Surface-Enhanced Raman Scattering of Bacterial Cell CultureGrowth Media

NICOLE E. MAROTTA and LAWRENCE A. BOTTOMLEY*School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400

The application of surface-enhanced Raman spectroscopy (SERS) to

characterizing bacteria is an active area of investigation. Micro- and

nano-structured SERS substrates have enabled detection of pathogens

present in biofluids. Several publications have focused on determining the

spectral bands characteristic of bacteria from different species and cell

lines. In this report, the spectra of fifteen commonly used bacterial growth

media are presented. In many instances, these spectra are similar to

published spectra purportedly characteristic of specific bacterial species.

The findings presented herein suggest that bacterial fingerprinting by

SERS requires further examination.

Index Headings: Surface-enhanced Raman scattering; Surface-enhanced

Raman spectroscopy; SERS; Nanostructured silver surface; Bacterial

identification; Growth media.

INTRODUCTION

Surface-enhanced Raman scattering (SERS)1–5 is an emerg-ing analytical tool for pathogen detection and identification.Several recent publications have focused on determining thespectral bands characteristic of bacteria from different speciesand cell lines.6–26 The goal is to improve public health throughthe rapid identification of bacterial pathogens present in theenvironment, biological fluids, and in food products. DifferentSERS substrates, including silver and gold colloids, films, andnanoparticles, have been explored in classifying bacteria.

Recently, highly uniform silver nanorod arrays have beenused as SERS active substrates as they are easy to fabricate andprovide good signal enhancements in comparison to bulkanalyte.27 These arrays are fabricated using the physical vapordeposition at a glancing angle.28–33 This technique producesmetal thin films consisting of columnar microstructuresresulting from atomic shadowing. These substrates are suitablefor detection of viruses34–39 and bacteria.25,40

Initially, we set out to explore the applicability of silvernanorod arrays as SERS substrates for distinguishing betweenbacterial types. Spectra were acquired on three differentbacterial species, two Gram-positive (Arthrobacter histidino-lovorans and Bacillus cereus) and one Gram-negative(Escherichia coli). We were pleased to find that our spectrafor the latter two were comparable to those previouslypublished for these bacteria. But, we were dismayed to find aremarkable similarity between the spectra for these bacterialspecies and that of the medium in which they were grown. Toresolve this apparent disparity, we began a systematicexamination of commonly used bacterial growth culture mediaand compared the SERS spectral bands characteristic of themedia with those previously reported as characteristic ofspecific bacterial species. This report documents our findings.

EXPERIMENTAL

Surface-Enhanced Raman Scattering Substrates. Sur-face-enhanced Raman scattering substrates were prepared byDr. HsiaoYun Chu of Prof. Yiping Zhao’s group in physics atthe University of Georgia by electron beam evaporation.Standard glass microscope slides (25 3 75 mm) were mountedon a sample holder and placed directly above the source. Thinfilms of Ti (50 nm) and Ag (500 nm) were deposited on thesample surface at normal incidence. Then stepper motorsrotated the substrate to an incident angle of 868 from normaland Ag nanorods (AgNRs) were deposited.35

To maximize the number of samples that can be analyzed persubstrate, polymer wells were patterned onto the substratesthrough contact printing using the stamp and procedurepreviously described.33 Following deposition and patterning,substrates were packaged in UniMailerTM slide holders (VWRScientific Inc., West Chester, PA) and stored in a desiccatoruntil use.

Substrates were characterized by scanning electron micros-copy (SEM) before and after application of analyte samples.Scanning electron micrographs were obtained using a ZeissSEM Ultra60 scanning electron microscope (Carl Zeiss SMTInc., Peabody, MA). The accelerating voltage was 5 eV fornanorod arrays and reduced to 2 eV for substrates containingbacteria samples. To minimize charging artifacts, slides wereattached to the holder using double-sided copper tape. SERSspectra were obtained with a Kaiser Optical Systems’ Holo-probe 785 spectrometer (Kaiser Optical Systems, Inc., AnnArbor, MI) using an excitation wavelength of 785 nm, a 103objective, an integration time of 10 s, and a surface power of;4.5 mW. The volume of analyte applied to each well was 5lL; a minimum of five spectra were recorded per well. In someinstances, spectra were acquired across a well at 25 lmintervals. Spectra were background corrected to remove thecharacteristic curvilinear baseline but not rescaled or normal-ized unless otherwise noted. The reader is referred to ourprevious publications for a detailed discussion of theperformance and stability of AgNRs as SERS substrates.

Cell Culture Growth Media. The following cell culturemedia were used as received from each supplier, listed inparentheses: Difco Nutrient Broth, Difco Meuller Hinton IIBroth, Difco Luria Broth, BBL Selenite-F Broth, BBL GramNegative Broth, BBL Cooked Meat Medium, BBL MotilityTest Medium, BBL Peptone Water, BBL Rapid Urea Broth,BBL Sabouraud Liquid Broth, Campylobacter ThioglycollateMedium, BBL LB Broth, and BBL Standard Methods Agar(BD Diagnostics, Franklin Lakes, NJ), and Turbo Broth andSuperior Broth (Athena Environmental Sciences, Inc., Balti-more, MD). All stock solutions were prepared following thesupplier’s recipe. Cell culture media were serially diluted from‘‘stock’’ solutions in nanopure water.

Bacteria and Cell Culture Preparation. Bacterial samples

Received 22 October 2009; accepted 11 March 2010.* Author to whom correspondence should be sent. E-mail: Bottomley@gatech.edu.

Volume 64, Number 6, 2010 APPLIED SPECTROSCOPY 6010003-7028/10/6406-0601$2.00/0

� 2010 Society for Applied Spectroscopy

A. histidinolovorans, B. cereus, and E. coli were provided byRobert Martinez and Prof. Patricia Sobecky (Biology Dept.,Univ. Alabama, Tuscaloosa). A. histidinolovorans and E. coliwere grown at 30 8C to an optical density (OD) of 0.2 and B.cereus was grown to 1.0 OD, all at 600 nm. Cell populations of;108 cfu mL�1 were achieved for all three bacterial strains.Bacteria samples were diluted serially in nanopure water or inthe growth medium Nutrient Broth.

RESULTS

Nutrient broth is commonly used for culturing E. coli andStaphylococcus aureus. The upper panel of Fig. 1 presentsspectra obtained for Nutrient Broth. The spectrum of stockNutrient broth (trace a) is featureless apart from thecharacteristic fluorescent background. The lack of spectralbands for stock medium agrees with previous SERS studiessensing bacteria.25,41 The lower panel of Fig. 1 presents a SEMimage acquired of the evaporated Nutrient Broth on thenanorod array substrate. The dried broth deposits within and ontop of the nanorods. Figure 1 also presents the SERS spectrumobtained on a sample of the broth diluted 1:100 (v/v) withwater (trace b) following background correction. This spectrumcontains several well-defined bands that are characteristic ofthe components of this growth culture medium. Dilutionreduces the amount of growth medium deposited onto the

AgNRs by evaporation, improves the collection efficiency ofthe SERS signal, and decreases the fluorescent background.

Figure 2 presents three SERS spectra: one acquired on B.cereus at a concentration of ;108 cfu mL�1 in Nutrient Broth(trace a), one acquired after 1:100 dilution of this solution inthe growth medium (trace b), and one acquired after 1:100dilution in water (trace c). Traces a and b are identical to thatobtained for undiluted Nutrient Broth; trace a has been offset inintensity for clarity. Trace c contains spectral features similar tothose obtained by others for B. cereus.15,24,42,43

B. cereus is a Gram-positive bacterium. The spectral featuresare expected to be those representative of the peptidoglycancell wall layer. The outer wall structure of Gram-negativebacteria is composed of lipopolysaccharides. Thus, differencesin spectral bands characteristic of the type of bacteria areexpected. A schematic of the structural differences betweenthese two types of bacteria is depicted in Fig. 3. To test thishypothesis, spectra were also acquired on another Gram-positive bacterium (A. histidinolovorans) as well as on a Gram-negative bacterium (E. coli). Cell colonies for both were grownin Nutrient Broth to a stock concentration of ;108 cfu mL�1.Figure 4 presents the SERS spectrum acquired on all threebacterial species following 1:100 dilutions of their stocksolutions in nanopure water. For comparison purposes, allspectra displayed in this figure were normalized to the peak ofhighest intensity and offset along the y-axis for clarity. Thespectrum obtained for E. coli is in close agreement with thatreported by others.12,15,16,19,20,23,25,42–44 The spectrum of thediluted Nutrient Broth is also included in this figure. The fourspectra are strikingly similar in appearance. The similarity inthe spectra obtained on Gram-positive and Gram-negativebacterial cells is troubling.

To verify that spectra presented in Fig. 4 were indeedcharacteristic of the bacterium, two additional experimentswere performed. First, SEM images were acquired on thenanorod arrays to verify that the bacterial cells were intact andspatially distributed throughout the well. The image obtainedfor A. histidinolovorans is presented in the upper panel of Fig.5. This bacterium has a rod-like structure typically 1 to 2 lm inlength. The inset presents an image whose features areconsistent with this dimension. The SEM images show thatthe bacteria are not uniformly distributed across the well.

FIG. 1. (Top) The SERS spectrum of Nutrient Broth (a) stock, and (b) diluted1:100 (v/v). The spectrum of the diluted sample was background corrected butnot rescaled. (Bottom) The SEM image of stock Nutrient Broth on the Agnanorod array. The scale bar is 10 lm.

FIG. 2. SERS spectrum of B. cereus: (a) 108 cfu mL�1 in stock Nutrient Broth;(b) 106 cfu mL�1 in stock Nutrient Broth; and (c) 106 cfu mL�1 in dilutedNutrient Broth prepared by 1:100 (v/v) dilution in nanopure water. Trace (a)was offset along the intensity axis for clarity, and trace (c) was backgroundcorrected.

602 Volume 64, Number 6, 2010

Rather, they are clustered in random locations typically with 2–5 cells per cluster on top of the nanorod array. Next, SERSspectra were acquired across the well in 25-lm steps, with alaser spot size of 20 lm (diameter), depicted in Fig. 5 as acircle. Comparison of spectra acquired across the well showedno significant changes in the Raman bands; the relativeintensity of spectral bands was dependent upon location. TheSERS intensity of the most intense peak in the spectrum (725cm�1) was plotted as a function of position across the well andis presented in the lower panel of Fig. 5.

Similar findings were obtained with B. cereus. Thisbacterium also has a rod-like structure with dimensions thatare four to five times larger than that of A. histidinolovorans.The SEM images and SERS spectra acquired across the wellare presented as Supplementary Material to this paper(available on-line). Since (1) the Raman shifts are unchangedin spectra acquired across the well, (2) the bacteria are too largeto fit between the nanorods in the array, and (3) the spatialdistribution of bacteria across the well is non-uniform, weconclude that the Raman shifts observed are due to growthmedium adsorbed onto the nanorod array. The troubling aspect

FIG. 3. Schematic of the bacterial cell wall structure.

FIG. 4. SERS spectra of (EC) E. coli, (BC) B. cereus, and (AH) A.histidinolovorans in Nutrient Broth diluted 1:100 (v/v) in nanopure water to afinal concentration of 106 cfu mL�1. The spectrum of diluted Nutrient Broth(NB) is also included for ease of comparison. All four spectra were normalizedto the peak of highest intensity and offset along the intensity axis for clarity ofpresentation.

FIG. 5. (Top) SEM image following evaporation of a 5 lL aliquot of A.histidinolovorans at 106 cfu mL�1 prepared by diluting the cells grown in stockNutrient Broth by 1:100 (v/v) in nanopure water. The scale bar is 10 lm. Thewhite circle denotes the size of the laser spot. Arrows denote the location of twobacterial cell clusters. The inset depicts a SEM image at higher magnification.The scale bar in the inset denotes 2 lm. The size of the bacterial cell relative tothe nanorods is clearly visible in the zoomed image. (Bottom) SERS peakintensity for the ;725 cm�1 band as a function of position followingbackground correction. Spectra were acquired in 25 lm step increments acrossthe well prior to acquiring the SEM images shown in this figure.

APPLIED SPECTROSCOPY 603

of this conclusion is the similarity of our spectra to thosepreviously published for the bacteria investigated here-in.7,8,13–16,19,21,23–26,41–43,45,46

This compelled us to investigate the spectral signatures ofother bacterial growth culture mediums. Figure 6 presents thespectra acquired for 14 different media; all were diluted innanopure water. The specific dilutions are identified in thecaption. All spectra presented were normalized to the peak ofhighest intensity and offset along the y-axis for clarity. Many ofthe bands found in these spectra have been reported elsewhereas fingerprint bands for various bacteria.

DISCUSSION

Bacterial cell growth media generally contain a source ofamino acids, a carbon source (e.g., glucose or dextrose), water,and salts to promote bacterial growth. The amino acid sourcesare typically protein hydrolysates, often referred to as peptones.Peptones are classified by the source from which they arederived, e.g., animal-free, meat, or casein and whey. Somegrowth media contain peptones from multiple sources.47 Forexample, Nutrient Broth, the growth medium used to culturethe three bacteria studied herein, contains 0.3% (w/v) beefextract and 0.5% pancreatic digest of casein. Some media areoptimal for culturing a specific bacterial cell line. For example,Mueller-Hinton II Broth is commonly used to cultureStaphylococcus aureus, Enterococcus faecallis, and Pseudo-monas aeruginos, whereas Standard Methods Agar is used ingrowing Bacillus subtilus, Bacillus stereothermophilus, andEnterococcus hirae. Some bacterial cell lines can be cultured ina variety of growth media. For example, E. coli can be grownin several of the media studied herein.47

The spectral bands we observed for diluted media areconsistent with the components making up the medium. Manypossessed spectral features that were similar to those of othermedia. For example, the spectra presented in Fig. 6 for NutrientBroth, Standard Methods Agar, Mueller-Hinton II Broth,Cooked Meat Medium, and Sabouroud Liquid Broth Modifiedall contained an intense Raman band between 725 and 730 cm�1

as well as a band between 1325 and 1330 cm�1 whose intensityvaries among the five media. The 725–730 cm�1 band has beenattributed in the literature to adenine from flavin,10,48 therocking vibration of CH2,14 and the C–O–O� bend.41 The 1325–1330 cm�1 band has been attributed to a C–H deformation forguanine or protein6,13,48–50 and specifically the amino acidtyrosine.14 Most of the spectra contained bands at 1000 and1030 cm�1, a region typically attributed to aromatic componentssuch as phenylalanine11,14,48,50 and between 960 and 965 cm�1,a band often attributed to the C–N stretch. All of these bands areexpected for protein hydrolysates.

Interestingly, these bands have also been assigned assignatures of specific bacteria (e.g., B. cereus,15,24,43,48,51

Bacillus lichenformis,20 Bacillus megaterium,19,20 Bacillussubtilus,14 and E. coli13,16,19,20,45,51–53). Spectral characteristicspresent in Raman and SERS spectra have been assigned tocomponents of the protein coat and cell wall for variousbacteria. Some of the designated peaks for bacterial identifi-cation are also peaks observed in diluted media spectrapresented in this work.�

Efrima and Zeiri26 have previously commented on the factthat published SERS spectra acquired from the same type ofbacterium vary widely from group to group while the normalRaman spectra are quite similar. Differences in SERS spectrahave been attributed to variations in sample and substratepreparation, excitation wavelength, and the type and shape ofthe SERS substrate (e.g., nanoparticles, nanorods), as well asthe laser spot location and diameter relative to the bacteria. Inthis context, we have compiled the spectra previouslypublished for E. coli (Fig. 7) and B. cereus (Fig. 8) andcompared them with our spectra. The spectrum we report for E.

FIG. 6. SERS spectra acquired on bacterial growth media. All media, preparedaccording to the manufacturer’s recipe, gave spectra similar in structure to thatpresented in Fig. 1a. Each medium was diluted in nanopure water in varyingratios. The spectra presented in the figure were normalized to the peak ofhighest intensity and offset along the intensity axis for clarity of presentation.Abbreviations and dilution factors: (RU) Rapid Urea Broth, diluted 1:100 (v/v);(SF) Selenite-F, diluted 1:10,000; (CT) Campylobacter Thioglycollate Medium,diluted 1:1,000; (TB) Turbo Broth, diluted 1:1,000; (LB) Luria Broth, diluted1:10; (GN) Gram-negative Broth, diluted 1:10,000; (LBB) LB Broth, diluted1:100; (PW) Peptone Water, diluted 1:100; (MM) Motility Medium, diluted1:10; (NB) Nutrient Broth, diluted 1:100; (CM) Cooked Meat Medium, diluted1:1,000; (SL) Sabouroud Liquid Broth Modified, diluted 1:10; (MH) Meuller-Hinton II Broth, diluted 1:100; and (SM) Standard Methods Agar, diluted1:100.

� The reader is directed to the table included in the Supplementary Materialfor a more detailed listing of peaks observed in various growth media.Also included in this table is a comparison of these spectral bands to thosereported for both E. coli and B. cereus.

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coli, located at the top of Fig. 7, has features that are commonto all other published spectra for this bacterium. Similarly, thespectrum we report for B. cereus, located at the top of Fig. 8,also has features that are common to three of the four publishedspectra for this bacterium. Careful examination of the spectrapresented in these two figures reveals spectral features that areconsistent throughout the set. For example, the band at ;725cm�1 is present in every spectrum in the figure. Its relativeintensity is, in most instances, the strongest in the spectrum.Other spectral features are inconsistent from spectrum tospectrum. For example, the 930–1130 cm�1 band, previouslyassigned to membrane phospholipids,54 is expected in thespectrum of Gram-positive bacteria. However, it is observed inspectra of both E. coli and B. cereus with varying intensities.Relative intensities in SERS are known to vary with incidentbeam wavelength, substrate composition, and structure, as well

as orientation of the scattering moiety on the substrate and itsposition within the plasmon field.55–64

Principle component analysis (PCA) has been used to assessspectral differences between different bacterial types, species,and strains.14,24,25,65–68 Often, classifications were based onsubtle differences in spectra. Heretofore, it has been widelyassumed that washing of the bacterial sample with watereffectively removes the growth medium and that the spectrumobtained on the sample is that of the cell wall.� Thisassumption follows from published spectra of the media thatare featureless. The results presented in Figs. 1 and 2 show thatthe spectrum of diluted media is far from featureless. Thus, bywashing bacterial cell samples, previous workers may havebeen inadvertently diluting the medium. This holds for bothnutrient broths and nutrient agars. We suspect that the resultanthierarchical clustering may have been influenced more byresidual growth medium in the sample rather than bydifferences in the components of the bacterial cell wall. Weinvite experts in multivariate analysis of SERS spectra to testthis hypothesis by challenging the spectra of diluted growthmedia against the existing hierarchical clusters for bacteria.

Regardless of whether the above hypothesis is disproven,our findings provide strong impetus for a re-investigation ofprotocols commonly used in discriminating between bacterialcell type, species, and strain by SERS. Given (1) the structuraland compositional similarities in the bacterial cell wallstructure, (2) the lack of consensus on the spectral attributesof a specific bacterial species, (3) the similarity in spectralfeatures between the medium and the bacterium, and (4) thevariability in sample and substrate preparation methods, thereexists a clear need for acquisition of SERS spectra on bacterialsamples devoid of growth medium. We have definitively

FIG. 7. Published SERS spectra acquired on E. coli. The spectra presented inthe figure were taken from the reference corresponding to each spectrum label.Spectra were offset along the intensity axis for ease of comparison.

FIG. 8. Published SERS spectra acquired on B. cereus. The spectra presentedin the figure were taken from the reference corresponding to each spectrumlabel. Spectra were offset along the intensity axis for ease of comparison.

� The extent of growth medium dilution by the traditional washing methodis a function of the volume of the decant, mass of the pellet, and volume ofbuffer used to resuspend the pellet. Thus, washing bacteria dilutes thecomponents of the growth medium and may or may not remove them,depending upon the specific protocol used.

APPLIED SPECTROSCOPY 605

demonstrated that small quantities of medium present in thesample give rise to a strong SERS response. This responsereflects the affinity of medium components for the substratesurface and their favorable mass transport characteristics,relative to that of the intact bacterial cell. Should thepreparation of a medium-free, intact, and viable bacterialsample prove elusive, then spectra characteristic of a givenbacterial type, species, and strain will be obtainable usingeither confocal Raman8,11,45,50,53,69 or optical tweezer11,70

based Raman techniques.

ACKNOWLEDGMENTS

We thank Mr. Robert Martinez and Prof. Patricia Sobecky (University ofAlabama, Tuscaloosa) for providing us with bacterial samples for analysis. Wealso thank Prof. Mohan Srinivasarao and Mr. Min Sang Park from the School ofPolymer and Textile Fiber Engineering at Georgia Tech for extended use of theirRaman spectrometer as well as Dr. HsiaoYun Chu and Prof. Yiping Zhao of theUniversity of Georgia for providing us with the Ag nanorod substrates. Financialsupport of this research was provided by the Georgia Tech/UGA BiomedicalResearch program and the Georgia Research Alliance VentureLab program.

SUPPLEMENTAL MATERIAL

The Supplemental Material mentioned in the text is availablein the on-line version of the Journal at http://www.s-a-s.org.

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