Experiment 1: Agar Plate Preparation and Bacterial Inoculation

Growth media is used to grow and maintain microorganisms. In this experiment, you will aseptically prepare nutrient agar plates and inoculate microorganisms with bacterial samples obtained from you and your environment.

Procedure

Prepare the Agar Plates:

1. Loosen or remove the cap on the Nutrient agar bottle. Place the bottle 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 agar bottle is liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the heat.
   Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. After boiling has stopped, use a hot pad protecting your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!
2. Gently swirl the bottle to mix the solution a final time.
3. Slowly pour the liquefied agar solution into the bottom half of five petri dishes so that it covers the entire bottom of the dish. It is important that the entire bottom is coated and that the agar is given time to spread out over the dish.
4. Place the lids onto the petri dishes and let them sit at room temperature until the plates are solidified and cooled. The plates can be used immediately or stored in the refrigerator for later use.
5. If you have stored the plates in the refrigerator, remove them and allow them to sit at room temperature for at least one hour before use.

Inoculate the Prepared Plates:

6. Look around for three surfaces to swab microorganisms from. Things such as shoes, a table, teeth, mouth or throat, bathroom doors, and shopping carts can be great sources. Try to avoid surfaces that are frequently cleaned with antibacterial detergents and soaps.
7. Use a permanent marker to label the bottom of one of your petri dishes with the title of the surface you select (e.g., if you choose a table, label the dish as “Table”).
8. Put on a pair of gloves, and begin collecting your samples by rubbing a cotton swab on the location you select. Be sure to get good coverage on the cotton swab; and, use a new cotton swab for every sample.
   Note: It helps if the cotton swab has been lightly moistened with sterile water prior to collecting your samples. The extra water helps promote visible, well-developed bacterial growth.
9. Remove the lid from the agar plate but hold it closely over the top of the plate to use as a shield to prevent airborne contamination of the plate.
10. Carefully streak the cotton swab onto the gelled medium (agar). To do this, start at the top and work down in a zigzag motion. Ensure that you do not press too hard when streaking the swab on the agar to prevent cutting into the agar surface.
11. 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).
12. Place the plate upside down in a warm area to incubate.
13. Repeat steps 6 - 11 for two additional surface areas and agar plates.
14. Use a permanent marker to label the bottom of the fourth petri dish “Control”. Take a sterile cotton swab and rub it onto the control agar plate. Do not rub your sterile cotton swab on any surface. This will test for your accuracy in keeping the plates and swabs sterile while performing the experiment.
15. Repeat steps 10 - 11 on the Control petri dish.
16. Use a permanent marker to label the fifth petri dish “Airborne Contamination”. Remove the lid from the plate and expose the agar to the air for 1 hour. It may be helpful to place the dish close to an airregister or fan to increase the air circulation surrounding the dish.
17. Repeat Step 11 for the Airborne Contamination petri dish.
18. Let all five of the plates incubate in a warm area for 3 days and then observe the growth. Record their growth in Table 1.
    **Answer questions 1 - 3 in the Post-Lab after you have recorded your growth observations.**
19. Set the plate with the most bacterial growth aside for the next experiment.
20. Pour approximately 5 mL of the 10% bleach solution onto the surface of the remaining agar plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the sink with running water.
21. Seal the petri dishes with Parafilm™ and dispose of them in the trash.