Moldy bread, experimental design, and you

For many semesters, my nonmajors biology lab did a lab called chicken wing microbiology. You can download it here, from the wonderful Association for Biology Laboratory Education website (Walvoord and Hoefnagels, 2006). In the lab, students devised a method to kill the bacteria on chicken wings, carried out an experiment (including serial dilutions) to test their proposed method, collected plate count data the following week, and wrote a short lab report on their results.

One semester, a vegetarian student notified me that he would refuse to work with chicken, so we agreed that he could try the same lab with potatoes in place of chicken wings. It worked, and what was once a lab that consumed dozens upon dozens of chicken wings, plus nearly 1000 test tubes and petri dishes, became a lab that consumed a lot of small potatoes — but still used the same number of test tubes and petri dishes. It was still very resource- and labor-intensive to set up. Plus, if you have ever tried incubating 1000 petri dishes containing wild bacteria for a week, you know they stink.

So last summer, I was looking around for a substitute that would be cheaper and easier to set up and that would use less space to complete than 1000 petri dishes. I happened upon a lab in Biology Brought to Life by Handelsman, Houser, and Kriegel. If you are not familiar with that book, it’s a unique product that focuses on active learning. The book has a lab called “Bread, mold, and environment: a lesson in biology and the environment.” Its focus is the Host – Pathogen – Environment disease triangle, with bread standing in for the host and bread mold standing in for the pathogen (while acknowledging that bread isn’t alive and that bread mold doesn’t cause disease). The lab challenges students to “develop a hypothesis about the environmental factors that might affect the ability of Penicillium to grow on bread and design an experiment to test your hypothesis.”

That turned out to be the inspiration I needed for our new lab. Sarah (my capable undergraduate assistant) and I unleashed the Summer of Mold to figure out whether we could have students test predictions about mold growth by spraying spore suspensions or sterile water control solutions on various baked goods. The first step was to figure out what type of baked goods would work the best. Sarah scoured the stores and came back to the lab with a huge variety of treats, including cookies, Twinkies, muffins, sweet breads, sandwich breads, garlic breads, and more. We sprayed, and then we waited. As you can see in the photos below, some baked goods got moldy. A lot of them did not.

One major finding was that the easiest baked goods to interpret were plain slices of sandwich bread, and further pilot testing revealed that quarter slices of bread would work just fine.


Fresh bread from a bakery got moldy, even after being sprayed with sterile water. Photo by M. Hoefnagels.

So for week 1 of the final lab, we decided to have students pretend they were opening a bakery but unable to decide whether or not their bakery’s bread should contain artificial preservatives. We let them think about how they might test the effect of preservatives on mold growth if they were given a squirt bottle containing mold spores and bread slices of two types: store-bought whole wheat bread with artificial preservatives, and fresh, bakery-bought bread without artificial preservatives. Once a TA approved their methods, they went at it. We had plenty of space in the back of the classroom to incubate all of the ziploc baggies at room temperature.

During week 2, each group had to figure out how to quantify the mold growth on their bread slices. We gave them plastic transparencies marked with 1-cm2 grids and asked them to figure out how to use the grids to generate their data (without opening their baggies). Once a TA approved their proposed methods, they collected their data, produced their graphs, and wrote their reports.

Good news: It worked! Freshly baked bread without preservatives typically got very moldy indeed, whereas store-bought bread with preservatives hardly had any mold spores at all. Below is an example of a graph submitted with a student’s report.

2019-01-18 12.46.13 pm

Sample graph; used with permission. In the color key, “Mold” refers to the mold spore suspension; “Water” refers to sterile water. No mold grew on store-bought bread sprayed with sterile water.

If you’re thinking of doing a lab like this, it does take some planning. A couple of weeks before the lab, you’ll need to sterilize several liters of water (and quite a few squirt bottles). You’ll also need to buy a Penicillium culture and some extra plates of potato dextrose agar, inoculate the plates with your Penicillium, and let the plates grow for a week or two — long enough to generate the spores you’ll wash into the squirt bottles as you prepare for the lab. You’ll also need to teach students about aseptic technique and make sure they know to spray INSIDE the plastic bags, not outside — this is an important precaution that maximizes safety and minimizes cross-contamination.

Once it’s time for week 1, the materials needed are simple: bread, Lysol, plastic knives (for cutting bread into four pieces), cutting boards, squirt bottles containing sterile water, squirt bottles containing Penicillium spores, ziploc baggies (for incubating the slices for a week), and Sharpies. During week 2, you’ll need transparencies pre-printed with a 1-cm2 grid and water-soluble marking pens. For safety’s sake, you’ll also need to remind students never to open ziploc baggies containing moldy bread.

What did the students think? They liked it! It was not overly complicated, but it was reasonably challenging. The TAs told me that students would have liked more freedom to test different types of natural preservatives, but I confess that I am not sure how to do that without introducing confounding variables. However, it would not be difficult to add refrigeration as a treatment to substitute for (or add to) store-bought bread with artificial preservatives. In our July pilot studies, refrigeration substantially inhibited mold growth.

Anyway, it’s an open-source lab so if you want a copy of the lab and prep notes, leave a comment below and I’ll send them to you.

This entry was posted in Engaging students, fungi, Laboratory activities, Microbiology and tagged , , , , , , , , , . Bookmark the permalink.

15 Responses to Moldy bread, experimental design, and you

  1. beanm5 says:

    I loved the idea. I did something similar but testing hamburguer from a fast-food restaurant and an hamburguer from a butcher shop, but I’d really like to try this one with my students.

  2. Elizbeth Larson says:

    I love this combination of moldy bread with experimental design, and I would really like to try it with my non-majors biology students:)

  3. Alana Shockley says:

    I would love a copy of this!

  4. buggirly says:

    Can you send me a copy of this? Looks fun!

  5. Scott Lawson says:

    Would love a copy of the lab if possible! Thanks

  6. Jo says:

    Hi, excellent! I have already begun a similar experiment with year 5’s STEAM comparing mould/microorganism growth from various sources around our school.on bread in snap lock bags. Our agar plates grew mould, in the fridge, before we even started!
    What I am interested in is the collating of data and the method you used with the transparencies of squares and leading towards graphing? could you share the process?

  7. Hi, thanks for your comment! We actually let the students decide how they want to quantify mold coverage on the bread. One way to approach it is to say that any square with any mold colony counts, which would lead to an overestimate of mold coverage. Another is to count a square that’s half-covered or more as one square, and one that’s less than half covered as non-moldy. Yet another is to estimate the percentage of each square that has visible mold. That’s the most accurate but also the most time-consuming. I hope this helps!

    • Jo Clements says:

      Yes it does thanks.
      I will let them problem solve and come up with a soloution, knowing that is the more accurate estimate. 🙂

  8. Nia Bauer says:

    I would love your notes to see if this is a good fit for us. How long did you dedicate to the project? How long are your labs? Thanks!

    • Hi, thanks for the comment! I will email you the materials shortly. Our labs are just under 3 hr long, and we dedicate two lab periods to the project. Students get out a little bit early from the first period (setup), and data collection (in the second period) does not take long at all. We use that time to help students begin to write their lab reports.

  9. STEM FUN says:

    I’m looking for new labs for our nonmajors class and this sounds like fun and could be something new for us when addressing experimental design and the scientific method. I’d love to see them “let loose” and then try to figure out what went wrong (too many variables, where’s the control?, etc). Thank you for sharing!

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s