Vivian Tran

Project title: Validation of a Sponge Processing Method for Characterizing Microbes in the Bullitt Center

Degree: MS | Program: Environmental Health (EH) | Project type: Thesis/Dissertation
Completed in: 2015 | Faculty advisor: John Meschke


Background: The Bullitt Center is a six-story, 50,000 square-foot green building that emphasizes energy efficiency and environmental performance based on design and operation. Microorganisms found in indoor surface environments most often come from human sources or by transport from the outdoor environment. Quantity and location of surface microbes can change due to re-aerosolization with human activity and building air movement. Few studies have been performed on surface microbes in green buildings. The goals are 1) to validate a sponge processing method based on literature reviews and 2) to characterize microbial populations from surfaces in the Bullitt building over a one-year period.

Methods: Three processing methods (shaker table, hand massage, and stomacher 400 circulator) were evaluated for microbial recovery from 3MTM sampling sponges with 10 ml neutralizing buffer. Sponges were spiked with 1.06E8 to 2.88E8 E. coli B cells and cells recovered by each method. DNA from recovered cells was extracted (MO BIO PowerSoilTM DNA Isolation Kit), quantified by nanodrop (ND-3300) using picogreen, and E. coli specific targets amplified by qPCR (with fluorescent probe). The best processing method was determined by examining both percent recoveries of CFU and C(t) values. The Bullitt surface samples were processed by the most efficient elution method followed by DNA extraction, nanodrop quantification of nucleic acid, and Illumina 16S metagenomic sequencing to characterize the microbes that were present.

Results: Hand massage was determined to be the most effective elution method based on the highest and most consistent calculated percent recoveries of CFUs ranging on average from 51.5-56.2%. C(t) values gave inconsistent results using undiluted nucleic acid, but tighter results using 10-fold dilutions of the sponge extractions ranging from (14.7-18.4% for shaker table, 12.5%-36.6% for hand massage, and 20.8-28.3% for stomacher) suggesting the presence of inhibitors. Throughout the study, nucleic acid results ranged from (0-5121.5 ng/ml). The highest nucleic acid values were observed in June ranging from (78.1-5121.5 ng/ml). Results from Illumina 16S (>10% OTUs) showed changes in microbial populations over time and by location.

Conclusion: Validation of hand massage as a sponge processing method was the best compared to the shaker table and stomacher methods and can be used for future studies to characterize microbial populations. Nanodrop and Illumina 16S results provided information in the change of quantity and location of surface microbes in the indoor environment. Such knowledge may help facilitate an understanding of the indoor microbiome and ultimately reduce human exposure to bacteria that cause adverse health effects.