Abstract

Objective

In this science project you'll isolate nematodes from a variety of soil samples to determine which types of soil are the best nematode habitats.

Introduction

Nematodes, also called roundworms, are the most abundant multicellular animals on Earth. There are more than 15,000 identified species of nematodes, and scientists are still discovering new ones! Most nematodes are 1-5 millimeters (mm) long, but scientists have identified a few very long nematodes, too, like Placentonema gigantisma, an 8-meter-long nematode found in the placenta of a sperm whale. All nematodes have fairly simple body plans. These animals are tube-like in shape; have an outer body wall, called a cuticle; and a digestive track that runs most of the body length. Because of this relatively simple body plan, nematodes are sometimes described as "a tube inside a tube." Here are two very different nematodes in Figure 1.

Figure 1.a. Caenorhabditis elegans is a bacterivorous soil nematode that is approximately 1 mm long as an adult. (WormBook, 2006.)	Figure 1.b. Ascaris lumbriocoides is a parasitic nematode that can live in the human intestine and grow to be 15-30 centimeters (cm) long. (CDC.)

Despite a simple body plan, nematodes are complex animals. Scientific research using a variety of nematodes has been important in understanding ecology, medicine, and basic biology. One of the most well-studied nematodes is Caenorhabditis elegans. C. elegans are approximately 1 mm long, are found in soils all over the world, and feed on bacteria. These small worms are used to study a variety of biological phenomena. In fact, research using C. elegans has been so important that several scientists who used C. elegans in their experiments have won the Nobel Prize!

Where can you find nematodes? Just about everywhere. They're residents of many different habitats, including soil, plants, freshwater, and saltwater. Some nematodes are even parasitic, meaning they live, grow, and reproduce inside other organisms at the expense of their host's health. Such a wide range of habitats also means that among the various nematode species, there is a lot of diversity in what they eat. Soil nematodes include bacterivores (bacteria eaters), fungivores (fungus eaters), algivores (algae eaters), and herbivores (plant eaters). In this science project, you will isolate bacterivorous nematodes from soil samples in your own backyard, or from any other local soil areas you choose.

Terms, Concepts, and Questions to Start Background Research

• Nematodes
• Body plan
• Caenorhabditis elegans
• Parasitic
• Bacterivore
• Fungivore
• Algivore
• Herbivore
• Species

Questions

• Where can you find nematodes and what do they look like?
• What kinds of food do nematodes eat?
• Would you expect to find nematodes in your backyard? If so, what species of nematodes?

Bibliography

One of the best places to start your bibliographic research is an encyclopedia. Try looking under Nematoda, which is the phylum name for nematodes. Other good resources include:

• Parker, Steve. Nematodes, Leeches & Other Worms. Minneapolis, MN: Compass Point Books, 2006.
• University of California Museum of Paleontology Community. (n.d.). Introduction to the Nematoda. Retrieved February 29, 2008 from the University of California Museum of Paleontology's website: http://www.ucmp.berkeley.edu/phyla/ecdysozoa/nematoda.html
• McSorley, Robert. (2007). Featured Creatures: Phylum Nematoda. Retrieved February 29, 2008 from http://creatures.ifas.ufl.edu/nematode/soil_nematode.htm

Materials and Equipment

• Live E. coli culture
  • It is commonly available in labs, or you can purchase it from an online supplier, such as Carolina Biological Supply Company, catalog #124400, or Science Kit, catalog #WW6740200.
    Note: Science Kit does not sell bacterial cultures to the general public. You will need your school teacher, or a sponsor from a research laboratory or biotech company, to order from this company.
  • If you decide to grow your own E. coli culture, you will need:
    • Plate of E. coli colonies
    • Clean pipette (1)
    • Sterile test tube (1)
    • Loria Broth (LB) or Nutrient Broth (NB); both can be purchased from online science supply companies. For example, the Nutrient Broth is available at Carolina Biological Supply Company, catalog #826150, or Science Kit, catalog #WW0175016.
    • Inoculating loop, or a sterile toothpick or sterile cotton swab (from an unopened package counts as sterile)
    • Incubator that can hold at 37°C
• Nutrient agar plates, three for every soil location (minimum of nine plates). These can be purchased from an online supplier such as Carolina Biological Supply Company, catalog #821862, or Science Kit, catalog #WW6564600.
  Note: Science Kit does not sell agar plates to the general public. You will need your school teacher, or a sponsor from a research laboratory or biotech company, to order from this company.
• Sterile cotton swabs (from an unopened package counts as sterile)
• Tablespoon measuring spoon
• Plastic baggies
• Permanent marker
• Soil samples (minimum of three locations, each with a different type of soil); here are some ideas of different sample soils to test:
  • Sandy soil
  • Compost
  • Garden soil
  • Playground dirt
  • Soil underneath a fruit tree
• Water
• Magnifying glass
• Bleach
• Lab notebook

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Experimental Procedure

1. To start this science project, you will need a live E. coli culture. If you have ordered it online, it will be ready to use when it arrives. Make sure to use it within 24 hours of arrival. If you have access to a plate of E. coli colonies and are going to grow your own liquid culture, you will need to use sterile technique:
   1. Using a clean pipette, add 2 milliliters (mL) of media to a sterile test tube. E. coli will grow in many different media. Two common media used are Loria Broth (LB) and Nutrient Broth (NB).
   2. Using your inoculating loop (or sterile toothpick or cotton swab), pick a single bacterial colony from the plate. Put the tip of the inoculating loop into the test tube of media. Swish the inoculating loop gently from side to side to transfer some of the E. coli cells into the media.
   3. Cap the test tube and put it in a 37°C incubator, shaking, for 12–24 hours. The media should be cloudy, indicating that the bacteria have replicated.
2. Make your E. coli plates:
   1. Dip a sterile cotton swab in the E. coli culture.
   2. Gently wipe a swab across the surface of the first nutrient agar plate. Spread the bacteria in a circle, encompassing the middle 50% of the agar plate.
   3. Use a new cotton swab for each nutrient agar plate. When you are done, you should have three swabbed plates for every soil type you want to test.
   4. Dispose of used cotton swabs properly (see Bacterial Safety below).
   5. Incubate the plates, with their lids on, for 72 hours at room temperature for the E. coli lawn to grow. Avoid keeping the plates in direct sunlight or they will dry out. Alternatively, if you have access to an incubator that will maintain a temperature of 37°C, the E. coli lawn will grown within 24 hours if the plates are placed in there.
3. Using a tablespoon, collect 2 Tbsp. each of several soil samples, keeping them all separate in plastic baggies. Using a permanent marker, label each baggie with respect to the type of soil sample and replicate. For example, Playground A. Try diverse locations and/or soil types. Make sure to collect the samples in triplicates so that you have replicates for your experiment. For example, if you collect compost soil and playground dirt, you should have compost soil samples A, B, and C, as well as playground dirt samples A, B, and C. At least one of your samples should be a nutrient-rich soil, like compost or fertile garden soil.
4. Once you are back inside, slightly dampen each soil sample with ¼ teaspoon of water, while the soil is still in the baggies.
5. Using a permanent marker, label each agar plate with respect to the type of soil sample and replicate. For example, Playground B. (Note: always label the bottom or sides of agar plates, rather than the lids. That way you won't get your samples mixed up if you take off the lids of more than one plate at once.) Distribute the soil samples in a ring around the E. coli lawns. Each sample should go on a separate agar plate.

   Figure 2. In this nematode isolation setup, the damp soil is placed around the E. coli lawn. If there are any nematodes in the soil sample, they will crawl toward the E. coli. (Wormbook, 2007.)

6. After 30 minutes, examine the plates using the magnifying glass. Which plates have nematodes on the E. coli lawn? How many nematodes do you see per plate? Using your lab notebook, record your findings in a data table like this:

   Soil Sample	Replicate	# of Nematodes
   Playground	A
   	B
   	C

7. From your three replicates, calculate the average number of nematodes per soil sample. Advanced students may want to calculate the standard deviation. Which types of soil or soil locations have larger populations of nematodes?

Bacterial Safety

Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders. You can also see the Microorganisms Safety Guide for more details. Additionally, many science fairs follow ISEF Rules & Guidelines, which have specific guidelines on how bacteria and other microorganisms should be handled and disposed of.

• Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
• Make sure to wash your hands thoroughly after handling bacteria.
• Proper Disposal of Bacterial Cultures
  • Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
  • Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
  • After bleach treatment is completed, these items can be placed in your normal household garbage.
• Cleaning Your Work Area
  • At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
  • Be aware of the possible hazards of disinfectants and use them carefully.

Variations

• Does soil depth have an effect on nematode abundance? Compare the number of worms isolated from different soil depths. Remember to change only one variable at a time!
• Do different species of nematodes live in different soils? Try classifying the nematodes you find in each soil sample based on physical traits. You'll need a microscope to see the worms in more detail. You'll also need to research different species of soil nematodes.
• Not all soil nematodes are bacterivorous. Some species are plant parasites and others consume fungi. Design an experiment to examine plant- or fungi-eating nematodes.
• Soil is only one of the many habitats nematodes are found in. Design an experiment to examine marine or freshwater nematodes.

• For more science project ideas in this area of science, see Zoology Project Ideas.

Credits

Sandra Slutz, PhD, Science Buddies

This project was adapted from: Barrière, A. and Félix, M.-A. Isolation of C. elegans and related nematodes (July 17, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.115.1, http://www.wormbook.org. Retrieved February 29, 2008 from http://www.wormbook.org/chapters/www_nematodeisolation/nematodeisolation.html

Last edit date: 2008-03-25 12:00:00