Experiment 1: Testing the Environment

Experiment 1: Testing the Environment

Bacteria is found in almost all environments. Most are harmless, but there are those that cause disease. In this experiment, you will be test different environments for the presence of bacteria. You may be amazed to see how many different species are present on surfaces you come in contact with every day!

Materials 30 mL 8.25% Bleach Solution 4 Cotton Swabs (Sterile) 125 mL Nutrient Agar 60 cm Parafilm® Permanent Marker (4) 9 cm Petri Dishes	*Hot Pad *Microwave or Boiling Water Bath *Refrigerator *Scissors (to cut the Parafilm®) *You Must Provide

Notes About Working With Agar Plates…

• Prepared agar dishes should be stored upside-down in the refrigerator until used. This will prevent condensation from disrupting the growing surface.
• After inoculating, replace the cover on the dish, seal with Parafilm®, and store upside-down in a warm location (not to exceed 37.7 °C or 100 °F).
• You should see growth within a few days. The plate will start to smell once microorganisms are growing.
• Before disposing plates, kill the microorganisms by pouring bleach solution onto the agar surfaces and let sit for 20 minutes.

Procedure

1. Loosen or remove the cap on the Nutrient agar bottle. Place in the microwave (if you do not have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat until the entire bottle of agar is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
2. If you notice the liquid boiling over, STOP the microwave and let the bottle cool down before handling. With a hot pad protecting your hands, remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!
3. Gently swirl the bottle to mix the solution. Pour enough of the liquefied agar solution into the bottom half of 4 petri dishes so that it covers the entire bottom of the dish. Place the lids onto the dishes and CAREFULLY transport these plates into a refrigerator. Allow the plates to sit for 24 hours.
4. After 24 hours, remove the four agar plates from the refrigerator and allow them to sit at room temperature for at least one hour.
5. After the plates have warmed to room temperature, locate a surface to swab for bacteria. Things such as shoes, a table, teeth, bathroom doors, and shopping carts can be great sources. Try to avoid surfaces that are frequently cleaned with antibacterial detergents and soaps. On the bottom of the plate, write the name of the surface to be swabbed with your permanent marker.
6. Remove the lid from the agar plate. Unwrap one sterile cotton swab and smear the surface, being sure to roll the cotton swab to cover all sides.
7. Carefully streak the cotton swab onto the surface of the gelatinous medium (agar), being sure to start at the top and work down in a zigzag motion.
8. Place the lid onto the agar plate and seal it with a strip of Parafilm (hold one end of the Parafilm firmly against the side of the petri dish and stretch the other side to cover the entire perimeter). Place the plate upside down in a warm area to incubate.
9. Repeat this for two additional surface areas and agar plates.
10. For the last plate, label it “control.” Do not rub your sterile cotton swab on any surface, but rather take it straight from the package and streak it onto the plate. This will test for your accuracy in keeping the plates and swabs sterile while performing the experiment.
11. Let the plates incubate in a warm area for three days and then observe the growth. Answer the questions below.
12. After the experiment and after answering questions 1 - 3, set aside the plate with the most bacterial growth for the next experiment. Pour the bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Let the plates sit untouched for 10 - 20 minutes. Then, seal plates with Parafilm® and dispose appropriately.

Post-Lab Questions

1. In the space provided below, draw your plates and indicate the number of different colonies and identify the colony shape.

1. Which plate grew the most bacterial species? Was this a surprise? Why or why not?

A: The plate that grew the most bacterial species was 1.

1. Was your control plate free of bacterial colonies? If not, how do you think the swabs were contaminated?

A: Yes, my control plate was free of bacterial colonies.