Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Using a graduated pipet, add 0.5 mL of tap water into a microcentrifuge tube.
2. Cap the tube and turn it cap-side down. Mark the waterline with a permanent marker (see Figure 6).

3. Pour the water out of the microcentrifuge tube. Cut the microcentrifuge tube in half along the waterline mark using scissors. Note: Use sharp scissors.
4. Keep the end with the cap. This is the 0.5-cc (mL) soil scoop. The pointed end may be thrown in the trash.

Safety Precautions

Follow all laboratory safety rules. Be sure to follow standard sterile protocol when working with the soil samples and Petrifilm. Work surfaces should be wiped down with bleach or 70% ethyl alcohol after performing the experiment. Wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Petrifilm should be sterilized before disposal. Please follow Flinn Suggested Disposal Method for Type I biological materials as outlined in your current Flinn Scientific Catalog/Reference Manual.

Teacher Tips

• This kit contains enough materials for three classes of 30 students working in pairs. Enough Petrifilm is included for 150 tests or 50 dilution series.
• This activity introduces an exciting area of environmental science. It is an easy topic for field studies, and learning about the significant concepts of basic ecology (e.g., biogeochemical cycles or predator–prey relations) is much more real for students when they can see these things happening firsthand. These protocols for isolating and quantifying the major soil yeast and mold will allow students the opportunity to engage in real scientific exploration, gain the necessary understanding of soil chemistry and the role yeast and mold plays in soil health. Students can create their own hypotheses, design and perform their own experiments, analyze and interpret their own data, and as a consequence, learn to understand and appreciate how the microecology of the soil influences all the multi-cellular organisms in an ecosystem.
• Students should consult their textbooks or search the Internet for further background information pertaining to yeast and mold.
• The sterile water used needs to be boiled for 12 minutes to make it sterile enough for the serial dilutions. 50-mL centrifuge tubes (with caps) may be used in a central location for collection of sterile water. This cuts down the risk of contamination that occurs if beakers are used (chemical residue can kill the organisms being studied). Clean, unused plastic cups also work well to distribute the necessary water to student workstations.
• Following is a sampling of possible questions students might ask about yeast and mold and their role in the soil.
  1. What is the population density of microbes in the soil in various areas of the school’s campus?
  2. What is the population density of microbes at different soil depths?
• Students should collect soil from the top 15 cm of the location they are investigating and should place and store each sample in its own separate clean plastic sandwich or freezer bag (do not reuse any bag to avoid contamination). Soil augers make the task of soil sampling easier.
• It is very important that students collect all the samples they want to study on the same day at the same time because the soil is still going to be “alive” in the plastic bag. Only by collecting samples concurrently will the changes in the environment (such as a heavy downpour between one class session and the next) be controlled.
• Explain to students that this procedure is used for estimating yeast and mold population levels. There is no possible way to know the exact number of yeast and mold in a cubic centimeter of soil, and students need to keep this fact in mind when analyzing their data.
• Since yeast and mold population levels are estimates and because the quantity of microbes in the soil is so enormous, contamination is never really a problem with these protocols. Strict sterile procedure is not necessary, and the only materials which students need to dispose of after using them are the transfer pipets and Petrifilm plates. The dilution tubes and soil scoops may be reused without any autoclaving as long as students have cleaned them thoroughly with Alconox^® (or its equivalent).

Sample Data

References

Special thanks to David Brock and Mariel Torres, Roland Park Country School and Katie Loya, University of Maryland at College Park for presenting Flinn Scientific with this activity.

This lesson has its origins in a curriculum developed and supported by monies from the following institutions and programs:

• Institute for Ecosystem Studies
• Paul F-Brandwein Institute
• ReliaStar/Northern Life “Unsung Heroes” Program
• Toshiba America Foundation
• Captain Planet Foundation, Inc.

Introduction

Is there really yeast and mold in soil? If so, in what concentration? Perform this activity and find out.

Concepts

• Yeast and mold
• Population
• Decomposers
• Serial dilutions

Background

It may be hard to believe but a single gram of soil may contain over one million yeast and mold! Yeast and mold belong to the kingdom Fungi. Most fungi resemble a mass of tangled threads called a mycelium. The threads, called hyphae, contain and release spores that are considered the “seeds” of the fungi. Most of the fungi in soil must be examined under a microscope, but some fungi, such as mushrooms, may be much larger. Fungi lack chlorophyll and the type of roots and vascular system typical of plants. They must either feed upon decaying organic material (saprophytic) or on other organisms (parasitic) because they cannot photosynthesize their own food.

Along with bacteria, fungi act as the main decomposers in soil. Fungi can attack matter that resists breakdown by bacteria because the hyphae can grow directly into the material. Many fungi are plant parasites but others are beneficial to plant growth. Mycorrhizae are fungi that form a symbiotic relationship with plant roots. These fungi infest the roots of plants to obtain food and nutrients and in return provide many benefits to the host plant. The host plant’s roots are better able to absorb phosphorous, water and other nutrients, such as zinc and copper. Roots infected with mycorrhizae also live longer than uninfected ones and are also protected from various diseases.

One of the last “frontiers” in science is the very soil beneath our feet. The yeast and mold that inhabit the soil are crucial for the health of any terrestrial ecosystem. In this activity, yeast and mold isolated from the soil will be cultured on Petrifilm™ to determine their quantity.

Materials

Safety Precautions

Follow all laboratory safety rules. Be sure to follow standard sterile protocol when working with the soil samples and Petrifilm. Work surfaces should be wiped down with bleach or 70% ethyl alcohol after performing the experiment. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Performing a Serial Dilution to Count Yeast and Mold

1. Obtain one Aerobic Yeast and Mold 3M^® Petrifilm™ plate. Using scissors, cut the plate into thirds lengthwise (see Figure 1). Be sure not to lift the plastic cover off the sheet while cutting.

2. Label the top of the first third of the sheet with a sample number and a dilution factor of 10⁰. Label the second third of the sheet with a sample number and the dilution factor of 10^–1. Label the remaining third of the sheet with a sample number and a dilution factor of 10^–2.
3. Using a clean graduated pipet, transfer 5 mL of sterile water into a 10 mL culture tube using the measurements on the side of the tube as a guide. Label this pipet “SW” for “Sterile Water” with a permanent marker. Set aside for future use.
4. Use the soil scoop (see Prelab Preparation) to fill one level scoop with soil from the sample being tested. Empty a scoop of the soil into the 5 mL of sterile water in the first culture tube. This is the 10⁰ diluted solution. Note: You may have to open the cap and use a clean glass stirring rod to push any remaining soil out. Be sure to wash (with sterile water) and dry the soil scoop and glass stirring rod between each sample.
5. In the second culture tube, use the “Sterile Water” pipet from step 3 to fill the tube with 4.5 mL of sterile water (use the measurements on the side of the tube to measure 4.5 mL).
6. Cap the culture tube containing the 10⁰ diluted solution and shake it vigorously until the soil is evenly dispersed in the water.
7. Take a second, unused graduated pipet and label it “DP” for “Dilution Pipet” with the permanent marker. Use it to remove 0.5 mL of the 10⁰ diluted solution from the first tube and place it into the second tube (see Figure 2). The second tube is now the 10^–1 diluted solution.

8. Stop the dilution process at this point and use a clean, new pipet to collect 0.1 mL of the solution from the 10⁰ tube. Place this sample on the designated third of the Yeast and Mold Petrifilm strip from step 2 by lifting the cover of the sheet and distributing the 0.1 mL in a series of small drops of the solution down the center of the sheet until all of the solution is out of the pipet (see Figure 3).

9. Now lower the Petrifilm cover back down over the drops and press down on the sheet with a finger to distribute the solution across the plate (see Figure 4). Note: Control the spread of the solution to keep it within the edges of the sheet.
10. Empty the contents of the 100 tube and rinse thoroughly. (It is not necessary to wash with soap and water.)
11. Once the culture tube is cleaned, use the pipet labeled “SW” and add 4.5 mL of sterile water to the tube. This will become the tube containing the 10^–2 diluted solution (see Figure 5).

12. Using the pipet labeled “DP” from step 7, transfer 0.5 mL of the solution from the tube containing 10^–1 diluted solution to the 10^–2 dilution tube.
13. Stop the dilution process at this point and use clean, new pipets to collect 0.1 mL of the solution from the 10^–1 and 10^–2 tubes. Repeat steps 8 and 9 for the 10^–1 and 10^–2 dilutions, respectively. Be sure to place the correct dilutions on the appropriate Petrifilm strips.
14. Empty the 10^–1 and 10^–2 dilution tubes and wash with soap and water thoroughly. Be sure to rinse very thoroughly to remove any soap. These tubes may now be reused for any other samples being tested.
15. Let Yeast and Mold Petrifilm plates sit at room temperature for 48–72 hours.
16. Yeast and mold colonies may be distinguished on the Petrifilm strips by using the following table:

17. Determine the most diluted sample on which at least five but no more than 30 colonies can be found. Record the type and number of colonies and the corresponding dilution level of that strip in the Data Table. Note: A magnifying glass may aid in this process.
18. To determine the density of the yeast or mold, use the data from the Petrifilm strip in the following equation:

Number yeast and mold in 1 cc of soil = Number of Colonies on strip x 10² • 10 ^{|dilution #| at which these colonies were found}

19. Consult the instructor for disposal procedures.

Student Worksheet PDF

10596_Student1.pdf