas necesarias para producir y utilizar con eficacia las armas biolgicas, son realmente modestas. Los costos de produccin son bajos y los equipos comerciales de dispersin por aerosol se pueden adaptar fcilmente para la difusin de las armas biolgicas. Estas condiciones han desarrollado un creciente inters en el uso de algunas tcnicas fisicoqumicas analticas para la rpida deteccin e identificacin de microorganismos. La espectroscopia de Infrarrojo con transformada de Fourier (FTIR), la espectroscopia Raman y la espectroscopia de dispersin Raman amplificada por superficie (SERS), requieren una mnima preparacin de la muestra para una identificacin rpida de microorganismos. El uso de estas tcnicas para la caracterizacin de la huella espectroscpica de estos agentes y de sus simulantes ha aumentado considerablemente, debido al hecho de que estas tcnicas se pueden adaptar fcilmente para la deteccin de estos a distancias considerables. Adems, estas tcnicas muestran alta sensibilidad, selectividad y los tiempos de deteccin son bastante cortos. Esta investigacin se enfoca en establecer las bases para la deteccin y diferenciacin espectroscpica de Staphylococcus spp., Pseudomonas spp., Bacillus spp. y Enterobacteriaceae spp., junto con la identificacin de sus especies. Para alcanzar el objetivo propuesto, se han desarrollado protocolos para el manejo, cultivo y anlisis de las especies bacterianas. Se han encontrado semejanzas y diferencias espectroscpicas marcadas para los espectros de FTIR, Raman y SERS usando nanopartculas de plata. Para evaluar la eficacia de las tcnicas espectroscpicas en la diferenciacin de las especies bacterianas puras, se utiliz, anlisis de componentes principales (PCA), anlisis de factor discriminante (DFA) y por ultimo un anlisis jerrquico de agregados (HCA). El primer DFA y HCA para las tres tcnicas espectroscpicas mostr baja discriminacin entre las especies bacterianas. Sin embargo, un segundo anlisis realizado para cada tcnica, muestran una alta capacidad de discriminacin. El estudio se podra ampliar a las esporas producidas por algunos de estos microorganismos, puesto que estos son ms propensos a ser utilizados como agentes biolgicos, debido su fcil manejo y transporte.

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Copyrightby TatianaLunaPineda 2006

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To my loves: God, my parents, my brother, my chocolate candy (J. Camilo) and Morty.

ACKNOWLEDGEMENTS
I want to thank for God to accompany itself at all the moments of my life. I want to start expressing a sincere acknowledgement to my advisor, Dr. Samuel Hernandez-Rivera because she gave me the opportunity to research under her guidance and supervision. I would like to thank Dr. Carlos Rios-Velazquez for allowing me to use the lab facilities and for the great interest that he has shown in my work. To my graduate committee: Dr. Nairmen Mina and Dr. Julio Briano, for their collaboration during this period. To Kristina Soto, thanks for helping me all the time and the lab partners: Edwin De La Cruz, Leonardo Pacheco, Gabriel Perez and Indira Jerez by its collaboration in this work. To my friends: Edwin De La Cruz, Andrea Cabanzo, Myrna Reyes, Liliana Rizo and Celia Osorio. Thanks to Morty for his love and unconditional support. Words do not exist to thank for everything what it gives me. To my family, Ramiro Luna, Luz Marina Pineda, Javier R. Luna, J. Camilo Luna, Ana Julia de Luna, Dario Pineda, Graciela Navas, and the Pineda Princess: Laura, Jesica, Diana and Leydi, for their unconditional love and inspiration. Collaboration with the Center for Chemical Sensors Development of the University of Puerto RicoMayagez sponsored by the Department of Defense, University Research InitiativeMultidisciplinary University Research Initiative (URIMURI) Program, under grant number DAAD19-02-1-0257.

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Finally, I would like to acknowledge the University of Puerto Rico, Mayagez Campus and department of Chemistry for the opportunity to study in Puerto Rico.

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TABLE OF CONTENT

ABSTRACT ................................................................................................................................................. II RESUMEN ..................................................................................................................................................III ACKNOWLEDGEMENTS ...................................................................................................................... VI 1. INTRODUCTION .................................................................................................................................... 1 2. EXPERIMENTAL BACKGROUND ..................................................................................................... 6 2.1 BACTERIAL CELLS .............................................................................................................................. 6 2.1.1 Surface Structures....................................................................................................................... 6
2.1.1.1 Flagella ................................................................................................................................................. 6 2.1.1.2 Pili (Fimbriae)...................................................................................................................................... 7 2.1.1.3 Capsules ............................................................................................................................................... 7

2.1.2 Important Chemical Components of Surface Structures........................................................... 7

2.1.2.1 Cell Wall Peptidoglycans .................................................................................................................... 7 2.1.2.2 Teichoic Acids ...................................................................................................................................... 7 2.1.2.3 Lipoteichoic Acids ............................................................................................................................... 8 2.1.2.4 Lipopolysaccharides ............................................................................................................................ 8 2.1.2.5 Wall-Less Forms.................................................................................................................................. 8

2.1.3Cytoplasmic Structures ................................................................................................................ 8

2.1.3.1 Plasma Membrane............................................................................................................................... 8 2.1.3.2 Organelles ............................................................................................................................................ 9 2.1.3.3 Endospores ........................................................................................................................................... 9

2.1.4 Gram-Positive .............................................................................................................................. 9

2.1.4.1 Bacillus spp. ......................................................................................................................................... 9 2.1.4.1.1 Bacillus subtilis ............................................................................................................................ 9 2.1.4.1.2 Bacillus cereus ........................................................................................................................... 10 2.1.4.1.3 Bacillus thuringiensis ................................................................................................................ 10 Bacillus thuringiensis generally occurs in the soil. Pathogenicity for larvae of Lepidoptera and the production in the cell of a protein parasporal crystal [Bergeys Manual, 1984]. ....................................... 10 2.1.4.2 Staphylococcus spp. ........................................................................................................................... 10 2.1.4.2.1 Staphylococcus epidermidis ....................................................................................................... 10 2.1.4.2.2 Staphylococcus aureus ............................................................................................................... 10

2.1.5 Gram-Negative .......................................................................................................................... 11

2.1.5.1 Enterobacteriaceae spp. ..................................................................................................................... 11 2.1.5.1.1 Escherichia coli .......................................................................................................................... 11 2.1.5.1.2 Salmonella tennessee ................................................................................................................. 11 2.1.5.1.3 Klebsiella pneumoniae ............................................................................................................... 11 2.1.5.1.4 Enterobacter aerogenes ............................................................................................................. 12 2.1.5.1.5 Proteus mirabilis ........................................................................................................................ 12 2.1.5.2 Pseudomonas spp. .............................................................................................................................. 12 2.1.5.2.1 Pseudomonas aeruginosa .......................................................................................................... 12

2.2 BASIC PRINCIPLES OF OPTICAL SPECTROSCOPY ............................................................................ 13 2.3 INFRARED SPECTROSCOPY (IR) ....................................................................................................... 14 2.3.1 FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY .......................................................... 16 2.3.1 Advantages of FTIR spectroscopy ............................................................................................ 18 2.4 RAMAN SPECTROSCOPY.................................................................................................................... 19 2.5 SURFACE ENHANCED RAMAN SPECTROSCOPY (SERS) .................................................................. 21 2.5.1 Introduction to SERS ................................................................................................................ 21 2.5.2 SERS mechanisms and SERS enhancement factors ............................................................... 22 3. PREVIOUS WORK ............................................................................................................................... 28 4. METHODOLOGY ................................................................................................................................. 31

4.1 REAGENTS AND CHEMICALS ............................................................................................................. 31 4.2 ORGANISMS SELECTION ................................................................................................................... 31 4.3 INSTRUMENTATION ........................................................................................................................... 32 4.4 GROWTH KINETICS OF BACTERIAL ................................................................................................. 33 4.5 SYNTHESIS OF SILVER COLLOIDS ..................................................................................................... 34 4.6 SEM IMAGES OF BACTERIAL CELLS................................................................................................ 34 4.7 BACTERIAL SAMPLE PREPARATION ................................................................................................. 34 4.7.1 Raman Sample Preparation ...................................................................................................... 35 4.7.2 SERS Sample Preparation ........................................................................................................ 35 4.7.3 FT-IR Sample Preparation ....................................................................................................... 36 4.8 DATA ANALYSIS ................................................................................................................................ 36 5. RESULTS AND DISCUSSION............................................................................................................. 37 5.1 CHARACTERIZATION OF SILVER COLLOIDS .................................................................................... 37 5.1.1 Plasmon Absorption Band for Silver Colloids ......................................................................... 37 5.1.2 Silver Colloid used for adenine detection ................................................................................. 38 5.2 BACTERIA SPECIES DATA ANALYSIS ................................................................................................ 38 5.2.1 Growth curve ............................................................................................................................. 38 5.2.2 SEM IMAGES .................................................................................................................................. 39 5.3 RAMAN AND SERS SPECTRA............................................................................................................. 40 5.3 FTIR SPECTRA .................................................................................................................................. 48 5.4 STATISTICAL DATA ANALYSIS .......................................................................................................... 51 CONCLUSIONS......................................................................................................................................... 64 FUTURE WORKS ..................................................................................................................................... 66 REFERENCES ........................................................................................................................................... 67 APPENDIX A ............................................................................................................................................. 70

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List of Figures Figure 1. Typical energy-level diagram showing the ground state and the first excited stated. ........................................................................................................................ 14 Figure 2. The electromagnetic spectrum........................................................................... 16 Figure 3. FTIR spectrometer. a) Block scheme of the basic components of an FTIR spectrometer. b) Working principle of a Michelson interferometer consisting of a light source, beam splitter, fixed mirror, moving mirror, detector and sample (upper panel). A single frequency light source is transformed to the interferogram (lower panel)......................................................................................................................... 17 Figure 4. Simple schematic diagram for understanding the concept of electromagnetic SERS enhancement................................................................................................... 23 Figure 5. SERS-active colloidal. Silver particles in different aggregation stages, demonstrating the fractal nature of these structures together with the appropriate extinction curves [kneipp, K. et al., 2002]................................................................ 26 Figure 6. Renishaw Raman Microspectrometer RM-2000. .............................................. 32 Figure 7. FTIR spectrometer, Bruker IFS 66v/S............................................................... 33 Figure 8. UV-VIS spectra of colloidal silver nanoparticles used in this research: ........... 37 Figure 9. Raman Spectra: a) adenine 1x10-4 M with silver colloid; b) silver colloid; c) adenine 1x10-4 M. ..................................................................................................... 38 Figure 10. The growth behavior of bacteria: I. a) B. subtilis, b) B. cereus, c) B. thuringiensis; II. d) S. epidermidis, e) S. aureus. III. a) E. coli, b) S. tennessee, c) K. pneumoniae, d) E. aerogenes, e) P. mirabilis and IV. P. aeruginosa. ................................................................................................................................... 39 Figure 11. SEM images of Bacillus. a)B. subtilis, b)B. cereus and c)B. thuringiensis .... 39 Figure 12. SEM images to Enterobacteriaceae species and Pseudomonas aeruginosa: a) E. coli, b)S. tennessee, c) K. pneumoniae, d) E. aerogenes, e) P. mirabilis; f) P. aeruginosa................................................................................................................. 40 Figure 13. Bacillus thuringiensis Normal Raman spectrum (30x) (blue) and the SERS spectrum (fuchsia)..................................................................................................... 42 Figure 14. Escherichia coli Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia) .................................................................................................................... 42 Figure 15. Representative Raman spectra of Gram-positive species: a) B. subtilis; b) B. cereus; c) B. thuringiensis; d) Staphylococcus epidermidis; and e) S. aureus........................................................................................................................ 43 Figure 16. Representative Raman spectra of each Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes; e) P. mirabilis; f) P. aeruginosa................................................................................................................. 43 Figure 17. Representative SERS spectra of Gram-positive species: a)B. subtilis; b) B. cereus; c) B. thuringiensis; d) S. epidermidis; and e) S. aureus. ......... 44 Figure 18. Representative SERS spectra of Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes;, e) P. mirabilis; f) P. aeruginosa................................................................................................................. 44

Figure 19. The averaged FTIR spectra of Gram-positive species: a) B. subtilis; b) B. cereus; c) B. thuringiensis; d) Staphylococcus epidermidis; and e) S. aureus........................................................................................................................ 48 Figure 20. Averaged FTIR spectra of Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes; e) P. mirabilis; f) P. aeruginosa. ................... 48 Figure 21. PC-DFA plot of Raman spectra of the all bacterial species analyzed in this research. .................................................................................................................... 53 Figure 22. PC-DFA plot of raman spectra of B. subtilis, B. cereus, S. epidermidis and E. aerogenes .................................................................................................................. 53 Figure 23. Dendrogram resulting from HCA showing the relationship among all 11 bacteria species analyzed by Raman......................................................................... 54 Figure 24. Dendrogram resulting from HCA showing the relationship among four bacteria species analyzed by Raman Spectroscopy. ................................................. 55 Figure 25 PC-DFA plot of SERS spectra of the bacterial species analyzed in this research. .................................................................................................................... 56 Figure 26 PC-DFA plot of SERS spectra of the gram positive species of bacteria used in this research. ............................................................................................................. 57 Figure 27 PC-DFA plot of SERS spectra of the gram positive species of bacteria used in this research. ............................................................................................................. 57 Figure 28. Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by SERS. ...................................................................................... 58 Figure 29. Dendrogram resulting from HCA showing the relationship among grampositive bacteria species analyzed by SERS............................................................. 59 Figure 30 Dendrogram resulting from HCA showing the relationship among gramnegative bacteria species analyzed by SERS. ........................................................... 60 Figure 31 PC-DFA plot of FTIR spectra of the eleven bacterial species analyzed in this research ..................................................................................................................... 61 Figure 32 PC-DFA plot of FTIR spectra of the then bacterial species analyzed in this research ..................................................................................................................... 61 Figure 33 Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by FTIR. ....................................................................................... 62 Figure 34 Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by FTIR. ....................................................................................... 63

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List of Tables

Table 1 Approximate number of bacterial cell analyzed with Raman and SERS, and CFU/L for each bacterial specie. ............................................................................ 41 Table 2 Raman and SERS bands observed in spectra of gram-positive bacterial cells and tentative assignment.................................................................................................. 46 Table 3 Raman and SERS bands observed in spectra of gram-negative bacteria studied and tentative assignment. .......................................................................................... 47 Table 4 FTIR bands observed in spectra of gram-positive bacterial cells and tentative assignment................................................................................................................. 49 Table 5 FTIR bands observed in spectra of gram-positive bacterial cells and tentative assignment................................................................................................................. 50 Table 6 Bacterial species used in bacterial discrimination studies. .................................. 52

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APPENDIX LIST Figure A1. Bacillus subtilis Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.0x104. ................................................................ 70 Figure A2 Bacillus cereus Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 3.0x104. ................................................................ 70 Figure A3Staphylococcus aureus Raman spectrum (40x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 3.0x104. ................................................................ 71 Figure A 4Staphylococcus epidermidis Raman spectrum (30x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104. ................................................ 71 Figure A 5Salmonella tennessee Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104. ................................................................ 72 Figure A 6 Klebsiella pneumoniae Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104. ................................................................ 72 Figure A 7Pseudomonas aeruginosa Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104. ................................................ 73 Figure A 8 Enterobacter aerogenes Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 2.0x104. ................................................................ 73 Figure A 9Proteus mirabilis Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.0x104. ................................................................ 74

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1. INTRODUCTION

Analytical technologies are currently in demand for Homeland Security and National Defense applications. Domestic preparedness requires a wide array of detection capabilities for a range of potential attack scenarios. For example, a single location could be attacked by placing an explosive package, carrying an explosive on a person, releasing chemical/bacterial dispersion into the water supply, or an armed gunman. The threat then is expanded by considering the different types of explosives, chemical warfare (CW) agents, biological warfare agents (BWA) and toxins that need to be identified quickly and accurately. Today no single analytical technique is able to address all of the above possibilities; the future might bring a series of sensors that can be used to detect a series of hazardous materials in a rapid, multimode, reliable manner. As for the present time, a variety of analytical technologies is available to collectively detect and identify potential threats to the public in single mode operation.

Biological contamination is not only a medical problem. It also represents a hazardous threat for various food-related industries and plays a large role in pharmaceutical and biotechnology clean-room production environment. In these fields, a fast identification of pathogenic and no pathogenic microorganisms is necessary, because the time required for the identification of pathogens is an important determinant of infection-related mortality rates of hospitalized patients. Rapid identification techniques significantly reduce mortality and costs associated with infectious diseases. Identification and characterization of bacteria starts by inspecting the colony morphology when the cells have been cultured

on solid media, followed by microscopic analysis of gram-stained preparations [Rosch, P. et al., 2005]. Bacteria can be divided into groups based on their cell shape, arrangements and cell wall characteristics. Based on these morphological characteristics, a series of tests can be performed to examine the biochemical, physiological, and nutritional properties of an organism, leading to its identification. Generally, the tests are combined in a series of solid and/or liquid media, which are inoculated with bacteria and interpreted after a certain incubation period. A large number of different tests are often needed for a definitive identification of the bacteria. These tests require turnaround times from 24 h to up to 5 days between receipt of material and positive identification results by the clinician [Maquelin, K. et al., 2002]. Thus, empirical treatment with broad-spectrum antibiotics is often started while awaiting further identification of the bacteria. It has been reported that, as a result of this, 10-30% of patients suffering form bloodstream infections in intensive care units (ICUs) do not initially receive the correct antibacterial therapy. Mortality rates in this group have been reported to be 30-60% higher than in the group that promptly receives appropriate therapy [Ibrahim, E. et al., 2000]. Apart form the risk that the empirical treatment may not be effective at all, this practice may lead to adverse toxic side effects and it is known to aggravate problems with resistance to antibacterial agents [Maquelin, K. et al., 2003].

An alternative approach to the analysis of microorganisms is the application of vibrational spectroscopic techniques: infrared (IR) absorption, Normal Raman (NR) Scattering and Surface Enhanced Raman Scattering (SERS)], which have a long tradition

since the vibrational spectrum displays fingerprinting information of the chemical composition of each bacterium. While an IR spectroscopic investigation of bacterial cells requires a few hundred cells from controlled cultivation conditions for an analysis and drying steps, this is not necessary when applying Raman spectroscopy. In particular, when only a small sample amount is required when SERS spectroscopy is used [Rosch, P. et al., 2005].

In recent years, much effort has been invested in the development of new techniques for the identification of microorganisms, which include molecular methods, such as mass spectrometry, electrospray ionization and matrix-assisted laser desorption ionization, Fourier Transform Infrared (FTIR) and Raman spectroscopy. Among these methods, vibrational spectroscopy (FTIR and Raman) is a reagentless/solvenless procedure in which there is no need to add chemical dyes or labels for identification. The characteristics of the absorbed or scattered light depend on the molecules found within the sample and the environment in which the molecules found. Another advantage of vibrational spectroscopy is that it is a rapid, specific, and a noninvasive analytical method, which provides a promising, means for the identification of a single bacterium. When compared to FTIR, which is for measuring the IR absorption of the sample, Raman spectroscopy measures the inelastically scattered light following excitation. The advantages of Raman spectroscopy are suitability for aqueous samples analysis, the intense IR absorption of water is avoided and spectral bands are sharper and narrower and more distinguishable.

Vibrational spectra of bacterial cells consist of signal contributions of all components in the cells and therefore reflect their overall molecular composition. Nauman et al. (2000), using FTIR spectroscopy, showed that such spectra could serve to identify a wide range of microorganisms. More recently, Raman spectroscopy has been shown to have similar capabilities [Maquelin, K. et al., 2003]. The high discrimination power of vibrational spectroscopy is illustrated by the accurate differentiation of closely related bacterial species such as E. faecium, E. hirae, E. faecalis, E. gallinarum [Krisker, K. et al., 2003]. Various publications have confirmed the applicability of these methods to discriminate between bacteria at the species level and even at the strain level [Curk, M. et al., 1994]. Simplicity of sample preparation and speed of analysis are important advantages when vibrational methods are compared to classical microbial identifications tools [Pupples, G., 1999]. Biological molecules such as nucleic acids, proteins, lipids, and carbohydrates all generate specific Raman spectra, which provide biochemical information regarding the molecular composition, structure, and interactions in cells. The molecular composition and structure are different among different bacterial species. Therefore, from the wholecells spectra, single microorganisms could be identified and discriminated. Thus, the aim of the present research was to combine FTIR, Normal Raman and SERS spectroscopies with multivariate analysis for the reliable discrimination of bacteria. Eleven bacterial species were used to demonstrate the capability of spectroscopy techniques in the identification and discrimination of bacterial cells between various species.

This research started the discrimination of bacterial species at stationary phase with multivariate analysis technique of Principle Component Analysis (PCA). Then the spectroscopic discrimination of bacterial species were accomplished using other techniques such as: Discriminate Factor Analysis (DFA) and hierarchical cluster analysis (HCA).

2. EXPERIMENTAL BACKGROUND 2.1 Bacterial Cells All bacteria, both pathogenic and saprophytic, are unicellular organisms that reproduce by binary fission. Most bacteria are capable of independent metabolic existence and growth, but species of Chlamydia and Rickettsia are intracellular organisms. Bacterial cells are extremely small and are most conveniently measured in microns (0.1-6 ). They range in size from large cells such as Bacillus anthracis (1.0 to 1.3 m x 3 to 10 m) to very small cells such as Pasteurella tularensis (0.2 x 0.2 to 0.7 m). Mycoplasmas (atypical pneumonia group) are even smaller, measuring 0.1 to 0.2 m in diameter. Bacteria therefore have a high surface-to-volume ratio: about 100,000 [Beveridge, 1983; Ghuysen, 1994]. Prokaryotes have a nucleoid (nuclear body) rather than an enveloped nucleus and lack membrane-bound cytoplasmic organelles. The plasma membrane in prokaryotes performs many of the functions carried out by membranous organelles in eukaryotes. Multiplication is by binary fission.

2.1.1 Surface Structures 2.1.1.1 Flagella The flagella of motile bacteria differ in structure from eukaryotic flagella. A basal body anchored in the plasma membrane and cell wall gives rise to a cylindrical protein filament. The flagellum moves by whirling about its long axis. The number and arrangement of flagella on the cell are diagnostically useful.

2.1.1.2 Pili (Fimbriae) Pili are slender, hair like, proteinaceous appendages on the surface of many (particularly Gram-negative) bacteria. They are important in adhesion to host surfaces. 2.1.1.3 Capsules Some bacteria form a thick viscous polysaccharide gel outer capsule of high molecular weight; others have more amorphous slime layers. Capsules confer resistance to phagocytosis.

2.1.2 Important Chemical Components of Surface Structures 2.1.2.1 Cell Wall Peptidoglycans Both gram-positive and gram-negative bacteria possess cell wall peptidoglycans, which confer the characteristic cell shape and provide the cell with mechanical protection. Peptidoglycans are unique to prokaryotic organisms and consist of a glycan backbone of muramic acid and glucosamine (both N-acetylated), and peptide chains highly crosslinked with bridges in gram-positive bacteria (e.g., Staphylococcus aureus) or partially cross-linked in gram-negative bacteria. The cross-linking transpeptidase enzymes are some of the targets for-lactam antibiotics. 2.1.2.2 Teichoic Acids Teichoic acids are polyol phosphate polymers bearing a strong negative charge. They are covalently linked to the peptidoglycan in some gram-positive bacteria. They are strongly antigenic, but are generally absent in Gram-negative bacteria.

2.1.2.3 Lipoteichoic Acids Lipoteichoic acids as membrane teichoic acids are polymers of amphiphitic glycophosphates with the lipophilic glycolipid and anchored in the cytoplasmic membrane. They are antigenic, cytotoxic and adhesins (e.g., Streptococcus pyogenes). 2.1.2.4 Lipopolysaccharides One of the major components of the outer membrane of Gram-negative bacteria is lipopolysaccharide (endotoxin), a complex molecule consisting of a lipid A anchor, a polysaccharide core, and chains of carbohydrates. Sugars in the polysaccharide chains confer serologic specificity. 2.1.2.5 Wall-Less Forms Two groups of bacteria devoid of cell wall peptidoglycans are the Mycoplasma species, which possess a surface membrane structure, and the L-forms that arise from either Gram-positive or Gram-negative bacterial cells that have lost their ability to produce the peptidoglycan structures.

2.1.3Cytoplasmic Structures 2.1.3.1 Plasma Membrane The bacterial plasma membrane is composed primarily of proteins and phospholipids (about 3:1). It performs many functions, including transport, biosynthesis, and energy transduction.

2.1.3.2 Organelles The bacterial cytoplasm is densely packed with 70S ribosomes. Other granules represent metabolic reserves (e.g., poly-b-hydroxybutyrate, polysaccharide, polymetaphosphate, and metachromatic granules). 2.1.3.3 Endospores Bacillus and Clostridium species can produce endospores: heat-resistant, dehydrated resting cells that are formed intracellularly and contain a genome and all essential metabolic machinery. The endospore is encased in a complex protective spore coat.

2.1.4 Gram-Positive 2.1.4.1 Bacillus spp. Bacillus spp have straight rods shapes, measuring 0.5-2.5 m in diameter by 1.2-10.0 m in length. They produce endospores (very resistant to many adverse conditions), sporulation not repressed by exposure to air, flagella peritrichous, aerobic or facultative anaerobic, chemoorganotrophs [Bergeys Manual, 1984]. They exhibit an array of physiologic abilities that allow them to live a wide range of habitats, including many extreme habitats such as desert sands, hot springs and Arctic soils [Perreten et al., 2005]. 2.1.4.1.1 Bacillus subtilis Bacillus subtilis are round or irregular, surface dull; become opaque, aerobic, growth active at pH 5.5-8.5; endospores are widespread. They can ve generally can be found in the soil [Bergeys Manual, 1984]. B. subtilis is not considered a human pathogen [CDC, 2006].

2.1.4.1.2 Bacillus cereus The rods tend to occur in chains. Colonies have a dull or frosted glass appearance, and motile, extracellular products include hemolysin, soluble toxin lethal for mice and enzymes lytic for bacterial cells; spores are widespread [Bergeys Manual, 1984]. Multiplication has been observed chiefly in foods and may lead food poisoning [FDA, 2006]. B. cereus is the responsible for minority of the food borne illness [CDC, 2006]. 2.1.4.1.3 Bacillus thuringiensis Bacillus thuringiensis generally occurs in the soil. Pathogenicity for larvae of Lepidoptera and the production in the cell of a protein parasporal crystal [Bergeys Manual, 1984]. 2.1.4.2 Staphylococcus spp. Staphylococcus spp. have cells spherical of diameter: 0.5-1.5 m, occurring in single, pair or tetrads, nonmotile, facultative anaerobes, cell wall contain peptidoglycan and teichoic acid, chemoorganotrophs: metabolism respiratory and fermentative [Bergeys Manual, 1984]. 2.1.4.2.1 Staphylococcus epidermidis They form spheres, 0.5-1.5 m in diameter, slow growing and small colonies, cells occur dominantly in pairs and tetrads, colonies are smooth to mucous, raised, glistening, circular, entire and translucent to nearly opaque, chemoorganotrophic [Bergeys Manual, 1984]. 2.1.4.2.2 Staphylococcus aureus Spheres, 0.5-1.0 m in diameter, cells occur single and in pairs, colonies are smooth, raised, glistening, circular, entire and translucent, chemoorganotrophic metabolism

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respiratory and fermentative. [Bergeys Manual, 1984]. S. aureus cause minor skins infections, Toxic Shock Syndrome (TSS) [CDC, 2006].

2.1.5 Gram-Negative 2.1.5.1 Enterobacteriaceae spp. Straight rods, measurements 0.3-1.5 m in diameter by 0.6-3.0 m in length, motile by peritrichous flagella, pathogenicity, chemoorganotrophic: respiratory and fermentative metabolism, facultative anaerobic, parasitic on mammals and birds, plant [Bergeys Manual, 1984]. 2.1.5.1.1 Escherichia coli Cell diameter and length 1.1-1.5m x 2.0-6.0m, have polysaccharide capsules or microcapsules (LPS), motile by peritrichous flagella or non-motile, colonies are smooth, low convex, moist, gray, with a shiny surface and entire edge, chemoorganotrophic respiratory and fermentative metabolism. Occur in the lower part of the intestine of warm blooded animal [Bergeys Manual, 1984]. Insolated from extraintestinal sites it can be look upon as primarily opportunistic pathogen. Can be cause gastroenteritis [CDC, 2006]. 2.1.5.1.2 Salmonella tennessee Cell diameter and length 0.7-1.5 x 2.0-5.0m, usually motile with a peritrichous flagella, [Bergeys Manual, 1984]. Occur in the lower part of the intestine of warm blooded animal, can be cause gastroenteritis [CDC, 2006]. 2.1.5.1.3 Klebsiella pneumoniae Cell diameter and length 0.3-1.0m x 0.6-6.0m, capsulated, no-motile,

chemoorganotrophic: metabolism respiratory and fermentative [ATCC, 2006]. K.

11

pneumoniae is normally found in the intestine tract of the man and animals, but in low numbers. Pathogenicity: can be cause pneumonia, septicemia, infection in wouds or burns and infections in the urinary tract [CDC, 2006]. 2.1.5.1.4 Enterobacter aerogenes Cell diameter and length 6-1.0m x 1.2-3.0m, motile by peritrichous flagella, most strains of E. aerogenes are resistant to ampicillin and cephalosporins and sensitive to carbenicillin. Occur in water, sewage, soil, dairy products and the feces of man and animals [Bergeys Manual, 1984]. 2.1.5.1.5 Proteus mirabilis Cell diameter and length: 0.4-0.8 m x 1.0-3.0 m [Bergeys Manual, 1984]. Pathogenic: can be cause urinary tract infections; also secondary invaders, causing septic lesions at other sides of the body, occur in the intestine of humans and animals, also occur in manure, soil and polluted water some strains are hemolytic on blood agar, resistant to penicillins and cephalosporins [CDC, 2006]. 2.1.5.2 Pseudomonas spp. Straight or slightly curve rods, measurement 0.5-1.0m in diameter by 1.5-5.0 m in length, motile by polar flagella, strictly aerobic, chemoorganotrophs: metabolism is respiratory, never fermentative. 2.1.5.2.1 Pseudomonas aeruginosa Forms two main colony types (solid media): a) large, smooth, with flag edges and elevated center. b) Small, rough and convex. The bacteria can be insolated from the soil, and water, particularly from enrichment cultures for denitrifying bacteria. Pigments that the bacteria can be produce are: pyoverdin, pyocyanin and dark pigments [Bergeys

12

Manual, 1984]. In clinical cases can be insolated from would, burn and urinary tract infections. It is the causative agent of blue pus, origin of the synonym pyocyaneus. Occasionally pathogenic for plant [CDC, 2006].

2.2 Basic Principles of Optical Spectroscopy Spectroscopy is defined as the study of interaction of electromagnetic radiation with matter, excluding chemical effects. When an electromagnetic wave encounters a molecule, it can be either scattered (i.e., its direction of propagation changes) or absorbed (i.e., its energy is transferred to the molecule). If the electromagnetic radiation of the light is absorbed, the molecule is said to be excited. An excited molecule can posses any one of a set of discrete amounts of energy described by the laws of quantum mechanics. These amounts are called the energy levels of the molecule. The major energy levels are determined by the possible spatial distributions of the electrons and are called electronic energy levels, on these are superimposed vibrational levels which indicate the various modes of vibration of the molecule. All these energy levels are described by an energylevel diagram (Figure 1). A possible electronic transition between the ground state and the fourth vibrational level of the first excited is indicated bye long arrow. A vibrational transition within the ground state is indicated by the short arrow. The lowest electronic level is called the ground state and all others are excited states [Freifelder, D., 1982].

13

First excited state

ENERGY

Vibrational levels Ground state

Distance between electrons and nucleus or between atoms in a molecule.

Figure 1. Typical energy-level diagram showing the ground state and the first excited stated.

Etotal = Etransition+Eelectronic+Erotation+Evibration+Eelectron spin orientation+Enuclear spin orientation (Equation 1)

Energy can reside in molecules in a number of forms, of which the most important ones, are translational, electronic, vibrational, and rotational energy. Infrared spectroscopy is based on molecular vibrations and monitors the transition between vibrational energy level [Wilson et al., 1995]. Light is a electromagnetic wave. The energy of the wave is:
E=h.c/=h. (Equation 2)

In which h is Plancks constant, c is the velocity of light, is the wavelength and is the frequency [Freifelder, D., 1982].

2.3 Infrared Spectroscopy (IR) The IR region of the spectrum extends from the visible region until it overlaps the microwave, or very short radar, range at wavelengths of some millimeters (10-1-10-4 cm-1) (see Figure 2). The basic characteristic of this region is that is the number of waves per centimeter wavenumber, that is the number of waves per centimeter, is used to 14

characterize the radiation. The wavenumber unit is the reciprocal centimeter (cm-1) and, therefore, one is dealing with the convenient values of 10,000 cm-1 throughout the IR region. Also per convention, the IR region of the electromagnetic spectrum is subdivided into the Near Infrared Region (NIR), Mid Infrared Region (MIR) and Far Infrared Region (FIR) (Figure 2). An IR spectrum of a sample is obtained by scanning the intensity of IR radiation before and after passage of the IR beam through the sample. The IR spectrum is displayed by plotting the quantity:
T= Is/IR (Equation 3)

as a function of wavenumbers, where T is the transmittance, Is the intensity of the IR beam after and IR before passing through the sample. In most cases the absorbance A is used:
A= -log T (Equation 4)

Since the absorbance at a given wavelength is directly proportional to the concentration of a sample according to Beers law. The IR spectra of most materials consist of a large number of absorption (energy exchange) between discrete light quanta and mechanical motions (vibrational and rotational modes) of the molecules which are excited by the absorption of IR radiation. Since the constituents of a typical biological sample are present in a condensed phase (solids, liquids of solutions), only vibrational modes are observed. Consequently, IR spectra of biological specimens are only vibrational spectra [Naumann D., 2000].

15

1 10 100 1000 10 000

cm-1 s-1
-rays

108

1010

1012 1014 1018 1018

FIR MIR NIR FIR X-rays UV

Radio Waves

Figure 2. The electromagnetic spectrum.

2.3.1 Fourier Transform Infrared (FTIR) Spectroscopy With the advent of FTIR spectroscopy, major drawbacks of classical dispersive IR spectroscopy could be circumvented. Briefly, FTIR no longer measures one wavelength after the other and is not like dispersive spectrometers which are equipped whit prism and/or grating-monochromators where the light energy emanating from the IR source is strongly limited by several entrance and exit slits. FTIR, instead, applies interferometric modulation of radiation (Figure 3). As in dispersive instruments, a hot white-light source is used in the MIR (Figure 3a). A widespread type of interferometer used in FTIR is the so-called Michelson interferometer mounted with a KBr beam splitter coated with a germanium film to split the collimated IR beam into two parts [Figure 3. b) upper panel].

Waves

Micro Waves

Short

16

source

interferometer

sample

detector

amplifier

A/D Converter

Computer

A/D Converter

a)

Fixed mirror sample

Moving mirror

detector

SOURCE Single frequency

I( )

I(S)

S=1/2 Detector signal

White-light source

1
I(S)

S=1/4

I( )

Detector signal

b)

S=0

Figure 3. FTIR spectrometer. a) Block scheme of the basic components of an FTIR spectrometer. b) Working principle of a Michelson interferometer consisting of a light source, beam splitter, fixed mirror, moving mirror, detector and sample (upper panel). A single frequency light source is transformed to the interferogram (lower panel).

For rapid-scanning interferometers liquid nitrogen cooled mercury cadmium telluride (MCT) detectors are used. For slower scanning types of interferometer, pyroelectric detectors (e.g. a deuterated triglycine sulfate (DTGS) detector element) can bee used. The moving mirror of the interferometer (Figure 3 b, upper panel) yields a sinusoidal signal at the detector for each modulated frequency (Figure 3 b, central panel). For a white-light source all modulated signals superimpose to an interferogram as shown in Figure 3 b, lower panel. This interferogram is amplified and digitized by the A/D converter (Figure 2 a), computer stored, and finally transformed by fast Fourier transform (FT) techniques.

17

Thus, the auxiliary computer in case of FTIR is a virtual necessity. The interferometer can be thought of as a means of encoding the initial frequencies into a special form, which the detector can observe. The most important feature of the interferogram is that every individual data point of this signal contains information over the entire IR region. In essence, the detector is always observing all frequencies for the final representation of an IR spectrum. This, along with some other features of the interferometer, leads to several distinct advantages [Naumann, D., 2000].

2.3.1 Advantages of FTIR spectroscopy FTIR spectroscopy produces conventional spectrum and it has several important advantages. a) The most important advantage of FTIR spectroscopy for biological studies is that spectra of almost any biological material can be obtained in a wide variety of environments. b) The amount of sample is relatively small. c) FTIR method is a rapid and sensitive technique with sampling techniques that are easy to use. d) Since a computer is already used to obtain the Fourier transform, it is easy to perform many scans to improve the signal-to-noise ratio (noise adds up as the square root of the number scans, whereas signal adds linearly). e) The instrumentation is inexpressive compared to the cost of X-ray diffraction, NMR, ESR, and CD spectroscopic equipment and the operation of the equipment

18

is simple. Interpretation of the spectra is not particularly difficult and can be learned easily. f) Digital subtraction (that is, point-by-point substation of the separate spectra by a computer) can also be used to produce good difference spectra. This method has great advantages in obtaining infrared spectra in aqueous solutions. g) The FTIR has advantage in terms of spectral regions which originate from molecular vibrations and different molecular moieties. For example head group and hydrocarbon tails have spectral regions for membranes. h) There is no light scattering or fluorescent effects. [Mantsch, H. H. et al., 1996]

2.4 Raman Spectroscopy Raman spectroscopy is quite different from absorption spectroscopy in that it studies light scattered by a sample rather than light absorbed or emitted. Suppose a photon collides with a molecule in state a. If the energy of the photon is absorbed, the molecule makes a transition to the higher level. No matter what the energy of the photon is, the photonmolecule collision may scatter the photon, meaning that the photons direction of motion is changed. Although most the scattered photons undergo no change in frequency and energy (Rayleigh scattering), a small of the scattered photons exchange energy with the molecule during the collision. The resulting increase or decrease in energy of the scattered photons is the Raman Effect, first observed by C. V. Raman in 1928. Let 0 and scat be the incident photon and the Raman-scattered photon, respectively, and let Ea and Eb be the energies of the molecule before and after is scatters the photon. Conservation of energy gives:

19

h0 + Ea = hvscat Eb or E Eb Ea = h(0 - scat) h

(Equation 5)

The energy difference E is the difference between two stationary-state energies of the molecule, so measurement of the Raman shifts :

0 -scat

(Equation 6)

gives molecular energy-level differences. In Raman spectroscopy, one exposes the sample (gas, liquid, or solid) to monochromatic radiation of any convenient frequency 0. Unlike absorption spectroscopy, 0 need have no relation to the difference between energy levels of the sample molecules. Usually 0 lies in the visible or near-UV region. The Ramaneffect lines are extremely weak (only about 0.001 percent of the incident radiation is scattered, and only about 1 percent of the scattered radiation is Raman scattered). Therefore, the very intense light of a laser beam is used as the exciting radiation. Scattered light at right angles to the laser beam is focused on the entrance slit of a spectrometer, which disperses the radiation using a diffraction grating and records light intensity versus frequency, giving the Raman spectrum. Overtone and combination bands are usually too weak to be observed in Raman spectroscopy, so Raman spectra are simpler than IR spectra. As in IR spectra, vibrational Raman spectra of gases show rotational structure, but rotational structure is absent in Raman spectra of liquids and solids. It is difficult (but no impossible) to obtain IR spectra below 100 cm-1, but easy to obtain Raman spectra for the
w Raman shift in the range 10 to 100 cm-1, so Raman spectroscopy is the preferred w technique in this wavenumber range. For between 0 and 10 cm-1, the Raman-shifted

w lines are hidden by the strong Rayleigh-scattered peak at 0 .

20

An important advantage of vibrational Raman spectroscopy over IR spectroscopy arises form the fact that liquid water shows only weak vibrational Raman scattering in the Raman shift range 300-3000 cm-1 but has strong, broad IR absorptions in this range.

2.5 Surface Enhanced Raman Spectroscopy (SERS) 2.5.1 Introduction to SERS Surface Enhanced Raman Spectroscopy (SERS) is a Raman Spectroscopic (RS) technique that provides greatly enhanced Raman signal from Raman-active analyte molecules that have been adsorbed onto certain specially prepared metal surfaces. Increases in the intensity of Raman signal have been regularly observed on the order of 104-106, and can be as high as 108 and 1014 for some systems [Kneipp et al., 1999; Moskovits, 1985]. The importance of SERS is that it is both surface selective and highly sensitive where as RS is neither. RS is ineffective for surface studies because the photons of the incident laser light simply propagate through the bulk and the signal from the bulk overwhelms any Raman signal from the analytes at the surface. SERS selectivity of surface signal results from the presence of surface enhancement mechanisms only at the surface. Thus, the surface signal overwhelms the bulk signal, making bulk subtraction unnecessary. There are two primary mechanisms of enhancement described in the literature: an electromagnetic and a chemical enhancement. The electromagnetic effect is dominant, the chemical effect contributing enhancement only approximately an order or two of magnitude [Kambhampati et al., 1998]. The electromagnetic enhancement is dependent on the presence of the metal surfaces roughness features, while the chemical enhancement involves changes to the adsorbate electronic states due to chemisorptions of the analyte. The structural and molecular

21

identification power of RS can be used for numerous interfacial systems, including electrochemical, modeled and actual biological systems, catalytic, in-situ and ambient analyses and other adsorbate-surface interactions. SERS is observed primarily for analytes adsorbed onto coinage (Au, Ag, Cu) or alkali (Li, Na, K) metal surfaces, with the excitation wavelength near or in the visible region. Theoretically, any metal would be capable of exhibiting surface enhancement, but the coinage and alkali metals satisfy calculable requirements and provide the strongest enhancement. Metals such as Pd or Pt exhibit enhancements of about 102-103 for excitation in the near ultraviolet [Garrell, 1989; Moskovits, 1985]. The importance of SERS is that the surface selectivity and sensitivity extends RS utility to a wide variety of interfacial systems previously inaccessible to RS because RS was not surface sensitive. These include in-situ and ambient analysis of electrochemical, catalytic, biological, and organic systems. Zou, et al. (1998) discuss the alternative surface techniques (Sum Frequency Generation, Infrared Reflection Absorption Spectroscopy, and Electron Energy Loss Spectroscopy) whose limitations include a need for ultra high vacuum (UHV) conditions, low wavenumber range, low sensitivity, and bulk phase interference. SERS can be conducted under ambient conditions, has a broader wavenumber range, is quite sensitive and is surface selective [Campion et al., 1998]. 2.5.2 SERS mechanisms and SERS enhancement factors Figure 4 shows a simplified schematic diagram for understanding the concept of electromagnetic SERS enhancement. The metallic nanostructure is a small sphere with the complex dielectric constant () in a surrounding medium with a dielectric constant

0. The diameter of the sphere (2r) is small compared with the wavelength of light

22

(Rayleigh limit). A molecule in the vicinity of the sphere (distance d) is exposed to a field EM, which is the superposition of the incoming field E0 and the field of a dipole Esp induced in the metal sphere. The field enhancement factor A() is the ratio of the field at the position of the molecule and the incoming field

E ( ) 0 r A( ) = M E0 ( ) + 2 0 r + d

(Equation 7)

A() is particularly strong when the real part of () is equal to 20. Additionally, for a strong electromagnetic enhancement, the imaginary part of the dielectric constant needs to be small. These conditions describe the resonant excitation of surface plasmons of the metal sphere.

Molecule
d
r

EM = E0 + ESP
E : field of a point dipole in the center of the sphere

= '+i ' '

Metal
r

ESP = r 3

0.05

Sphere

0 1 E0 + 2 0 (r + d )3

Figure 4. Simple schematic diagram for understanding the concept of electromagnetic SERS enhancement

In an analogous fashion to the laser field, the scattered Stokes or anti-Stokes field will be enhanced if it is in resonance with the surface plasmons of the metal sphere. Taking into account enhancing effects for the laser and the Stokes field, the electromagnetic enhancement factor for the Stokes signal power G(S) can be written as:

Gem ( S ) = A( L ) A( S )

( L ) 0 ( S ) 0 r ( L ) + 2 0 ( S ) + 2 0 r + d

12

(Equation 8)

23

This equation is based on a very simple model already describes important properties and peculiarities of the electromagnetic SERS enhancement. It shows that the enhancement scales as the fourth power of the local field of the metallic nanostructure and that it is particularly strong when excitation and scattered fields are in resonance with the surface plasmons. This is the case for low-frequency Raman modes and explains that the scattering powers of different Raman bands in a spectrum fall off with increasing vibrational energy. Electromagnetic SERS enhancement does not require direct contact between molecule and metal but it strongly decreases with growing distance described by the decay of the field of a dipole over the distance [1/d]3 to the fourth power, resulting in [1/d]12. Maximum values for electromagnetic enhancement for isolated single colloidal silver and gold spheroids are on the order of 106107. Theory predicts stronger enhancement of electromagnetic fields for sharp features and large curvature regions, which may exist on silver and gold nanostructures. For example, it was shown that the electromagnetic SERS enhancement factor can be increased up to nearly 1011 when the sphere degenerates and becomes sharper at one edge. Also, closely spaced interacting particles can provide extra field enhancement. Electromagnetic enhancement factors up to 1011 have been estimated for the midpoint between two silver or gold spherical particles separated by a gap of 1 nm near the gap sites between two particles in proximity. In many experiments, SERS-active substrates consist of a collection of silver or gold nanoparticles exhibiting fractal properties, such as colloidal clusters formed by aggregation of colloidal particles or metal island films [Moskovits, 1978; Xu H. X. et al., 2000].

24

Figure 5 shows SERS-active colloidal silver particles in different aggregation stages. A comparison between the electron microscope view and the light microscope view shows strong similarities and demonstrates the fractal nature of these structures. In such colloidal cluster structures, the individual dipole oscillators of the small isolated particles couple, thereby generating normal modes of plasmon excitation that embrace the cluster and cover a wide frequency region from the visible to the near infrared (NIR). The broadening of the plasmon resonance when colloidal clusters are formed is demonstrated in figure 5, which shows the relatively narrow absorption spectrum of isolated silver colloidal particles centered at about 420 nm together with the broad extinction curve for silver colloidal clusters ranging from about 400 to 1000 nm. The excitation is not distributed uniformly over the entire cluster but tends to be spatially localized in so-called hot areas. Therefore, the surface of a fractal colloidal cluster structure shows a very inhomogeneous field distribution. Figure 5 illustrates the inhomogeneous field distribution on a silver cluster. This theoretical result was confirmed by near field measurements. The size of the hot areas can be as small as a few nanometres. Their locations depend strongly on the geometry of the fractal object and on the excitation wavelength and polarization of the optical fields. Particularly strong field enhancement for colloidal silver and gold clusters should exist in the NIR wavelength range. The strongly confined hot spots provide the opportunity to select single nano-objects spectroscopically within a larger population or to probe selectively parts of large molecules. When optical excitation is localized in such small hot spots, extremely large electromagnetic SERS enhancement (proportional to field enhancement to the fourth power!) up to 1012 was theoretically predicted for these areas[kneipp, K. et al., 2002].

25

Figure 5. SERS-active colloidal. Silver particles in different aggregation stages, demonstrating the fractal nature of these structures together with the appropriate extinction curves [kneipp, K. et al., 2002].

The order of magnitude of the enhancement factor has been confirmed in single-molecule SERS experiments, where the normal Raman signal of 1014 methanol molecules appears at the same level as the surface-enhanced Raman signal of a single molecule. In those experiments on colloidal silver and gold clusters, total nonresonant SERS enhancement factors on the order of 1014 have been observed, which can be understood by a superposition of a factor 1012 electromagnetic SERS enhancement and a factor 100 chemical enhancement. Electromagnetic enhancement factors approaching 1012 corresponding to field enhancement factors on the order of 103 has been also confirmed by surface-enhanced nonlinear RS, where signals nonlinearly depend on the enhanced local fields. The electromagnetic enhancement factors for Stokes, pumped anti-Stokes

26

and hyper-RS experiments scale as theoretically predicted from the appropriate nonlinear process [Kneipp, K. et al., 1999,2000]. All the experimental findings described above give compelling evidence for an electromagnetic field enhancement. If SERS were just an electromagnetic field enhancement effect, a strong SERS signal should exist for each molecule in the close enough vicinity of a silver or gold nanostructure. On the other hand, experimental observations such as dependence of the effect on the chemical nature of the molecule and a strong molecular selectivity provide clear indications for the existence of an (additional) chemical SERS enhancement. For example, methanol does not show any SERS enhancement. Other experimental observations which hint at mechanism(s) other than electromagnetic field enhancement include SERS enhancement measured from molecules on metal surfaces, which are flat on the nanometer scale, as well as the dependence of the enhancement factor on the electrode potential. Moreover, best electromagnetic SERS enhancement factors leave a gap of about two orders of magnitude to the best experimentally observed nonresonant SERS enhancement factors on the order of 1014, which suggests the existence of additional enhancement mechanism(s) accounting for the missing factors [Kneipp, K. et al., 1998].

27

3. PREVIOUS WORK The use of infrared spectroscopy as a means of differentiating and identifying bacteria was extensively reported as early as in the 1950s and 1960s [Nauman et al., 1991]. A critical review on this subject published in 1959 summarized that, although bacteria definitely eshibit IR-spectra that are unique for individual strains, the identification of bacteria via IR-techniques could not be regarded as a useful scheme as it is an impractical procedure. The revival of IR-spectroscopy as a means for characterizing microbial samples was initiated after the development of modern interferometric IR-spectroscopy, the availability of low-cost mini-computers and powerful new algorithms for multivariate statistical analysis and pattern recognition methodologies [Maquelin et al., 2002]. Compared to IR spectroscopy, Raman spectroscopy was neglected in the field of biological sciences. This has changed historically, ever since IR techniques were improved. In the 1950s and early 1960s, Raman spectroscopy gave similar information as IR spectroscopy but at higher cost, lower speed, much lower sensitivity and demanding relatively complicated instrumentation. Coinciding with the laser developments in the late 1960s and early 1970s, Raman spectroscopy was increasingly applied in biological studies. It was not until the late 1980s, however, that publications appeared in the literature reporting on the possibilities of Raman spectroscopy and SERS for identification purposes in microbiology. IR, Raman and SERS are complementary techniques. Different selection rules apply for IR absorption and Raman scattering by a molecule. Together, the three techniques provide a complete and highly specific vibrational spectroscopic fingerprint of cells [Nauman, et al., 1991]. In the last decade, these techniques have reached a level of

28

sensitivity that enables spectra to be obtained of even one single living cell [Puppels et al., 1991; Shucster et al., 2000]. SERS applications in the biochemical and biomedical field up to the middle of the 1990s are summarized in several review papers [Nabiev, et al., 1993]. The reviews discuss SERS experiments performed on amino acids and peptides, on purine and pyrimidine bases, but also on large molecules such as proteins, DNA and RNA. SERS was also applied to the study of many intrinsically coloured biomolecules such as chlorophylls and other pigments, as well as from larger molecules containing chromophores such as the heme-containing proteins. Other, more medical, applications include SERS detection of stimulating drugs and selective analysis of antitumour drug interaction with DNA [Nabiev, et al., 1991, 1995]. SERS is not only of interest as a method for ultrasensitive detection and structural characterization of biomolecules; the technique has been also applied to study biophysically interesting processes. For example, SERS can be used to monitor transport through membranes. The results show that SERS can discriminate between the movement of different molecules across a membrane and to observe different interfacial arrival times and concentration growth rates in the receiving (colloidal silver) solution [Wood, E. et al., 1997]. SERS also represents an interesting approach for studying charge transfer processes, for instance in cytochrome-c, which has been extensively investigated on bare and coated silver electrodes [Picorel, R. et al., 1998]. In particular, the selectivity of Surface Enhanced Resonance Raman Spectroscopy SERRS allows the determination of the interfacial potential-dependent equilibria and reactions by probing the vibrational spectra of the group of the adsorbed cytochrome-c exclusively. Recently, time-resolved SERRS was used to study the kinetics of the

29

heterogeneous electron transfer between cyt-c and a SAM-coated silver electrode, in particular the electric field dependence of the interfacial charge transfer [Murgida, D. H. et al., 2001].

30

4. METHODOLOGY

4.1 Reagents and Chemicals The following reagents and materials were used in this investigation: adenine (C5H5N5, 99%), Sigma-Aldrich; silver nitrate (AgNO3 99.99%), Strem Chemicals. Tri-sodium citrate hydrate (Na3C6H5O7.2H2O), nitric acid (HNO3, 70%), sulfuric acid (H2SO4, 98%), sodium chloride (NaCl, USP/FCC granular), glycerol (99.5%), LB broth Lennox (Dehydrate, Tryptone 10g, yeast extract 5g, sodium chloride 5g per liter of solution) were purchased from Fisher Scientific International.

4.2 Organisms Selection The following bacterial strains were obtained from the sources indicated and used in this research: a. Bacillus subtilis (ATCC number 6633), b. Bacillus cereus (ATCC number 14579), c. Staphylococcus aureus (ATCC number 6538), d. Escherichia coli (ATCC number 8789), e. Staphylococcus epidermidis (ATCC number 2228), f. Salmonella tennessee(ATCC number 93311), g. Klebsiella pneumoniae (ATCC number 3882), h. Pseudomonas aeruginosa (ATCC number 9721) were provided by Dr. Carlos Rios Velazquez and Magaly Zapata (Department of Biology, University of Puerto RicoMayagez Campus); i. Bacillus thuringiensis (ATCC number 35646); j. Enterobacter aerogenes (ATCC number 13048) and k)Proteus mirabilis (ATCC number 25933) were obtained from Fisher Scientifics. Pure cultures were stored at -800C in microvials

containing 20% glycerol (cryoprotectant) until use.

31

4.3 Instrumentation The silver colloids were characterized using techniques such as UV-Vis spectroscopy with a Varian Cary-100 UV-visible double beam. The growth kinetics of bacteria were obtained using techniques such as Visible spectroscopy with a 100 VIS

spectrophotometer to Buck scientific to 600 nm. The bacterial Images were obtaining in the Department of Biology (UPRM) with a Scanning Electron Microscopy (JEOL 540 LV ). A Renishaw Raman Microscope, model RM-2000 (Figure 6) was used to observe the scattering excited by a 532 nm diode laser. Typically, laser power was attenuated to 0.50 mW and the data acquisition time was in the 60s and the spectra were obtained in the range of 100-4000 wavenumber (cm-1) to obtain the Raman and SERS spectra of bacteria reported here. The frequency calibration was set by reference to the 521 cm-1 vibrational band of a silicon wafer. The Raman data was obtained with a 10x microscope Leica objective for Raman excitation/collection and SERS data was obtained with a 50x microscope Leica objective for Raman excitation/collection.

Figure 6. Renishaw Raman Microspectrometer RM-2000.

32

The FTIR used for the bacterial analysis was a Bruker model IFS 66v/S spectrometer (Figure 7) coupled to an infrared microscope equipped with grazing angle reflectance (GAR) objective of 15x magnification and a mercury-cadmium-telluride (MCT) detector and potassium bromide (KBr) beamsplitter. Bacterial spectra were collected using OPUS version 4.2, Bruker Software, over range 4000-100 wavenumber (cm-1) by averaging 50 scans with a resolution of 4cm-1 and absorbance mode. A background spectrum was obtained before each measurement session using a clean stainless steel plate at the same instrumental conditions used for bacterial spectra acquisition.

Figure 7. FTIR spectrometer, Bruker IFS 66v/S

4.4 Growth Kinetics of Bacterial All bacterial cells were grown in 5 mL of LB (Lennox) broth overnight with constant shake to 37.00C. After, each bacterial culture was diluted in 100 mL of LB (Lennox) broth with OD600 0.010-0.074 range. The optical density was taken each hour during 12 hours and 18 and 24 hours.

33

4.5 Synthesis of Silver Colloids One hundred milliliters (100 mL) of triple distilled water were placed in a 250 mL round bottom flask. The water was stirred vigorously, then heated up to approximately 90C and 0.024 g of AgNO3 was added. The solution was heated to almost boiling, and then a solution of sodium citrate (0.027g sodium citrate in 2.5 mL of triple distilled water) was added. The solution was then held at boiling for 45 minutes with a flow of nitrogen gas at 1 psi. After this time, the heat is removed and the solution is allowed to cool, stirring is still continued at this point and nitrogen is then passed through the solution once more for 15 minutes (Balaguera, M., 2006). The quality of colloids routinely assessed by collecting a UV-Vis spectrum fronts it and a solution of adenine (1x10-4M) was analyzed by SERS with a mix of 50 L of silver colloids, 10 uL of adenine and 5 uL of NaCl. Then 10 L of the mixtures were placed at one side of a microscope glass slide cover for analysis.

4.6 SEM Images of Bacterial Cells The protocol for the bacterial cells preparation for SEM was provided by Dr. Carlos Rios Velazquez from the Department of Biology at UPRM. The SEM images were taken in at the Microscopy Center from the Department of Biology at UPRM.

4.7 Bacterial Sample Preparation Bacterial cells were grown in 5 mL of LB (Lennox) broth for approximately 15 hours, centrifuged for 20 minutes at 6000 rpm. Subsequently for removing the broth from the obtained pellets were bacteria washed four times with 5 mL of NaCl 0.80% w/v.

34

Standard biosafety level 2 lab procedures were employed for handling the strains studied here even though they are nonvirulent. Spore-forming bacteria were destroyed by exposure to 95% ethanol for ~20 minutes and UV. Non spore-forming bacteria and contaminated glassware were autoclaved prior to disposal.

4.7.1 Raman Sample Preparation A small amount of the bacterial cells was placed on the cover glass slides of 0.02 mm wide using a loop. The Raman spectra were obtained with 10 mW of incident 532 nm excitation power, 100% laser power, 3 scan and 20 s of acquisition time.

4.7.2 SERS Sample Preparation The pellets were re-suspended in 50 L of NaCl 0.80% w/v. An aliquot of 10 L of bacterial cells suspension and 50 L of silver colloid were mixed in a micro tube. Ten L of suspension of bacterial cells was deposited on the cover glass slides and then placed on a gold coated microscope slide. The SERS spectra were obtained with 0.5 mW at 532 nm with 3 scans and 20 s of acquisition time. Spectra were acquired from 200 to 4000 wavenumber. Visual inspection of these spectra showed that the most information-rich area was between 400 and 1800 cm-1, and this region was used for data analysis. Calibration was periodically checked by recording the position of know Raman lines of silicon (521 cm-1). All data were obtained using the Renishaw, Inc. WIRE 2.0 software.

35

4.7.3 FT-IR Sample Preparation A small amount of the bacterial cells was placed on a stainless steel plate and then spread as evenly as possible across the surface using the straight edge of a Teflon sheet. Once the solvent evaporated, the spectra of the bacterial cells were collected immediately.

4.8 Data Analysis To assess the ability of SERS, Raman and FT-IR data discriminate between the different species, the spectra of each technique was analyzed by multivariate statistical techniques. This process involved three different analysis: principal component analysis (PCA) was employed to reduced the dimensionality of the data while preserving most of the variance. Discriminant factor analysis (DFA) was then used to discriminate between groups on the basis on the retained principal components (PCs) and finally the hierarchical cluster analysis (HCA). The PCA, DFA and HCA were all performed in Statgraphics software, version 9.0.

36

5. RESULTS AND DISCUSSION

5.1 Characterization of Silver Colloids 5.1.1 Plasmon Absorption Band for Silver Colloids The Ag nanoparticles display an optical absorption band peaked at 421 nm (~3 eV), which is typical of the absorption of metallic Ag nanocrystallites due to the surface plasmon resonance [Lee, P. C. et al., 1982]. The colloidal suspensions of silver particles exhibited a bright yellow-greenish color due to the intense bands around the excitation of the surface plasmon resonance as shown on Figure 8. The UV-VIS spectra revealed a plasmon absorption band at about max 418 nm to 440 nm which is typical for silver colloidal nanoparticles. a

Figure 8. UV-VIS spectra of colloidal silver nanoparticles used in this research: a) silver colloids max =419 nm; b) silver colloids max =424 nm.

Figure 8 a and b the silver colloids exhibited a major peak at 419 nm and 424 nm for the aqueous suspensions in the UV-VIS spectra, corresponding to the plasmon band [Krassimir, P. V. et al., 2003].

37

5.1.2 Silver Colloid used for adenine detection The vibrational modes observed in the SERS spectrum of adenine molecules adsorbed on silver thin colloids were 732, 953, 1024, 1115, 1327 and 1396 cm-1. These values were consistent with those found in Figure 9, the SERS spectrum for adenine with silver colloids by McNaughton et al. [Alvarez, R. A. et al., 2005].

732 cm-1
Intensity (a.u.)

a b c
500 700 900 1100 1300 1500 1700 1900

wavenumber (cm-1)

Figure 9. Raman Spectra: a) adenine 1x10-4 M with silver colloid; b) silver colloid; c) adenine 1x10-4 M.

5.2 Bacteria species Data Analysis 5.2.1 Growth curve Growth is an orderly increase in the quantity of cellular constituents, which depends on the ability of the cell to form new protoplasm from nutrients available in the environment. For a batch culture, the environments are obviously different at different growth phases. The figure 10 shows the growth curve of different bacterial species used in this study. Where we can see that the growth for these bacterial cells to fifteen hours were in the stationary phase. At this time of growth Raman, SERS and FTIR spectra were measured. 38

I
Absorbance

II
Absorbance

a b c
0

d e
0 3 6 9 12 15 Time (hours) 18 21 24

12 15 Time (hours)

18

21

24

III
Absorbance

IV
a b c d e
Absorbance 0

12

15

18

21

24

12

15

18

21

24

Time (hours)

Time (hours)

Figure 10. The growth behavior of bacteria: I. a) B. subtilis, b) B. cereus, c) B. thuringiensis; II. d) S. epidermidis, e) S. aureus. III. a) E. coli, b) S. tennessee, c) K. pneumoniae, d) E. aerogenes, e) P. mirabilis and IV. P. aeruginosa.

5.2.2 SEM images The SEM images were taken to verify the size and morphology of bacterial cells. Figure 11 shows the SEM images to Bacillus species, in this figure we can see that the three Bacillus shown rods with an approximate size between 0.3-1.0 x 2.0-3.0 m.

Figure 11. SEM images of Bacillus. a)B. subtilis, b)B. cereus and c)B. thuringiensis

The SEM images of Staphylococcus species was not as expected, for this reason, the gram-stain test was performed to validate the bacterial cells morphologic and size.

39

Figure 12. SEM images to Enterobacteriaceae species and Pseudomonas aeruginosa: a) E. coli, b)S. tennessee, c) K. pneumoniae, d) E. aerogenes, e) P. mirabilis; f) P. aeruginosa

The figure 12a show a SEM image of E. coli in this image show rods with size between 0.7-1.0x1.0-1.4 m. Figure 12 (f) shows a SEM image of S. tennessee where we can see rods with size between 0.5-0.7x1.2-2.0 m; the figure 12c show a SEM image of K. pneumoniae where we it shows with size between 0.3-0.5x1.2-1.5 m. The figure 12d is a SEM image of E. aerogenes, rods with a size between 0.5-0.7x1.7-2.0 m. The figure 12e corresponding to SEM imagine of P. mirabilis, rods with a size between 0.5x1.0-1.2 m; and the figure 12f show the SEM image of P. aeruginosa where we can see rods whit a size between 0.5x1.5-2.0 m, these sizes and morphologies complied with the expected. 5.3 Raman and SERS spectra Many bacterial cells samples were analyzed in Raman and SERS. In addition, the Colony Forming Unit (CFU) was calculated for each bacterial species by dilution to original suspension. Table 1, shows the approximate bacterial cells amounts analyzed with Raman and SERS techniques. The relation of bacterial cells analyzed between Raman - SERS is

40

approximately 17:1. For each species, three independent culture replicates were grown and three bacterial cells from each replicate culture analyzed.
Table 1 Approximate number of bacterial cell analyzed with Raman and SERS, and CFU/L for each bacterial specie.

Bacteria
Bacillus subtilis Bacillus cereus Staphylococcus aureus Escherichia coli Staphylococcus epidermidis Salmonella tennessee Enterobacter aerogenes Pseudomonas aeruginosa Bacillus thuringiensis Proteus mirabilis Klebsiella pneumoniae

CFU/L
4.2 x 106 4.4 x 106 4.8 x 106 6.9 x 106 4.9 x 106 2.1 x 106 2.3 x 106 3.3 x 106 2.6 x 106 4.1 x 106 4.6 x 106

Approximate number of bacterial cell analyzed RAMAN SERS 370 15 387 16 422 17 607 24 431 17 184 7 202 8 290 12 229 9 360 14 404 16

Raman cross-section enhancements due to the proximity of nanostructure metal surfaces are typically cited in the range of 103-106 per molecule from ensemble averaged SERS measurements depending on the nature of the SERS active substrate, excitation wavelength, and Raman chromophore electronic structure. Single molecule

enhancements at selected hot spots, however, as large as 1014 have been reported [Premasiri, et al, 2005]. To provide some quantitative measure of the magnitude of the bacterial SERS enhancement on the silver colloids substrates used here, the relative SERS and Normal Raman scattering cross-sections, i.e., scattered power per bacterium, normalized for data collection time and incident laser power, of Gram-positive, B. thuringiensis, bacteria were determined. If we compare the strongest vibrational band in each spectrum, the Raman cross-section of B. thuringiensis is found to be amplified by a factor of 2.0 x 104 on our silver colloid SERS substrate. The E. coli enhancement due to 41

our SERS substrate is found to be 1.5 x 104 for this Gram-negative specie. This amplification factor is slightly larger than that for B. thuringiensis. The observed corresponding normalized SERS and Normal Raman spectra are compared in Figure 13 for B. thuringiensis and in Figure 14 for E. coli. Additionally the other bacterial comparisons are shows in the figures A1 to A9.

Intensity / a.u.
400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure 13. Bacillus thuringiensis Normal Raman spectrum (30x) (blue) and the SERS spectrum (fuchsia).

Intensity / a.u. 400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure 14. Escherichia coli Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia)

42

Typical Raman and SERS spectra of the eleven Bacterial species studied in this work are shows in the figures 15, 16 and 17, 18 respectively.

Intensity / a.u. Intensity (a.u.)

d c b a

400

600

800

1000

1200

1400

1600

1800

Wavenumber (cm-1) Wavenumber / cm-1

Figure 15. Representative Raman spectra of Gram-positive species: a) B. subtilis; b) B. cereus; c) B. thuringiensis; d) Staphylococcus epidermidis; and e) S. aureus.

20000

f e d c

Intensity (a.u.) Intensity / a.u.

15000

10000

5000

b a
400 600 800 1000 1200 1400 1600 1800

-1-1 ) W a v e n u m b e/r cm (c m Wavenumber

Figure 16. Representative Raman spectra of each Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes; e) P. mirabilis; f) P. aeruginosa.

43

Intensity / (a.u.) Intensity a.u.

400

600

800

1000

1200

1400

1600

1800

Wavenumber / Wavenumber (cm-1)

cm-1

Figure 17. Representative SERS spectra of Gram-positive species: a)B. subtilis; b) B. cereus; c) B. thuringiensis; d) S. epidermidis; and e) S. aureus.

e
Intensity (a.u.) Intensity / a.u.

f d c b a
400 600 800 1000 1200 1400 1600 1800

Wavenumber / cm

W aven u m b er (cm -1) -1

Figure 18. Representative SERS spectra of Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes;, e) P. mirabilis; f) P. aeruginosa.

44

Simple visual inspection of the spectra (and definitely, of all spectra acquired) shows that they are qualitatively very similar. Nonetheless, on closer inspection, subtle quantitative differences can be observed. Several interesting bands appear in the spectra. Some tentative assignments are listed in detail in Tables 2 and 3. The bands match features reported in the literature for samples of larger amounts of bacterial biomass or its molecular components [Schuter, K. et al., 2005; Maquelin, K. et al., 2002]. Most bands correspond to functional groups in the main constituents of a microbial cell, protein, carbohydrates, lipids and nucleic acids. Some are more specific for smaller molecular compounds, like the very characteristic sharp band of phenylalanine at 964-1010 cm-1 , which is present in all protein-containing samples. It is obvious that such simple visual inspection of these spectra will not allow one to discover the relationship between the bacteria based on their Raman and SERS fingerprints, and alternative strategies need to be adopted. Therefore, multivariate statistical methods have to be used, and these included PCA, DFA and HCA.

45

Bacteria Assignment
C-O-C ring deformation guanine, tyrosine (nucleic acid) Adenine cytosine, uracil (nucleic acid) C-O-P-O-C (RNA backbone) (nucleic acid) tyrosine (in proteins) C=C deformation phenylalanine (in proteins) carbohydrates, mainly C-C (skeletal) DNA O-P-O- stretching (symmetric) =C-O-C= (unsaturated fatty acids in lipids) C-O ring Amide III (random) Amide III (protein), C-H deformation adenine, guanine (protein), CH deformation COO- stretching CH2 deformation amide II adenine, guanine (ring stretching) amide I >C=O ester stretching

B. subtilis Raman 540 668 SERS 540 660

B. cereus Raman 542 SERS 540 658 734

B. thuringiensis Raman SERS 662 736

S. aureus Raman 544 SERS 572 662 736

S. epidermidis Raman SERS 660 734 758

755 785

749

748 785

726 760

812 829 965 850 968 1041 860 967 1004 1044 1103 1132 1155 1225 1267 1318 1234 1287 1169 1253 1279 1323 1339 1397 1452 1523 1584 1672 1705 1137 1174 1254 1132 1173 1232 965 1006 1025 1039 1105 1135 965

965 1005

987

961 1034 1102

1131 1154 1171 1238

1135 1168 1249 1323

1130

1135 1169

1228

1297

1343 1398 1451 1585 1672 1714 1681

1337 1394 1454 1582 1632

1338 1399 1468 1572 1679 1704 1398 1440

1336 1396 1468 1544

1337 1398 1473 1583 1672

1341 1396 1455 1588 1682

1335 1397 1468 1557 1588 1632 1705

1468 1557

1708

Table 2 Raman and SERS bands observed in spectra of gram-positive bacterial cells and tentative assignment.

46

Bacteria Assignment
C-O-C ring deformation guanine, tyrosine (nucleic acid) Adenine cytosine, uracil (nucleic acid) C-O-P-O-C (RNA backbone) (nucleic acid) tyrosine (in proteins) C=C deformation phenylalanine (in proteins) carbohydrates, mainly C-C (skeletal) DNA O-P-O- stretching (symmetric) =C-O-C= (unsaturated fatty acids in lipids) C-O ring Amide III (random) Amide III (protein), C-H deformation adenine, guanine (protein), CH deformation COO- stretching CH2 deformation amide II adenine, guanine (ring stretching) amide I >C=O ester stretching

E. coli Raman SERS 660 730 754 809

S. tennessee Raman SERS 660 730 754 807 830 965 1005 1035 1060 1101

K. pneumoniae Raman SERS 663 734 754 808

E. aerogenes Raman SERS 663 734

P. mirabilis Raman SERS 665 738 753

P. aeruginosa Raman SERS 675 727 754

980

961 1004 1040

990 1005

966 1007 1034 1108

968 1005

984 1005

933 1005

830 968 1009 1041 1100

1084

1101 1142 1131 1174 1232

1134 1151 1234 1174 1234

1135 1162 1247 1295

1131 1171 1234

1129 1177

1132 1172 1232

1139

1135 1175 1238 1320

1137 1172

1172 1234 1320 1340 1398 1457 1587 1680

1229 1287

1220 1276

1339 1395 1464 1585 1694

1342 1398 1461 1513 1588 1677

1335 1398 1468 1585 1635 1705

1344 1398 1457 1588 1677

1334 1395 1464 1581 1675

1334 1398 1461 1585 1677

1337

1344 1398 1455 1589 1678

1338 1398 1459 1513 1593 1687 1396 1461 1587

1468 1585

1590

1703

Table 3 Raman and SERS bands observed in spectra of gram-negative bacteria studied and tentative assignment.

47

5.3 FTIR spectra The representative FTIR spectra of eleven bacteria in the 600-4000 cm-1 range acquired are shown below in Figures 19 and 20.

Absorbance

e d c b a
3600 3100 2600 2100 1600 1100 600 Wavenumber (cm-1)

Figure 19. The averaged FTIR spectra of Gram-positive species: a) B. subtilis; b) B. cereus; c) B. thuringiensis; d) Staphylococcus epidermidis; and e) S. aureus.

Absorbance

d f e c b a
3600 3100 2600 2100 1600 1100 600 Wavenumber (cm-1)

Figure 20. Averaged FTIR spectra of Gram-negative species: a) E. coli; b) S. Tennessee; c) K. pneumoniae; d) E. aerogenes; e) P. mirabilis; f) P. aeruginosa.

48

IR spectra of microbial specimens provide not only a number of absorption bands that describe molecular composition of the cells. Many of these bands are also sensitive to structural changes, various intra- and intermolecular interactions including membrane constitution and conformational states like different secondary structures of proteins. The physical state of the sample such as hydration or aggregation state, interaction with ions and so on has a strong influence on results. These facts necessitate the rigorous standardization of sampling, preparation, and data acquisition procedures [Nauman, D. 2000]. Tentative band assignments are shown in the tables 4 and 5.

Table 4 FTIR bands observed in spectra of gram-positive bacterial cells and tentative assignment. Assigment O-H stretching N-H stretching (amide) of proteins C-H stretching (antisymmetric) of CH3 in fatty acids C-H stretching (antisymmetric) of >CH2 C-H stretching of C-H in methine groups C-H stretching (symmetric) of -CH3 Amide I of -helical structures amide II C-H deformation of >CH2 C=O stretching (symmetric) of COOP=O stretching (antisymmetric) of PO2- phosphodiesters P=O stretching (symmetric) of >PO2B. subtilis 3302 B. cereus 3305 B. thuringiensis 3317 3101 2966 2931 2970 2933 2970 2933 2899 2875 1651 1554 1468 1404 1250 1097 2872 1655 1551 1475 1416 1252 1092 2875 1647 1549 1471 1408 1254 1097 2877 1654 1552 1470 1408 1255 1099 2868 1656 1552 1470 1404 1252 1097 2966 2935 2970 2933 S. epidermidis 3311 S. aureus 3307

49

Table 5 FTIR bands observed in spectra of gram-positive bacterial cells and tentative assignment. E. coli 3305 2974 2937 2873 S. tennessee 3307 2967 2933 2875 K. pneumoniae 3305 2976 2933 2870 E. aerogenes P. mirabilis 3313 2968 2929 2873 P. aeruginosa 3309 2970 2929 2873

Assigment O-H stretching C-H stretching (antisymmetric) of CH3 in fatty acids C-H stretching (antisymmetric) of >CH2 C-H stretching (symmetric) of -CH3 >C=O stretching of esters amide I of helical structures amide I of pleated sheet structures amide II C-H deformation of >CH2 C=O stretching (symmetric) of COOamide III band components of proteins C-O, C-C stretching, C-O-H, C-O-C deformation of carbohydrates

2960 2933 2875 1730

1653

1660

1660 1600 1554 1497 1417 1282

1654

1647

1552 1479 1423 1248

1552 1471 1421 1254

1552 1471 1414 1263

1552 1473 1419 1252

1551 1470 1412 1246

1097

1103

1101

1101

1093

The region between 4000-3100 cm-1 is dominated by rather broad spectral features resulting from OH (3300 cm-1). The region between 3100 and 2800 cm-1 exhibits the C-H stretching vibrations of CH3 and >CH2 functional groups and hence, is generally dominated by the spectral characteristics of fatty acid chains of the various membrane amphiphiles and by some amino acid side-chain vibrations. Complementary information can be deduced from the region between 1470-1350 cm-1 where the various deformation modes of these functional groups are found. The region between 1800 and 1500 cm-1 is 50

dominated by the conformation-sensitive amide I and amide II bands, which are the most sensitive bands in the spectra of all bacterial samples. Complex absorption profiles are observed between 1300-1500 cm-1 arising predominately from >CH2 bending mode of lipids and proteins. A characteristic, but weak feature is often to the symmetric stretching vibrations of COO- functional groups of amino acid side chains or free fatty acids. Around 1250 bands typical of different >P=O double bond asymmetric stretching vibrations of phosphodiester, free phosphate and monoester phosphate functional groups are observed. The spectral region between 1200-900 cm-1 is generally dominated by the symmetric stretching vibration of PO2- groups in nucleic acids and a complex sequence of peaks mainly due to C-O-C and C-O-P stretching of various oligo- and polysaccharides [Garp, S., 2005; Naumann, D. 2000]. For the characterization and identification of microorganisms based on vibrational spectroscopy techniques, it is, however, not necessary to identify all band intensities, frequencies, and bandwidths in a spectrum and assign them to specific molecular compounds. Spectra can be grossly evaluated as spectroscopic fingerprints of the sample studied. Multivariate statistical techniques were employed for the analysis of the total complex spectrum, in a similar manner as is happening in pattern recognition procedures. 5.4 Statistical Data Analysis To determine whether Raman, SERS and FRIT microscopy could discriminate between the bacterial strains, all spectra were analyzed using multivariate methods, each technique individually. Initially, all data spectra were normalized and then PCA was performed and the 7 principal components was retained to obtained de DCA, and HCA using Wards Method and Eucledian distances. The DCA and HCA shows the relationships between

51

the whole collections of bacterial species analyzed in this research. The labels used in the plots for the eleven bacterial species are listing in Table 6.
Table 6 Bacterial species used in bacterial discrimination studies. Identifier on Plots Bacteria ATCC Number

a b c d e f g h i j k green oval blue oval

Bacillus subtilis Bacillus cereus Staphylococcus aureus Escherichia coli Staphylococcus epidermidis Salmonella tennessee Klebsiella pneumoniae Pseudomonas aeruginosa Bacillus thuringiensis Enterobacter aerogenes Proteus mirabilis gram-positives species gram-negative species

6633 14579 6538 8789 2228 93311 3882 9721 35646 13048 25933

For Raman data the PCA was calculated using a range between 600 to 1800 cm-1 and seven principal components were used to obtained the DCA (Figure 21) and HCA (Figure 23). This first step, shows the discrimination between S. aureus, S. tennessee, K. pneumoniae, P. aeruginosa, B. thurindiensis, E. aerogenes and P. mirabilis, but B. subtilis, B. cereus, S. epidermidis and E. coli species were not discriminated. Thus, a second PCA, DCA (Figure 22) and HCA (Figure 24) of these four species was performed in the range of 1000 to 1800 cm-1, this with the objective of reduce the variability of data and concentrate in the most representative region, for this spectra. In this second analysis, the discrimination between this four species was successful, this demonstrate that Raman microscopy is a technique that it may used to identify between the eleven bacterial species used in this study.

52

Plot of Discriminant Functions Bacteria a b c d e f g h i j k Function 1

Figure 21. PC-DFA plot of Raman spectra of the all bacterial species analyzed in this research.

Function 2

PlotPlot of Discriminant Functions of Discriminant Functions

5.6 3.6 1.6 -0.4 -2.4 -4.4 -6 -2 2 6 Function 1 1 Function 10 14 Bacteria a b e j

Figure 22. PC-DFA plot of raman spectra of B. subtilis, B. cereus, S. epidermidis and E. aerogenes

Function 2 2 Function

53

d c g k i h f a b j b e
Figure 23. Dendrogram resulting from HCA showing the relationship among all 11 bacteria species analyzed by Raman

54

Figure 24. Dendrogram resulting from HCA showing the relationship among four bacteria species analyzed by Raman Spectroscopy.

55

In first instance the SERS data spectra was analyzed equal to Raman data spectra, that is to say in the equal range and used the data of eleven bacterial species. In this first analysis, low discrimination between all species was achieved, but a discrimination among Ggram-negative and Gram-positive species were observed. The top of PC-DCA plot shown the Gram-negative species and in the bottom the Gram-positive species was show. A second analysis was performer, the Gram-positive species and Gram-negative species were analyzed separately, and the discrimination between species, was observed. Of this form, we can conclude that the SERS spectroscopy may to used to discriminate between eleven bacterial used in this research, in two step. First, the data sample is analyzed with all set data to find it is Gram-negative or Gram-positive. The second step is located the data corresponding to find the species of sample. The PC-DCA and HCA of this analysis are shows in the figures 25-30.

Plot of Discriminant Functions Bacteria a b c d e f g h i j k

Function 1

Function 2

Figure 25 PC-DFA plot of SERS spectra of the bacterial species analyzed in this research.

56

Plot of Discriminant Functions Bacteria Function 2

Function 1
Figure 26 PC-DFA plot of SERS spectra of the gram positive species of bacteria used in this research.

Plot of Discriminant Functions

5 3 1 -1 -3 -5 -7 -9 -5 -1 3 7 11 15 Bacteria d f g h j k

Function 2

Function 1
Figure 27 PC-DFA plot of SERS spectra of the gram positive species of bacteria used in this research.

57

d j g f k h a b i e c

Figure 28. Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by SERS.

58

Distance
10 20 30 40 50 0
e c b a i

e e e e e c c c c c b b b b b a a a a a i i i i i

Figure 29. Dendrogram resulting from HCA showing the relationship among gram-positive bacteria species analyzed by SERS.

Ward's Method,Euclidean

Dendrogram

59

Figure 30 Dendrogram resulting from HCA showing the relationship among gram-negative bacteria species analyzed by SERS.

The FTIR data spectra were analyzed in the range between 600 to 3600 cm-1. In this analysis E. aerogenes, S. aureus, S. epidermidis, B. thuringiensis, P. aeruginosa and K. pneumoniae could be discriminated, but the other species, show a significant agglomeration. Then, a second analysis was to carried out, to exclude to E. aerogenes

60

because this specie shown a large difference with the other species in the first analysis. In the second analysis, the range used was 600 to 1800 cm-1. In this analysis we can see that the others bacterial species was discriminated. The PC-DCA and HCA obtained for this two analysis are shown in Figures 31 to 34.

Plot of Discriminant Functions Bacteria

a b c d e f g h i j k

Function 2

Function 1
Figure 31 PC-DFA plot of FTIR spectra of the eleven bacterial species analyzed in this research

Plot of Discriminant Functions Bacteria a b c d e f g h i k Function 1

Figure 32 PC-DFA plot of FTIR spectra of the then bacterial species analyzed in this research

Function 2

61

Distance

a, b, f

k b c e f h j g d i j

Figure 33 Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by FTIR.

62

b a f e g

k h i c d

Figure 34 Dendrogram resulting from HCA showing the relationship among all 11 bacteria analyzed by FTIR.

63

CONCLUSIONS

The vibrational spectroscopy techniques FTIR, Raman and SERS were used for the differentiation of eleven bacterial species in stationary phase of growth, using a multivariate analysis. The results of this study reflect the high discriminatory power of these techniques that allows accurate differentiation of closely related bacterial species such as Bacillus. The HCA obtained for FTIR data shows a relation between B. subtilis, B. cereus and S. tennessee, but the HCA of SERS data shows a separation among Gramnegative and Gram-positive species, therefore a large differentiation between the Bacillus and S. tennessee was observed for the HCA of SERS. The differentiation and relation of the bacterial species shown for the HCA and DCA of SERS, is as expected according to morphology, because shown a great differentiation between Gram-positive and Gramnegative species, but a closely relation between each group of bacteria. Furthermore, a comparison between FTIR, Raman and SERS clustering showed that there were other considerable differences between these methods, since very different classification schemes were obtained. This is most encouraging considering that these techniques see the total cell composition and structure on the basis of different molecular vibrational modes. In addition, the use of these three spectroscopic techniques proved to be capable of discriminating accurately at the strain level, which opens the door for using these physicochemical techniques as tools for epidemiological studies. This study illustrates the enormous potential of vibrational spectroscopic methods for rapid microbial identification. The developed methods are of practical interest, as they require very simple sample preparation consisting of washing the cells with saline

64

solution and placing under the spectroscopic microscope. No chemical reagents are necessary to specifically mark cell components; with the advantage that these techniques are non destructive and non invasive for microbial analysis. Therefore, these techniques may be a useful tools for classifying bacterial cultures, without requiring strict standardization of growth conditions. The spectra contain multidimensional information on all major substance classes present in bacterial cells. This work also demonstrated that when the SERS technique is used, an enhancement of signal by 104 is observed compared with the Normal Raman. Furthermore, this study clearly demonstrates the potential for SERS to produce spectra that can be used for rapid whole-organism fingerprinting, and in conjunction with a simple multivariate analyses. The present contribution is very promising, because it demonstrates that the developed methods are capable of assessing the heterogeneity, providing spectral information on chemical composition within a bacterial cell population.

65

Future Works The spectroscopic study may be extended to examine the spores produced by selected strains since these are more prone to be used as Biological Warfare Agents (BWA) due to their increased mobility and possibility of airborne transport. Furthermore, is important to extend this study to other bacterial species and to develop a research focused in finding the differences between each phase of growth. Finally, study the bacterial species without a washing the cells. Also, it is necessary to develop a quantitative analysis of the components of each bacteria specie using the FTIR technique.

66

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APPENDIX A

Intensity / a.u.
400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A1. Bacillus subtilis Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.0x104.

Intensity / a.u.
400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A2 Bacillus cereus Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 3.0x104.

70

Intensity / a. u. 400

600

800

1000 1200 1400 Raman shift / cm-1

1600

1800

2000

Figure A3Staphylococcus aureus Raman spectrum (40x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 3.0x104.

Intensity / a.u. 400

600

800

1000

1200

1400

1600

1800

2000

Raman shift /cm-1

Figure A 4Staphylococcus epidermidis Raman spectrum (30x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104.

71

Intensity / a.u.
400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A 5Salmonella tennessee Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104.

Intensity / a.u.
400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A 6 Klebsiella pneumoniae Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104.

72

Intensity / a.u.

400

600

800

1000

1131 1178

754

1200

1319 1348 1380 1403

1400

1600

1590

1800

2000

Raman shift / cm-1

Figure A 7Pseudomonas aeruginosa Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.5x104.

Intensity / a.u. 400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A 8 Enterobacter aerogenes Raman spectrum (20x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 2.0x104.

73

Intensity / a.u. 400

600

800

1000

1200

1400

1600

1800

2000

Raman shift / cm-1

Figure A 9Proteus mirabilis Raman spectrum (10x) (blue) and the SERS spectrum (fuchsia), Factor enhancement is 1.0x104.

74