Materials Included In Kit

Additional Materials Required

Prelab Preparation

Making a Soil Scoop

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

3. Pour the water out of the micrcentrifuge tube. Cut the microcentrifuge tube in half along the waterline mark using scissors. Note: Be sure to use sharp scissors.
4. Keep the end with the cap. This is the 0.5-cc (mL) soil scoop.

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Be sure to follow standard sterile protocol when working with the soil samples and Petrifilm. Work surfaces should be wiped down with 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 is a Super Value Kit and contains enough materials for five classes of 30 students working in pairs. Enough Petrifilm is included for 3 different soil samples per student group.

• 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 bacteria will allow students the opportunity to engage in real scientific exploration, gain the necessary understanding of soil chemistry and the role bacteria 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 bacteria.
• 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 work stations.
• Following is a sampling of possible questions students might ask about bacteria 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?

• The first time the serial dilutions are performed, the instructor may want to plate all the dilution levels. The instructions here are for plating the most common levels where the correct number of bacteria are found, but soils can vary and levels of dilution contain the best numbers for your specific geological location may need to be determined.
• 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 bacteria population levels. There is no possible way to know the exact number of bacteria in a cubic centimeter of soil, and students need to keep this fact in mind when analyzing their data.
• Because bacteria 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 that students need to dispose of after using them are the transfer pipets. 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

Bramble, Judith E. (1995) Field Methods in Ecological Investigation for Secondary Science Teachers. St. Louis: Missouri Botanical Garden.

Cothron, Julia H.; Giese, Ronald N. & Rezba, Richard J. (2000) Students and Research: Practical Strategies for Science Classrooms and Competitions, 3rd ed. Dubuque: Kendall/Hunt Publishing Company.

Environmental Science Summer Research Experience for Young Women (2003) Available [online] http://faculty.rpcs.org/brockda/essre.htm

Hall, Geoffrey S., ed. (1996) Methods for the Examination of Organismal Diversity in Soils and Sediments. Paris: CAB INTERNATIONAL.

Nardi, James B. (2003) World Beneath Our Feet: A Guide to Life in the Soil. New York: Oxford University Press.

Samuels, Myra L. (1989) Statistics for the Life Science. Englewood Cliffs: Prentice Hall.

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 money 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 bacteria in the soil? If so, in what concentration? Perform this activity to find out.

Concepts

• Bacteria

• Serial dilutions
• Population
• Decomposer

Background

Bacteria are simple, one-celled organisms that are the most abundant inhabitants of soil. In fact, up to 100 million bacteria may live in one teaspoon of soil! Common bacteria are rod shaped, though many assume other shapes such as cocci (round) or spiral as well. Bacteria are very small organisms-roughly 1/25,000 inches wide. While they are single celled, many bacteria cling together to form chains. Bacteria commonly grow and can be found in small colonies on the surface of soil particles.

Most bacteria are saprophytic—meaning they feed upon dead material. Bacteria comprise one of the groups most responsible for breaking down organic material in soil. A few species are pathogenic and cause disease to other organisms. Others are actually primary producers or autotrophs and obtain their energy from chemical reactions within the soil.

One of the last “frontiers” in science today is the very dirt beneath our feet. Bacteria that inhabit the soil are responsible for making it possible for plants to grow and are crucial to the very health of any terrestrial ecosystem. In this activity, bacteria that inhabit soil will be grown on Petrifilm to determine their quantity.

Materials

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Be sure to follow standard sterile protocol when working with the soil samples and Petrifilm™. Work surfaces should be wiped down with 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 Bacteria

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

2. At the top of the first third of a sheet, label it with the sample number and a dilution factor of 10^–3. Label the second third of a sheet with the sample number and a dilution factor of 10^–4. The remaining third of sheet may be used for any future dilutions. Set sheets aside for later plating.
3. Using a clean graduated pipet, transfer 5-mL of sterile water into a 15 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 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 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. Empty the contents of the tube containing the 10⁰ diluted solution, and rinse thoroughly.
9. 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 3).

10. 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 cleaned 10^–2 dilution tube.
11. Clean the contents of the 10^–1 dilution tube as per directions in step 8.
12. Use the pipet labeled “DP” to place 4.5 mL of sterile water into the newly cleaned culture tube. This will become the tube containing the 10^–3 diluted solution (see Figure 4).
13. Shake the contents of the 10^–2 dilution tube and using the pipet labeled “DP”, transfer 0.5 mL of solution from the tube containing the 10^–2 diluted solution into the 10^–3 tube.
14. Now empty the contents of the 10^–2 dilution tube as per directions in step 8.
15. Into the newly cleaned culture tube, place 4.5 mL of sterile water using the pipet labeled “SW”. This will become the tube containing the 10^–4 diluted solution (see Figure 4).
16. Cap and shake the 10^–3 dilution tube and then using the pipet labeled “DP”, transfer 0.5 mL of the solution from the tube containing the 10^–3 diluted solution into the 10^–4 dilution tube (Figure 4). These two tubes are the final dilution tubes.

17. Stop the dilution process at this point and use a clean, new pipet to collect 0.1 mL of the solution in the 10^–3 tube (Figure 2). Plate this sample on the designated third of the 3M Petrifilm plate 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 (Figure 4).
18. Now lower the cover back down over the drops and press down on the sheet with a finger to distribute the solution across the plate (see Figure 5). Note: Control the spread of the solution to keep within the edges of the sheet.
19. Next, repeat steps 17–18 with the 10^–4 dilution tube and the designated third of the 3M Petrifilm plated (labeled 10^–4).
20. Empty the 10–3 and 10–4 dilution tubes and wash and clean 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.
21. Let 3M Petrifilm plates set at room temperature for 48–72 hours.
22. Bacteria will be indicated as a red colony. Determine the most diluted sample on which at least 5 and no more than 30 colonies can be found. Record the number of colonies and the corresponding dilution level of that sheet in the Data Table. Note: A magnifying glass may aid in this process.
23. To determine the density of the bacteria, use the data from the Petrifilm strip in the following equation:

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

24. Consult the instructor for disposal procedures.

Student Worksheet PDF

10595_Student1.pdf