Reflected scatterometry for noninvasive interrogation of bacterial colonies

Abstract

A phenotyping of bacterial colonies on agar plates using forward-scattering diffraction-pattern analysis provided promising classification of several different bacteria such as Salmonella, Vibrio, Listeria, and E. coli. Since the technique is based on forward-scattering phenomena, light transmittance of both the colony and the medium is critical to ensure quality data. However, numerous microorganisms and their growth media allow only limited light penetration and render the forward-scattering measurement a challenging task. For example, yeast, Lactobacillus, mold, and several soil bacteria form colorful and dense colonies that obstruct most of the incoming light passing through them. Moreover, blood agar, which is widely utilized in the clinical field, completely blocks the incident coherent light source used in forward scatterometry. We present a newly designed reflection scatterometer and validation of the resolving power of the instrument. The reflectance-type instrument can acquire backward elastic scatter patterns for both highly opaque media and colonies and has been tested with three different bacterial genera grown on blood agar plates. Cross-validation results show a classification rate above 90% for four genera.

Development of a Smartphone-Integrated Reflective Scatterometer for Bacterial Identification

“…Therefore, extensive research has been conducted to develop a rapid, reliable, reagent-free, and user-friendly system. We and others have demonstrated optical techniques that utilize the light scattering or diffraction pattern of a bacterial colony grown on agar plates for bacterial identification [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. The comparison of these technologies are summarized in the Supplementary Materials (Table S1) .…”

“…Therefore, a reflective (backscattering) ELS technique was introduced to differentiate the colonies grown in such conditions. The feasibility was tested on four different bacteria, resulting in over 95% of classification accuracy [ 9 ]. Because of the wide reflection angle, instead of a lensless image sensor, a high-resolution DSLR (digital single lens reflex) camera and a screen were utilized to capture patterns.…”

Generalized spectral light scatter models of diverse bacterial colony morphologies

“…However, the single‐wavelength model has a limitation in classifying organisms at lower hierarchical levels . For several years, researchers have worked on developing innovative BARDOT approaches, resulting in the introduction of multispectral and reflective BARDOT instruments .…”

“…Follow‐up studies performed to expand the application of the scatter model have shown promising results in describing the nature of the forward light‐scattering pattern produced from a bacterial colony. According to the model, the scattering pattern is a function of the morphological structure of the colony, including the colony height, diameter and refractive index . Bae et al demonstrated that the size of the pattern is dependent on the aspect ratio of a bacterial colony—the ratio of a colony's center elevation to its diameter.…”

Angular Photodiode Array-Based Device to Detect Bacterial Pathogens in a Wound Model

“…BARDOT systems have been shown to provide different results due to changes in agar or nutrient concentration as well [28]. Recent advances in BARDOT systems have eliminated the need for an optically transparent culture surface through the use of reflection, but still require bacteria to be cultured [29]. Our group has previously demonstrated that Mie scatter can be used to detect the presence of bacteria on ground beef [30].…”