ED-STEEP: Education Solutions to Environmental
and Economic Problems
Soil Bacteria
Subjects
Biological Sciences, Environmental Sciences, Agricultural Sciences
Introduction
Soil Bacteria are the major decomposers in most terrestrial ecosystems, and are the basis for a
complex food chain in soils. In a cupful of soil, there are often more bacteria than all the people
on Earth. A gram (ca. one teaspoon) of soil can easily contain 100 million to 1 billion bacteria.
In this lesson, students will learn about the important function of soil bacteria in major ecological
processes, and will isolate and calculate the amount of bacteria in soils. It’s an excellent lesson
in experiment methodology and students will learn to use a variety of techniques and equipment.
Relevant STEEP Research Projects



Researchers at Washington State University and with the U.S. Natural Resources
Conservation Service looked at the effects of no-till cropping on soil bacteria. They
found that soil organic matter and microbial activity were greatest in fields under direct
seeding compared with convention tillage.
The amount of bacteria in soil is directly related to the amount of soil organic matter,
which is the main food source for the bacteria.
Under no-till, soil pH tends to decrease. Adding lime to the soil increases the soil pH
and, as a result, increases microbial activity and microbial biomass.
Bezdicek, D.F., J. Hammel, M. Fauci, D. Roe and J. Mathison. 1999. Impact of long-term no till
on soil physical, chemical, and microbial properties. 1999 STEEP Research Reports.
Bezdicek, D., S. Albrecht, M. Fauci, M. Flury and J. Hammel. 2001. Impact of direct seeding on
crop water use efficiency, soil physical and microbial properties and quality of soil organic
matter, 2001 STEEP Research Reports.
Bezdicek, D., S. Albrecht, M. Fauci, M. Flury, J.P. Fuentes, and J. Hammel. 2002. Impact of
direct seeding on crop water use efficiency, soil physical and microbial properties and
quality of soil organic matter, 2002 STEEP Research Reports.
Objectives



Students will discuss the role of bacteria in terrestrial ecosystems
Students will isolate soil bacteria using microbiological techniques
Students will estimate the number of bacteria in soil
Major Concepts
Terrestrial Ecosystems; Microbiology; Experiment Design and Scientific Methodology
Standards
AAAS Benchmarks
Idaho State Science Standards
Oregon State Science Standards
Washington State Science Standards
Materials
Print Resources
 Scientific Experiments and Lab Report Format
 Constructing Bar and Line Graphs
 Student Handout
Web Resources
 Bacteria, Chapter 3, Soil Biology Primer, NRCS Fact Sheet prepared by E.R. Ingham.
Bacteria Isolation











A cupful of different soil types; one soil per group
Balance, weighing paper, spatula for weighing soil
Nutrient agar plates - 5 plates/group
1, 100-ml flask containing 99 ml of sterile water per group
6 test tubes per group; each containing 9 ml of sterile water; with caps (or foil)
Tape for labeling flasks and test tubes
1 test tube rack per group (groups can share racks), or a beaker for holding the test tubes
Vortex machine (optional)
1-ml sterile pipettes with bulbs
“hockey sticks” – bent glass rods for spreading out liquid on the agar plates
alcohol and alcohol lamps for sterilizing equipment (optional)
Procedures
Student preparation
1. Have student read the NRCS Fact Sheet on Bacteria, by Elaine Ingham.
2. Discuss the form and function of soil bacteria.
3. Ask the students how they can estimate the number of bacteria in a gram of soil.
Preparing soil
4. Collect a small baggy (cupful) of soil from 1-5 cm below the soil surface; store in a
refrigerator until needed. Moist soil works well.
5. Break up the soil into fine pieces so it can be suspended in water.
6. Weight out 1 g of soil per group.
Serial dilutions
7. Students will need to practice using the 1-ml pipettes.
8. Label flasks and test tubes.
 100 ml flask – 10-2
 test tube # 1 – 10-3
 test tube # 2 – 10-4
 test tube # 3 – 10-5
 test tube # 4 – 10-6
 test tube # 5 – 10-7
 test tube # 6 – Control
9. Place 1 g of soil into the beaker with 99 ml of water. This beaker contains 0.01 gram of
soil/ml of water). Mix thoroughly so all the soil is suspended in solution.
10. Using a sterile 1 ml pipette, transfer 1 ml of the soil suspension from the flask into tube # 1
and mix thoroughly (use a vortex if available). This tube contains 0.001 g of soil/ml.
11. Transfer 1 ml of soil solution from tube #1 to tube #2 and mix. This tube contains 0.0001 g
of soil/ml.
12. Transfer 1 ml of soil solution from tube #2 to tube #3 and mix. Tube #3 contains 0.00001 g
of soil/ml.
13. Transfer 1 ml of soil solution from tube #3 to tube #4 and mix. Tube #4 contain 0.000001 g
of soil/ml.
14. Transfer 1 ml of soil solution from tube #4 to tube #5 and mix. Tube #5 contain 0.0000001 g
of soil/ml.
Culture Bacteria
15. Set up 5 Nutrient Agar plates and label (10-4 , 10-5 , 10-6 , 10-7 , and Control).
16. Add 0.5 ml of water in the control tube to the Control plate.
 Use Only 0.5 ml
17. Using the same pipette, add 0.5 ml of soil suspension from tube 10-7 to plate 10-7.
18. Using the same pipette, add 0.5 ml of soil suspension from tube 10-6 to plate 10-6.
19. Using the same pipette, add 0.5 ml of soil suspension from tube 10-5 to plate 10-5.
20. Using the same pipette, add 0.5 ml of soil suspension from tube 10-4 to plate 10-4.
21. Using the hockey stick, carefully spread the soil suspension evenly over the agar plate (start
with the control, then the most dilute, to the least dilute).
22. Incubate the plates upside down at room temperature for 2 days.
23. Choose a “readable” plate and count the number of colonies. Each colony is from a single
bacteria.
24. Calculate the number of bacteria per gram of soil.
# bacteria/g soil = (# colonies/amount of soil on the plate)x2
Evaluation
Formal Lab Report
Student Handout
Notes on Sterile Technique:
This lab offers a great opportunity to introduce the students to the subject of sterile technique.
Sterile technique makes the students much more aware that our world is filled with bacteria.
Typically, sterile technique involves the following:
 Using an autoclave to sterilize equipment
 Disinfecting surfaces with either 70% ethanol or 10% bleach
 Using sterile equipment
 Flaming the lips of test tubes and flasks before poring liquids (this is actually done to
prevent contaminants from entering the glassware)
 Flaming loops and other equipment
 Minimizing exposure of agar plates (use the lid to shield the plate when pouring agar;
inoculating plates quickly)
Most secondary school science classroom do not have an autoclave to sterilize agar and
equipment. If a pressure cooker is available, equipment can be sterilized by running it for 15
minutes at about 15 psi. However, for the bacteria lab, it generally isn’t necessary to have
absolute sterile conditions. The nutrient agar can be dissolved in water that has been boiled for
several minutes. Once dissolved, the liquid agar can be heated gently for several minutes (it will
tend to “boil over”). Other equipment can be rinsed or wiped with 70% ethanol, and wrapped in
aluminum foil until used. Bench surfaces can be wiped down with alcohol or dilute bleach right
before the lab. Loops and hockey sticks can be flamed, then cooled by touching the surface of
agar plates. I would not recommend having any open containers of alcohol around to sterilize
equipment.
Notes on Soil Bacteria:
It will not be possible to identify easily the bacteria isolated from the soil. However, typical soil
bacteria contains species of Bacillus, Pseudomonas, Streptomyces, and Arthrobacter. The latter
two are actinomycetes.