Investigation of the presence of rod-shaped bacteria on food surface via elastic light scattering

Euiwon Bae, Huisung Kim, Helen A. McNally, Arun K. Bhunia, E. Daniel Hirleman

Department of Electrical and Computer Engineering Technology, Purdue University, West Lafayette, IN, 47907, USA.
Molecular Food Microbiology Laboratory, Department of Food science, Purdue University, West Lafayette, IN, 47907, USA.
School of Engineering, University of California-Merced, Merced, CA, 95343, USA.
School of Mechanical Engineering, Purdue University, West Lafayette, IN, 47907, USA.

DOI: 10.4236/abb.2012.34050                

Abstract

We investigated the presence and related signal-to-noise ratio (SNR) of rod-shaped bacteria on a produce surface using elastic scattering. The theoretical noise was defined as a scattering signal from a rough produce surface while the signal was defined as a scattering signal from the increasing numbers of rod-shaped bacteria on the produce surface. In this research, we measured the surface topography of a tomato using BioAFM to provide the quantitative nature of the surface roughness which was, in turn, modeled with the discrete dipole approximation (DDA) for an accurate estimation of the background scattering signature. Then we included the DDA model of rod-shaped bacteria and calculated the combined elastic scattering signature in the upper hemispherical space with different polarizations, wavelengths, and incident angles. The total scattering cross-section (TSC) and partial scattering cross-section (PSC) were both computed on six predefined aperture locations. The results indicate that, upon proper selection of the wavelength and incident angle, it was possible to provide the minimum number of bacteria (~32) to provide a differentiable elastic scattering signal from the produce surface.

Keywords

Scattering; Roughness; E. coli O157; Food Surface; Diffraction

1. INTRODUCTION

Recent outbreaks from various agricultural products such as spinach, tomato, and Jalapeño remind us that the food supply system is susceptible to contaminations from pathogenic bacteria and imposes a great risk on public health. The consumption of fresh produce (fruits and vegetables) is of great concern because these are grown in open fields, where the chances of exposure to the pathogenic bacteria are many. Further, the breach in sanitary practices during processing and packaging may also increase the opportunity of bacterial contamination. In addition, the economic loss due to the recall of the foods could cost in millions of dollars to the already suffering agricultural companies from the impact of the hike of the raw material price.

Therefore, for the food and healthcare industries, the rapid identification and isolation of bacteria such as pathogenic Listeria monocytogenes and Escherichia coli in products are very important [1]. In the realm of methodologies, various techniques have been developed, including morphological, immunological, and proteomics/ genomics [2]. Genome based molecular methods have been modified to reduce time-consuming enrichment steps for bacterial detection, such as real-time PCR using molecular beacons to detect E. coli O157:H7 (>10 CFU) [3], recirculating immunomagnetic separation (RIMS) coupled with real-time PCR, and standard culture methods (0.1 CFU/g of spinach) [4]. In addition, the piezoelectric-excited millimeter-size cantilever (PEMC) sensor immobilized with an antibody has been used for detection of E. coli O157:H7 [5]. Various sensing mechanisms, including light scattering, Fourrier transformed infrared (FTIR) spectroscopy, and Raman, have been developed to detect microbes based on their morphological/structural/chemical properties directly without the need for any labeling reagents. These methodologies typically utilize lasers to generate and differentiate transmission or reflection signals from the incoming probing beams depending upon the shape, thickness, or color of the sample under testing [6-8].

Light scattering has been used for feature detection and identification in areas as diverse as the biological, astrophysical, and semiconductor industries due to its speed and accuracy. When the incoming primary wavelength and outgoing secondary wavelength are identical, this method is called elastic scattering, while if there exists a wavelength shift, it is referred to as inelastic scattering. In the elastic scattering method, angular scatter measurements have been utilized for various quantitative studies of micro-particles like bacteria in flow [9] and red blood cells [10] through the use of a scatter-model based approach. Very recently, angle-resolved back scattering has been used to identify micro colonies of such bacteria in flow, by matching their known surface textures [11], instead of their individual size, shape, or refractive indices. Bronk presented an extensive description of light-scattering experiments on rod-shaped bacteria such as E. coli [12]. However, if the morphological difference was minute or the background scattering was significant, the specificity of the overall scattering signature started to lose their uniqueness. Recently, it was shown that light scattering signatures from colonies were indeed reproducible and differentiable without applying any specific labels [13-18]. Inelastic light scattering such as Raman or SERS (surface enhanced Raman spectroscopy) is another method of interrogating microorganisms. Fluorescence is the most widely-used inelastic light scattering method in flow Cytometer and confocal microscopic measurement. The microorganism of interest inherently contained certain proteins or molecules that reacted to a specific wavelength or the researchers artificially infused the fluorophore either by tagging a molecule or via genetic engineering. Even though the incoming light was incident on the microorganism, the localized areas with the specific fluorophore would only react to the light and emit photons for detection. Historically, most of the optics-based quality control of the produce was targeted at discriminating the firmness or sugar content using single wavelength [19-22] or multi-spectral methods [23- 29] rather than directly detecting the source of the contamination. Recently, some researchers have suggested a laser induced fluorescence (LIF) measurement to detect the animal feces on an apple surface [30]; this method was still categorized as an indirect method although there was a correlation between the animal feces and the existence of pathogenic bacteria. In addition, an indirect method is known to limit the types of pathogenic bacteria that could be correlated via the LIF since they might be originating from different types of sources. In general, the signal strength of the inelastic scattering is several orders of magnitude lower than the elastic scattering; therefore, a more sophisticated and costly measurement system is actually required. In addition, their specificity of incident wavelength and certain proteins limits their applicability to broader applications. Therefore, here we have investigated the optimum selection of physical parameters to detect the presence of bacteria on the rough surface via computational electromagnetic codes. First, we selected the tomato as a representative produce example and analyzed the surface characteristics via atomic force microscopy (AFM). Based on this result we provided the micro-scale model to more accurately predict the light scattering responses of both the blank tomato and the tomato with a certain number of bacteria and also provided whether light scattering distribution was differentiable.

2. MATERIAL AND METHODS

2.1. DDSURF

To predict the light scattering characteristics from the tomato surface without any labeling, we applied the discrete dipole approximation (DDA) technique for modeling the scattering [31]. In DDA, particles were subdivided into volume elements, and the optical characteristics of each element were approximated by modeling the element as a single electric dipole. The dimensions of the volume elements represented by the dipoles must be much smaller than the wavelength of the incident light (typically < λ/10). Typically, the DDA modeled the light scattering problem in free space while a new technique was reported that modeled the scattering from particles on the surface using iterative solvers [32,33]. This code, called DDSURF, was crucial to appropriately model multiple bacteria on the tomato surface. As is shown in Figure 1, the total electrical field at any dipole was composed of the electrical field incident upon the cells and the vector sum of the electric fields incident from all other dipoles making up the neighboring cells, including the reflections off of the substrate [34,35].

When a laser source was incident upon the dipole array, the dipole moment at dipole was related to the electromagnetic field shown as:

, (1)

Figure 1. Modeling of rod-shaped bacterial using discrete dipole approximation. Bacteria was modeled with many dipoles and placed on top of a random rough surface. The interbacteria (E_direct) and bacteria-surface (E_reflection) interactions were all included for accurate computation. The figure on the right insert shows an example of 7 bacteria on the surface (top) and their scattering pattern on the hemispherical surface.

where was the dipole polarizability tensor and was the total electric field present at the dipole. In the presence of a surface, the electric field incident on the receiving dipole also included components from the surface-reflected incident shown as:

, (2)

where was the direct interaction at the dipole and was the surface reflected interaction of the between. When we represented all N individual dipoles with Eq.2, we obtained a global matrix equation:

(3)

where D represented a diagonal matrix from polarizability, A was a square matrix from direct interaction, R was a square matrix from reflection interaction, P was the dipole moment matrix of , and was the incident beam matrix of. After iteratively solving the scattering problem in DDSURF, a residue was defined as:

, (4)

where was the residue at the iteration. Once the dipole moment of the all the location was computed by Eq.3, the far-field radiation pattern was computed by a summation of the scattered field radiated from each dipole as:

, (5)

where indices i and j are used for the i^th observation point and j^th dipole center location. and are the electric fields at dipole j due to the TM and TE incident plane waves with a wavevector k = −k_sca. To explore the differentiability of the light scattering pattern against the physical input parameters, we compared the reflection scattering signature of a control sample with and without bacteria on the surface and the total number of bacteria models were also varied to investigate the theoretical detection limit beyond the background scattering signal. We defined a differential scattering cross-section (DSC) as a power dissipated per unit solid angle and the DSC was formulated as [36]:

(6)

where I_sca was the scattered irradiance, A was the detection area, r was the distance from feature to the observation point, I_inc was the incident irradiance, and W was the detection solid angle. The total scattering cross-section (TSC) was defined as the integral of the DSC over the hemispherical distribution which integrated all the differentially scattered photons on the 2π radian and was defined as the total power scattered by the incident intensity which gives the unit of area (µm²). The far-field quantities which provide the quantitative indicator were the TSC and the partial scattering cross-section (PSC), as is shown in Figure 2. However, the TSC value provided limited information regarding the spatial distribution of scattered light. Therefore, we also defined the PSC as:

, (7)

where C_sc was the TSC, Ω was the solid angle, I_s was the scattered intensity, R was the distance from the scatterer to the detector, I_i was the incident intensity, and A was the aperture area. Therefore, the SNR was defined as:

(8)

where PSC_b and PSC_t were the cross-sections with and without the bacteria on the tomato’s surface.

2.2. Sample Measurement

To understand the surface scattering from bacteria on the surface of produce, it was critical to understand the surface roughness of the produce itself which played a role as a background noise in the scatter measurement (and eventually determined the Signal-to-noise ratio (SNR)). Here we performed the atomic force microscopy (AFM) measurement of a clean tomato and computed the surface statistics. The AFM was performed using the Bioscope II

Figure 2. Definition and variation of PSC in the Cartesian coordinate. P and S define the polarization of the incident light. AP1 to AP3 was when the detector was located at (θ, ϕ) of (75˚, 90˚), (75˚, 90˚), and (75˚, 90˚). For backscattering, AP4 to AP6 was located at (75˚, 0˚), (75˚, 0˚), and (75˚, 0˚). Inset shows the f/2 collection optics of PSC with f = 50 mm.

AFM and an associated controller (Veeco Instruments, Santa Barbara, CA). Surface topography measurements were conducted in air using a tapping mode and an OTESPA (Veeco) cantilever operating at 322 KHz where the scans were collected at a rate of 1 Hz. Dehydration issues were addressed by eliminating the optical microscopy light and minimizing the length of experiments. A fresh tomato was cut at approximately 1 × 1 mm and placed on the glass slide for AFM measurements. Six different samples were selected with an approximate 100 × 100 µm² area by use of the tapping mode, and both the root-mean-square (RMS) and average roughness (Ra) were computed. The measurement results were then converted to the DDA model which incorporated all the bacteria-tomato surface interactions in a near-field scattering. When the surface roughness was smooth such as with a silicon wafer, light scattering could provide a high signal to noise ratio since the scattering from the background was negligible. As shown in the inset of Figure 1, when rod-shaped bacteria were modeled on an optically flat surface, the background scattering was negligible so that it was sufficient to measure the TSC to detect the presence of bacteria. However, to perform a similar prediction on a real-food sample, situations changed dramatically. According to Table 1, the surface roughness of the food sample was on the same order of magnitude with the bacteria itself which made it difficult to discriminate in the light scattering signature as compared to the smooth surface scattering. In addition, the light distribution was now coupled with both bacteria and background scattering which now required a careful design of the specific position of the detector to maximize the signal level. The DDSURF code enabled us to directly model the tomato sample surface and to include in the inter-bacteria and bacteria-surface interactions which provided a theoretical prediction of which combination of physical parameters would have an optimum signalto-noise ratio (SNR) with a rough surface.

Figure 3(a) displays the surface characteristics of a tomato which demonstrated diamond shaped craters. The quantitative measurement results from the AFM were then converted to a DDSURF input file, as is shown in Figure 3(b), where a cluster of four bacteria was positioned at the center of the valley. The dotted circled area of approximately 40 μm in diameter was selected and programmed to provide a 3-D input dipole model as:

Conflicts of Interest

The authors declare no conflicts of interest.

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