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Biomedical Optics & Medical Imaging

Light scattering endows bacterial colonies with unique fingerprints

The light-scattering signatures of bacterial colonies, which depend on minute genetic differences, may provide a way to rapidly identify pathogens in the food supply.
16 February 2007, SPIE Newsroom. DOI: 10.1117/2.1200702.0557

Ultrasensitive and rapid bacteria detection methods are essential tools to control and prevent pathogens in food-processing facilities as well as to maintain the security of the food supply. But these methods need to become substantially more robust and less costly so that arrays of distributed sensors can be deployed at critical intermediate points in the food-processing cycle. Conventional culture methods followed by biochemical analyses such as the polymerase chain reaction (PCR) provide reliable results, but their analysis time (four to seven days), labor, and cost severely limit wide deployment.

Numerous researchers have tried to develop biosensors that can deliver the performance required. Many methods are based on reactions between antibodies and antigens, important components of the body's immune system. These methods are very specific to bacterial serotypes, subdivisions of a species based on their antigens, that cause food safety concerns. Examples of techniques include fiber-optic-based sensors, such as those using surface plasmon resonance effects, immunofluorescent assays (IFA), enzyme-linked immunosorbent assays (ELISA), and DNA microarrays. These methods require labeling, processing, and handling the wide array of antigens that target specific harmful bacterial serotypes as well as implicitly require advance knowledge of which bacteria could be present.

Methods to detect and classify bacteria by observing their interaction with laser light have been studied for some time. Most of these methods have attempted to detect bacterial cells in liquid suspension. This presents several challenges, however, as the liquid culture may contain other microorganisms or the arrangement of bacteria may be inconsistent. Our team1–3 has developed a rapid, noninvasive method that in effect measures the optical-physical properties of bacterial colonies on solid agar plates. Genetic differences between bacterial strains are manifested in subtle but repeatable differences in the microscopic and mesoscopic properties of the colonies they procreate. Our scattering instrument amplifies these subtle differences to produce remarkably different scattering phenomena.

This method requires an integrated combination of microbiological protocols for incubating the colonies, optical system design and optimization, and image-processing schemes with a physical basis that allow automated scattering fingerprint analysis. The bacteria are grown in brain heart infusion (BHI) broth, and ribotyping confirms the identities of the cultures. Culture dilutions are plated on surface BHI agar (∼100 colonies per plate) and incubated at 37° C for 18–36h or until the colony diameter reaches 1.2–1.5mm. As shown in Figure 1, the optical system consists of a source (laser diode module at 635nm wavelength), a sample holder, and an imager (640×480 pixel monochromatic IEEE-1394 CCD image sensor) that acquires the scattering images. Advanced image-processing methods facilitate automated analysis.2

Figure 1. Schematic of the optical system used to generate light-scattering patterns from bacterial colonies.

We studied colony growth characteristics and the corresponding evolution of scattering patterns for a wide variety of pathogenic bacteria relevant to food safety. Based on these observations, we developed a model that predicts the forward-scattering signatures we observe. We also studied colonies produced by acid-, heat-, and osmotically stressed bacteria and confirmed that, once resuscitated, the optophysical properties of the resulting colonies produce essentially the same forward-scattering fingerprints as nonstressed bacteria. Figure 2 demonstrates the power of our method. Scattering fingerprints from closely related serotypes of Escherichia coli show very distinctive patterns. Figure 3 presents scattering fingerprints from two colonies of the same bacteria, demonstrating typical colony-to-colony repeatability.

Figure 2. Forward-scattering fingerprints from a variety of Escherichia coli strains. (a) E. coli K12 is a nonpathogenic strain. (b), (c), and (d) These fingerprints correspond to pathogenic strains of E. coli serotype O157:H7. The E. coli O157:H7 G5295 in (b) was responsible for an outbreak at a daycare center in 1988. Panel (c) shows E. coli O157:H7 K6, and (d) E. coli O157:H7 EDL 933.

Figure 3. Two different colonies of E. coli serotype O157:H7 strain 505B grown under similar conditions exhibit the typical colony-to-colony repeatability of forward-scattering fingerprints obtained with our method.

In summary, we have found that bacterial species and serotypes generally have unique scattering fingerprints that can be used for rapid detection and classification of the bacteria. Our light-scattering method is fast, highly sensitive, and virtually reagentless. It shows great promise for identifying colonies of a wide range of bacteria relevant to infectious disease, homeland security, and food safety.

E. Daniel Hirleman, Eui Won Bae
School of Mechanical Engineering, Purdue University
West Lafayette, USA
Karleigh Huff, Priya Banada, Arun Bhunia
Food Science Department, Purdue University
West Lafayette, USA