Drs. Benoit and Wilson have long had a love affair with Bacillus thuringiensis (Bt) spores and their study. Their many papers on the subject have often centered on a germination assay that follows the conversion of dormant and resistant spores back to vegetative growth. The assay is based on watching a change in the optical density (absorbance) of spores using a spectrophotometer. It is fast and easy and dependable. At least it was until about 12 years ago when that assay, for some reason, stopped working.
The first indication something was wrong came in the form of an email from a researcher in New Zealand in the fall of 2001. The graduate student was studying Bt germination and ran into difficulty replicating the results Wilson and Benoit had reported. The email simply asked Dr. Wilson whether there were any special tricks involved in the assay. Since neither Wilson or Benoit was involved in research due to administrative responsibilities at the time and had not experienced similar problems, they had no advice to offer.
Fast forward a few years to the creation of the BIMS program and a new graduation requirement that every student must participate in capstone research, an Honors project, or an internship. The department needed research ideas, and Benoit and Wilson resurrected the germination assay as a means to engage students in studying new aspects of spores physiology. The number of unique projects this system would provide for future students was enormous. But the assay failed to work. Even with new facilities and new resources to support research, several student projects failed to recreate results from earlier papers. Nothing in the literature and no one in the field had an answer.
This summer, Honors student Heather Rawls became the most recent student to attempt the assay. Through the summer she tried differences in media, growth temperature, ways of collecting and processing spores, water quality being used, activation techniques, a variety of germinants, and at least five different spectrophotometers with no predictability or consistency in the results. If anything, fewer and fewer spores were germinating with each attempt.
In August, Heather and Dr. Wilson had a research pow wow and developed an alternate project for her Honors thesis. Time was running out to complete her research before starting the writing, and moving to something with a higher probability for success was needed. But Heather wasn’t ready to give up that easily. At an impromptu meeting a couple of weeks ago, a new thought emerged when it seemed every variable possible already had been tested. Glassware! In their graduate programs and during their prior research using the assay, Benoit and Wilson always collected the spores in glass containers and the germinants and all other chemicals used had been stored in glass containers. In our growing emphasis on research, McMurry had improved the funding of science programs so much that the use of disposable plastics was now the norm. Maybe the plastic centrifuge tubes used to collect spores and store germinants were coated with something inhibiting spore germination? Maybe some chemical was leaching out of the plastic?
Over the last two weeks, Heather switched to glassware for collecting spores and making reagents. Her results were dramatically different. We now are certain plasticware has an inhibitory effect on germination. A decade of frustration was caused by our affluence and the use of disposable labware rather than old-school glass.
With a working assay, Heather will turn back to the project she intended to complete this summer looking at the germination of a variety of genetically-engineered and wildtype strains of Bacilli. Another Honors student just beginning her work will investigate what the mystery chemical from the plastics might be and how it inhibits germination, or the range of spore-forming species affected. One mystery is solved, more are uncovered…
BIMS Honors students prove to us on a daily basis that they are among the best thinkers and hardest workers on campus. This commitment to uncovering the truth is what will drive them to become leaders in biomedical science and healthcare provision in the future.
Recently, three BIMS majors found out they will receive Bloomer and Beasley Research Fellowships for the coming year. All three are students of Dr. Gary Wilson and will be pursuing different projects investigating Bacillus thuringiensis spore properties as they pursue Honors research and write their Honors theses in the next year.
The Charles and Lisa Bloomer Research Fellowship is awarded to support research of promising students in the School of Natural and Computational Science (SNCS). This initiative of the Science and Math Advisory Board (SMAB) provides a research stipend for students as they work closely with McMurry faculty on a research project. Dr. Bloomer is a successful oral surgeon in Abilene who has generously and regularly supported the sciences at his alma mater. The biennial picnic the Bloomers host for SMAB members and SNCS faculty is a popular event building relationships and communicating the vision each holds for McMurry’s science future. The Beasley Research Fellowship is a new program supporting student research in the biological sciences. McMurry’s science alumni are spearheading an effort to create an endowment in memory of Dr. Clark Beasley, Distinguished Professor Emeritus from the Department of Biology who died this past summer. This represents the first year this fellowship has been awarded.
Recipients of this year’s awards are Heather Rawls, Miranda Nguyen, and Nicole McGunegle. Their projects will study wild type and genetically-engineered strains of Bacillus thuringiensis (Bt) and Bacillus cereus (Bc) grown in rich and poor media. Bt is a spore-former that produces an insecticidal toxin at the time of sporulation. Bc is a commonly encountered and well-studied spore-former closely related to Bt but generally harmless. The genetically-engineered strains include Bt strains that do not form crystals and Bc strains that have been engineered to produce Bt crystals. One project will look at how the presence or absence of the crystal in rich and poor media influences spore and crystal size and toxicity. A second project will look at how growth conditions impact spore dormancy and the process of activation and germination. It is possible an undiscovered variation of quorum sensing might be involved. The third project will explore UV and chemical resistance of wild type and genetically-engineered strains produced in rich and poor media. All projects fit the criteria for BIMS research: a complete project doable in a short time frame, certain discovery no matter the experimental outcome, publishable work.
Stay tuned for updates on how this work is progressing!
Senior BIMS major Luke Burcham has a fascination with fermentation. As an amateur brewmaster, his interest in the physiology of yeast fermentation has resulted in his choice for “life after McMurry”. He will be entering a graduate program at UC-Davis in January to study all things related to fermentation and brewing. From Borger TX to Davis CA is quite a move, but Luke is ready for the adventure.
With that in mind, Luke worked with Dr. Tom Benoit this semester to design a project that would give him a head-start on his graduate studies. After Luke reviewed some literature on the subject, he began to envision a project to investigate the impact of modifying the ion content of the growth medium for beer production to determine the best formulation. The experimental design began with a question on how calcium levels in the water used for the process might influence the physiology of the yeast in the batch. Could it be that increasing calcium ion content altered the growth characteristics of the yeast and alcohol content of the product? Would hard water result in a fundamentally different product than that produced in soft or distilled water?
Notes from research meetings and designs for experiments decorate the white board in the senior research lab (shown above). The final design Dr. Benoit and Luke settled on centered on using production of cell clumps and measurement of alcohol content as ways of monitoring physiological differences between batches. As yeast grow in a broth culture, they can form clumps of cells that fall out of suspension as nutrients are exhausted and the growth period comes to a close. This flocculation of yeast cells and cell debris is a necessary step to move from the cloudy active culture to a clear final (and commercially appealing) product. Could calcium concentration alter the dynamics of growth and flocculation?
After completing a variety of experiments this spring, the answer seems to be “yes”. Luke tells me that batches made with distilled/RO water averaged clumps of 17 cells (as measured microscopically using a hemacytometer), while batches made with added calcium chloride averaged clumps of 55 cells. In addition to having larger clumps, calcium enriched batches also produced less alcohol (as measured using a hydrometer). Luke is currently in the analysis process – explaining why clumps are larger and why larger clumps mean less alcohol produced – as he takes these findings and turns them into the final project poster for his capstone experience.
Luke’s project is a great example of the flexibility we provide our students to pursue senior projects in line with their interests and future plans. Luke’s future seems destined to be quite different from the one envisioned when he began his college life as a Biomedical Science major intent on dental school. However, the knowledge and skills and abilities gained in the BIMS program have application to his future and have equipped him well to succeed in his graduate work at UC-Davis.