Reflected scatterometry for noninvasive interrogation of bacterial colonies

Authors: KIM, HUISUNG - Purdue University; DOH, IYII-JOON - Purdue University; STURGIS, JENNIFER - Purdue University; BHUNIA, ARUN - Purdue University; ROBINSON, J - Purdue University; BAE, EUIWON - Purdue University

Submitted to: Journal of Biomedical Optics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2016
Publication Date: 10/24/2016
Citation: Kim, H., Doh, I., Sturgis, J., Bhunia, A.K., Robinson, J.P., Bae, E. 2016. Reflected scatterometry for noninvasive interrogation of bacterial colonies. Journal of Biomedical Optics. doi: 10.1117/1.JBO.21.10.107004.

Interpretive Summary: Rapid classification is important for detection of foodborne pathogens and bacterial colony morphology (form and structure)-based classification is one commonly used method. A recently developed laser scatter pattern-based bacterial colony classification system only works with transparent media and translucent colonies. However, many bacterial colonies or growth media are sufficiently dense that no light scan penetrate the bacterial colony on the plate. A new bacterial colony interrogation system was developed that can analyze colonies on opaque media. The new system utilizes a high power laser to reflect the incoming laser light and a digital imaging system to capture the whole image at once. Validity of the method was tested on Salmonella, Vibrio, Listeria, and E. coli which were all grown on sheep blood agar. The developed classification system successfully provided 90% or higher correct results on the tested samples. This result will greatly expand the utility of the laser based identification of bacterial colonies into clinical and veterinary fields where blood agar is the standard culture media, improving the ability to rapidly identify bacterial pathogens.

Technical 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.