Date of Award

Summer 8-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

First Advisor

Rebecca Giorno

Abstract

Bacteria of the Bacillus genera are capable of forming dormant and resilient cells called spores in response to starvation. Spores can break dormancy in the presence of nutrients in a process called germination. Historically, spores are exposed to sublethal heat treatments to increase the extent and rate of germination. This process is known as heat activation (HA). Previous studies on Bacillus species indicate the effects of HA are reversible after 72 hours. After this time period, the spores must be reactivated. However, spores of Bacillus anthracis, a member of the Bacillus family, have not been tested to see how long they remain activated.

In this study, B. anthracis spores were heat activated and germination was measured for seven days to see if they remained activated. B. anthracis spores were prepared by the exhaustion method in Difco Sporulation Media and extensively water washed. Each sporulation was split into three samples: no heat treatment (UH), heat activated on day one of the experiment (HA), and activation on the particular day relative to day one (Hn). Germination was initiated with 1mM L-alanine and 1mM inosine and was measured by the loss of optical density (OD) at 580nm. The assays were performed on days one, three, five, and seven.

As expected, heat activation had a positive impact on spore germination. On day one, HA and H1 spores had a final OD loss of 57.24% and 57.88%, respectively, while the UH spores had an OD loss of 34.69%. On day seven, HA spores had an OD loss of 54.16%, H7 had a 52.66% OD loss, and UH had a 27.49% OD loss. Although all reactions showed a decrease in germination on day seven compared to day one, a two- way ANOVA test showed this decrease to be statistically insignificant. The heated samples (HA, Hn) were shown to be significantly different from the UH sample on both day one and day seven. Interestingly, there was no significant difference in germination for HA spores between day one and day seven, suggesting that HA spores can remain activated over seven days. No significant difference was seen between HA and Hn for either day.

This study shows that HA impacts B. anthracis spores for seven days, much longer than originally expected. This changes our understanding of heat activation in B. anthracis. Further studies will help define the time it takes for spores to be inactivated as well as test the effects of storage conditions on activation.

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