Living Soil
Christa Van Treeck
Oshkosh West High School
July 20, 2007
Activity Description:
The purpose of this lab is to show that soil, which we usually think of as a non-living substance, really is living. Students will work in groups to analyze the physical and microbial characteristics of soil to determine how much life really is in soil. Students will learn how to extract and grow bacteria from the soil and test it for the ability to digest starch, which is necessary to decompose particles into soil.
Class:
Conservation and Wildlife, Conservation and Forestry
Grades 9-12
Unit in Class:
Habitat Requirements/Sustainability
Topics of human impact of the environment, forest ecosystems, land properties, etc.
Length of Activity:
Day 1: Discussion on soil components, physical observations, culture bacteria
Day 2-3: Observe results, continue discussion of components of soil, analyze types of bacteria
Wisconsin Science Model Academic Standards:
B.12.5 Explain how science is based on assumptions about the natural world and themes that describe the natural world
C.12.3 Evaluate* the data collected during an investigation*, critique the data-collection procedures and results, and suggest ways to make any needed improvements
F.12.1 Evaluate the normal structures and the general and special functions of cells in single-celled and multiple-celled organisms
F.12.7 Investigate how organisms both cooperate and compete in ecosystems
Special Considerations:
Little modification should be needed to accommodate all students. Allergies should be considered. Students with physical disabilities, depending on the severity, should be able to participate with some further assistance.
Evaluation:
Students will be evaluated on the basis of class participation, following precise lab procedures and submitting a lab report.
Outline
Teacher Instructions / Procedure/How? / Competencies/OutcomePrepare all materials for lab previous to class / See “Living Soil: Teacher Instructions”
Introduction
Start with discussion of what students think is in soil… living, etc?
— Can we see everything?
— Can we see anything?
How do we know that soil is living?
How can we prove that soil is living? / Class discussion
…write responses on board or
…give each student several notecards and have them write answers…read
See background information for discussion follow up
Hand out “Living Soil: Student Instructions” and Living Soil: Lab Report” / Students will be able to…
— Accurately make dilutions
— Have an understanding that soil contains many living components
— Be able to culture and analyze soil bacteria
— Understand the role soil plays in the ecosystem
— Realize that bacteria are decomposers in the soil
Lab Activity
Be sure to remind students about the lab report / Refer to “Living Soil: Student Instructions” for procedure
Wrap up
Is soil living?
How did we prove that?
What did we find?
Answer questions that students might still have / Refer to background information for information about specific bacteria
Evaluation / Have students turn in lab reports
Background:
Soil bacteria
Bacteria are some of the smallest and most abundant microbes in the soil. In a single gram of soil, there can be billions of bacteria. There are an estimated 60,000 different bacteria species, most which have yet to be even named, and each has its own particular roles and capabilities. Most live in the top 10cm of soil where organic matter is present.
Characteristics of bacteria
Some bacteria species are very fragile and can be killed by slight changes in the soil environment.
Other species are extremely tough, able to withstand severe heat, cold or drying. Some can lie dormant for decades waiting for favourable conditions. Others can extract nitrogen directly from the air or break down some toxic substances. Populations of microbes can boom or bust in the space of a few days in response to changes in soil moisture, soil temperature or carbon substrate. To gain advantage in this process, many microbes release antibiotic substances to suppress particular competitors. In this way some species can suppress other disease-causing microorganisms.
Types of bacteria
Decomposers
Bacteria play an important role in decomposition of organic materials, especially in the early stages of decomposition when moisture levels are high. In the later stages of decomposition, fungi tend to dominate. Bacillus subtilis and Pseudomonas fluorescens are examples of decomposer bacteria. Additions of these bacteria have not been proved to accelerate formation of compost or humus in soil.
Nitrogen fixers
Rhizobium bacteria can be inoculated onto legume seeds to fix nitrogen in the soil. These nitrogen-fixing bacteria live in special root nodules on legumes such as clover, beans, medic, wattles etc. They extract nitrogen gas from the air and convert it into forms that plants can use. This form of nitrogen fixation can add the equivalent of more than 100kg of nitrogen per hectare per year. Azotobacter, Azospirillum, Agrobacterium, Gluconobacter, Flavobacterium and Herbaspirillum are all examples of free-living, nitrogen-fixing bacteria, often associated with non-legumes. To date, inoculating the soil with these organisms has not proved an effective means of increasing nitrogen fixation for non-legume crops.
Disease suppressors
Bacillus megaterium is an example of a bacterium that has been used on some crops to suppress the disease-causing fungus Rhizoctonia solani. Pseudomonas fluorescens may also be useful against this disease. Bacillus subtilis has been used to suppress seedling blight of sunflowers, caused by Alternaria helianthi.
A number of bacteria have been commercialized worldwide for disease suppression. However, suppression is often specific to particular diseases of particular crops and may only be effective in certain circumstances.
Aerobes and anaerobes
Aerobic bacteria are those that need oxygen, so where soil is well drained aerobes tend to dominate. Anaerobes are bacteria that do not need oxygen and may find it toxic. This group includes very ancient types of bacteria that live inside soil aggregates. Anaerobic bacteria favour wet, poorly drained soils and can produce toxic compounds that can limit root growth and predispose plants to root diseases.
Actinobacteria
These soil bacteria help to slowly break down
humates and humic acids in soils. Actinobacteria
prefer non-acidic soils with pH higher than 5.
Sulfur oxidisers
Many soil minerals contain sulfides but this form of sulfur is largely unavailable to plants. Thiobacillus bacteria can covert sulfides into sulfates, a form of sulfur which plants can use.
Management of bacteria
Though largely unaffected by cultivation, bacteria populations are depressed by dry conditions, acidity, salinity, soil compaction and lack of organic matter. Except in the case of certain seed inoculations, it is very difficult to build desirable populations of bacteria just by adding them to the soil. If populations of soil bacteria are low, it is probably because conditions are unfavourable, so any new additions are likely to suffer the same fate.
A more effective approach to bacteria management is:
• address soil health problems such as acidity and compaction
• ensure that there is a good ground cover of grass or mulch
• build organic matter through practices such as green manure crops, mulching, strategic grazing and minimum tillage.
Each of these measures has multiple benefits and will also support healthy populations of soil bacteria. Poor drainage encourages undesirable anaerobic bacteria. Reducing compaction and building soil organic matter will improve water infiltration without compromising moisture storage and will discourage anaerobic bacteria.
Key points
• Populations of soil bacteria change rapidly depending on moisture, time of year, type of crop, mulching, etc.
• Healthy populations of soil bacteria are encouraged by ground cover and organic matter.
More information
Soil biology basics is an information series describing basic concepts in soil biology. For more detailed information we recommend the Australian book Soil biological fertility:A key to sustainable land use in agriculture (2003), edited by Lyn Abbott & Daniel Murphy.
NSWDPI has online soil biology information at
http://www.agric.nsw.gov.au/reader/soil-biology.
The University of WA has online soil biology
information at
http://ice.agric.uwa.edu.au/soils/soilhealth.
Written by Greg Reid and Percy Wong
©2005 State of New South Wales
Department of Primary Industries
The information contained in this publication is based on knowledge and understanding at the time of writing (2005). However, because of advances in knowledge, users are reminded of the need to ensure that information on which they rely is up to date, and to check the currency of the information with the appropriate officer of NSW Department of Primary Industries or the user’s independent adviser.
LAB: LIVING SOIL
Teacher Instructions
Modified from Leslie Brinson, J.H. Rose High School, Greensville, NC 27834
Materials (teacher preparation):
— Starch
— Sterile Petri dishes
— Iodine
— Potassium Iodide (KI)
— Balance/Scale
— Nutrient Agar
— Distilled Water
— Pressure Cooker
For Each Student Group
— 3 Petri dishes with starch agar
— Gram’s iodine
— Bacterial spreader/inoculating loops
— 3 sterile 1ml pipets
— 3 sterile capped bottles or test tubes
— Sterile water
— 95% ethanol
Teacher Preparations:
— Make starch agar
o Add 2.0 g of soluble starch to 400 ml of distilled water
o Add 9.2 g of nutrient agar and put in pressure cooker for 15 minutes at 15 psi of steam pressure. (Yield 20 plates)
— Make Gram’s Iodine
o Add 1.0 g of iodine and 2.0 g of KI to 100 ml of distilled water
o Above procedure could be done without making conditions sterile. Sterile technique is desired to show that bacterial growth truly comes from soil and not contamination.
— Have each student group bring in a soil sample
o Soil samples should come from a variety of sources (e.g. near garbage cans, forest floor, etc.)
Safety Considerations and Cleanup:
— When using sterile technique students need to be wearing safety glasses.
— Extreme caution should be used when using alcohol and fire together.
— When dealing with bacteria, one should always treat as a potential pathogen.
— Organisms can be disposed of by soaking the plates in a 20% chlorine bleach solution overnight.
Other Considerations/Extensions:
— May need to cover the topic of sterile technique, serial dilutions, pipetting, and bacteria growth
— Colonies can be isolated and cultured immediately after applying the iodine to further investigation
— Colonies can be grown for longer than 48 hours until all starch is digested to illustrate carrying capacity.
LAB: LIVING SOIL
Student Instructions
Name:______
Soil sample take from:______
Introduction:
The purpose of this lab is to show that soil, which we usually think of as a non-living substance, really is living. Students will work in groups to analyze the physical and microbial characteristics of soil to determine how much life really is in soil. Students will learn how to extract and grow bacteria from the soil and test it for the ability to digest starch, which is necessary to decompose particles into soil. You will look for the bacteria that have the enzyme amylase, which makes them capable of digesting starch. This is important to see that the microbes truly are decomposers.
Materials:
— 3 starch agar plates
— Gram’s Iodine
— Sterile inoculating loops
— 95 % ethanol
— 3 sterile 1 ml pipets
— 3 sterile capped bottles
— Sterile water
— Soil sample
Procedure:
— Weigh out 1 gram of soil and add this to 99 ml of sterile distilled water. Mix well. (This is a 1:100 dilution)
— Dilute this further by removing 1 ml of this mixture and adding it to another bottle which contains 9 ml of sterile distilled water. Mix by swirling. (This results in a 1:1,000 dilution)
— Dilute this further by removing 1 ml of this mixture and adding it to another bottle which contains 9 ml of sterile distilled water. Mix by swirling. (This results in a dilution of 1:10,000)
— Label each of the 3 petri dishes with the following: Your name, date, dilution (e.g. 1:100).
— Remove 0.1 ml of this mixture and put it on a Petri dish containing starch and agar. Take a sterile inoculating loop to spread the 0.1 ml sample evenly around the plate.
— Repeat previous step with other 2 dilutions. Be sure to use sterile pipets and inoculating loops.
— Be sure the covers are on each Petri dish. Turn them over and leave overnight at room temperature.
— The next day, examine the plates to see if clear areas are developing in the starch agar. Add a small amount of Gram’s iodine to stain the agar to more clearly see the starch eating bacteria.
— Be sure to dispose of your plates properly.
LAB: LIVING SOIL
Lab Report
Name:______
Soil sample take from:______
1. Why was it necessary to use sterile water to dilute the bacteria?
2. What dilutions of soil are represented on each of your 3 plates? Why did we make these dilutions? How did we prepare the dilutions?
3. Why was it necessary to make sure all measurements were accurate? What could have resulted?
4. Using words or diagrams take one of your plates and describe the pattern of bacterial growth- number of colonies, size, shape, etc.
5. What is the purpose of applying Gram’s iodine? What did it show?
6. Approximately what proportion of your bacteria appear to be producing amylase? Is there any consistency in the appearance of amylase-producing bacteria?
7. How does the bacterial growth of your soil sample compare to the rest of the class? What conclusions can you make?
References:
— Brinson, Leslie. Selecting Soil Organisms. J.H. Rose High School, Greensville, NC
— Heeter, Carrie. Microbe Zoo. Retrieved July 16, 2007, from http://commtechlab.msu.edu/sites/dlc-me/zoo/index.html
— Reid, Greg, & Wong, Percy. (2005). Soil bacteria. [Electronic version]. Soil Biology Basics.
— Supplies from Carolina Biological 1.800.334.5551
o Potassium Iodide (88-3808) $20.20
o Iodine (17-7020) $4.95
o Starch (89-2501) $11.65
o Petri Dishes, Sterile, Polystyrene Disposable (74-1246) $5.21
o Nutrient Agar (77-6366) $16.60
o Inoculating Loops, Disposable, Sterile (70-3037) $7.00
o Transfer Pipets, Calibrated 1 mL, Sterile (21-4551) $27.00
o Ethanol (86-1261) $7.95