Today, we’re going to do a little Microslide experiment where we investigate microbes on bread using impression sampling with Microslides. Our objective is to answer the following questions: Why is bread a microbe food? What kinds of microbes like bread? And finally, what can reduce microbe populations in bread? So let’s start off with a little bit of background information about bread!

Introduction

Food spoilage is caused by the enzymes produced by microbes. Food spoilage microbes need: organic nutrients (proteins, carbohydrates, fats), a convenient temperature, some moisture, in most cases, some air and a suitable pH.

  1. Nutrients
    • Breads are mostly a mixture of flour and water that is baked. Flour is a ground powder made from cereal grains (usually wheat and corn), seeds, or roots. The caloric content of flour is mostly carbohydrates (80%), proteins (15%), and fats (5%).
  2. Moisture Content (water activity)
    • Available moisture (water) is essential for microbe growth and survival. Food microbiologists generally describe the water requirements of microorganisms in terms of the water activity (aw) of the food or surrounding environment. The water activity (aw) of a food is the ratio between the vapor pressure of the food itself, when in a completely undisturbed balance with the surrounding air media, and the vapor pressure of distilled water under identical conditions. A water activity of 0.80 means the vapor pressure is 80% of that of pure water. The water activity increases with temperature. Bread has a water activity of 0.96. If breads are cut or placed into a closed container (e.g. plastic bag) they will “moisture equilibrate.”
Microbe Water Activity
Most bacteria >0.91
Most yeasts >0.88
Most fungi >0.80
  1. pH
    • In their natural state, most foods such as meat, fish, and vegetables are slightly acidic while most fruits are moderately acidic. A few foods, such as egg whites, are alkaline. Breads have a pH range of 5.0 – 6.2. Microbial cells must maintain their intracellular pH (pHi) above some critical level so that cellular proteins will not denature. Most harmful food microbes can survive within these pH ranges.
Microbes & pH
Most bacteria pH 4 to 9
Most yeasts pH 2 to 8
Most fungi pH 0 to 11

microbes on bread, dipslides, investigating microbes, microbiology experiment

  1. Temperature
    • Temperature is the most important factor directly affecting how fast microbes grow. Bread-spoiling microbes prefer a temperature range of 30˚C (86-113˚F) but can grow in temperatures as low as 5-15˚C (41-59˚F). As a rule of thumb, for every increase in temperature of 10˚C (50˚F), the activity increases two times. This rule is true within the temperature range of 32 to 60˚C.
  1. Time (Shelf Life)
    • Bakers address the concept of time as it relates to microbial growth when a product’s shelf life is determined. Shelf life is the time period from when the product is produced until the time of consumption. The “sell by” date must incorporate the shelf life of the product plus a reasonable period for consumption that consists of at least one-third of the approximate total shelf life of the perishable food product.
  1. Food Additives: Preservatives
    • Preservatives are added to prevent the growth of unwanted microorganisms, food spoilers and food pathogens. Most bakeries add one or more chemical compounds to their bread products to extend shelf life and reduce microbe contamination.

Now, let’s get to our investigation!

Materials

Procedure

microbes on bread, dipslides, investigating microbes, microbiology experiment

  1. Use a toothpick to carefully punch 4-6 small holes in a zip-lock bag.
  2. Place a bread slice into the bag and seal it. You may want to encourage bread microbes by adding moisture – a moistened paper towel or a quick spray of the bread slice with a water mister.
  3. Predict how long you think it will take to visibly see mold on the bread.
  4. Observe the bread slice carefully over time (5-9 days) and away from sunlight. Do any visible microbes appear? Was your prediction right? How long did it take for visible colonies to appear?
  5. To direct-sample growing microbes on breads, carefully open the plastic bag so that there is enough room for the paddle to be inserted to sample a contaminated surface.
  6. Twist to remove the paddle from the vial. Be careful not to touch the agar with your hands, or you will cross-contaminate the sample, leading to false results.
  7. Allow the paddle surface to come into physical contact with the contaminated bread area(s). Contact both paddle surfaces on various contaminated bread areas.
  8. Replace the paddle into the vial.
  9. Secure the paddle with transparent sticky tape. Wash your hands!microbes on bread, dipslides, investigating microbes, microbiology experiment
  10. Incubate the paddle for 5-7 days at room temperature away from sunlight.
  11. Monitor daily for signs of colony growth. Do not attempt to remove the paddle from the vial. Instead, make all observations through the vial. Use a magnifier to help you observe finer details of your microbe finds!
  12. Use the Counting Panels below to estimate bread microbe counts. Record this data in your Laboratory Notebook.
  13. Use the Free iPad app to see if you can presumptively (tentatively) identify some of the microbe colonies.

Counting Microbe Colonies

microbes on bread, dipslides, investigating microbes, microbiology experiment

NOTES: A colony Forming Unit (CFU) is a visible colony formed from usually one microbe cell or spore. TCC (total colony counts) is the total number of visible microbe colonies (growth areas) on a paddle. Paddle area is 10 cm2. 104 = 10,000; cm2 = square centimeter (area).

That’s it for today’s experiment! We hope you learned a little bit about bread microbes!