Escherichia coli detection via photobacterium leiognathi bioluminescence spectroscopy

Date of Publication

9-2023

Document Type

Bachelor's Thesis

Degree Name

Bachelor of Science in Premed Physics

Subject Categories

Physics

College

College of Science

Department/Unit

Physics

Thesis Advisor

Romeric F. Pobre
Glenn G. Oyong

Defense Panel Chair

Emmanuel T. Rodulfo

Defense Panel Member

Al Rey C. Villagracia
Marigold O. Uba

Abstract (English)

We designed and developed a new protocol for rapid detection of Escherichia coli (E. coli) bacteria concentration via Photobacterium leiognathi (P. leiognathi) bioluminescence spectroscopy. In this experimental study, the researchers employed a bioluminescence spectral technique for bacterial detection, focusing on E.coli since it is the primary contaminant in most water quality tests. The bioreceptor used in designing a bioluminescence spectral-based detection system was P. leiognathi, isolated by Ms. Jocelyn Pilante Luyon from the Molecular Sciences Unit Laboratory of the College of Science. The study involved a microplate fluorescence image readout of the assay containing one treatment group (P. leiognathi and E. coli in marine broth) and three control groups (marine broth only, P. leiognathi in marine broth, and replication of E. coli dilution). The presence of E. coli in marine broth solution induced a visible blue fluorescence emitted by P. leiognathi. To identify the optimal concentration of P. leiognathi that produces a significant fluorescence signal with an autofluorescence background signal, the researchers measured the bacterial concentration of E. coli indirectly using an optical density reader and monitored the fluorescence activity every five minutes for two hours. Spectroscopic results showed that E. coli concentration of 8.24 log (optical density) in CFU/mL unit of the sample displayed the optimal fluorescence signal, with peak luminescence activity observed at the 50-minute mark. To further validate the E.coli concentration of each sample, a quantitative Polymerase Chain Reaction was undertaken to verify the amount of E.coli. concentration. Statistical analysis between qPCR results and curve-fitted optical density of the bioluminescence spectroscopic signal led to a non-linear correlation log-log plot with an R-value of 0.59 that follows a quadratic downward opening uptrend with error bars ranging from 0.02 to 3.10 CFU/mL deviation. This quadratic curve would serve as the calibration curve in detecting E. coli concentration in terms of the optical density measurement from the water sample. Despite some notable deviations from the quadratic calibration curve, this study showed an earnest and promising step towards developing a more rapid, efficient, reliable, and accurate alternative technique of water quality tests using bacterial bioluminescence spectroscopic technique. Further refinements in the preparation and handling of water samples together with bioluminescence detection protocol need to be iterated and optimized for rapid and cost-effective water quality monitoring.

Abstract Format

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Abstract (Filipino)

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Abstract Format

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Language

English

Format

Electronic

Keywords

Escherichia coli; Bioluminescence

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Embargo Period

12-14-2023

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