Experiment 1: Fluid Thioglycollate Medium to Assess the Effect of Oxygen on Bacterial Growth

Fluid Thioglycollate Medium (FTM) is a basic medium that is suitable for growth of a wide variety of microorganisms. FTM reduces oxygen to water through the action of sodium thioglycollate and L-cystine, and it contains an oxygen indicator (resazurin) that is pink in the presence of oxygen and colorless in its absence. An oxygen gradient is formed from top (high) to bottom (none). This facilitates the growth of microbes ranging from obligate aerobes to obligate anaerobes. A pink zone that occupies more than onethird of the tube indicates that excess oxygen has diffused into the FTM; these tubes should be melted in a boiling water bath and re-cooled before use.

Procedure

Prepare the Nutrient 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.

Inoculate the Prepared Plates:

6. After the plates have warmed or cooled to room temperature, use a permanent marker to label the bottom of four of the petri dishes as “Skin”, “Nose”, “Throat”, and “Shoes”.
7. Put on a pair of gloves, and use a sterile cotton swabs to obtain a bacterial samples from your skin (such as your arm, cheek, etc.) .
8. 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.
9. Carefully streak the used cotton swab onto the gelled medium (agar). To do this, start at one end and work across the plate 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.
10. Place the lid onto the agar plate and set aside.
11. Place the plate upside down in a warm area to incubate.
12. Repeat steps 7 - 11 this for three of the remaining plates. However, use new, sterile cotton swabs to obtain samples from the inside of your nose, your throat, and the sole of your shoe. Inoculate the plates being sure to spread each sample on the corresponding plate, respectively.
13. Label the fifth plate “Control” and inoculate it with a sterile cotton swab.
14. Seal the plates with 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), invert them, and incubate them in a warm location (not to exceed 37.7 °C or 100 °F) for 2 - 3 days.
15. Label the FTM tubes with the same labels as on the Nutrient agar plates.
16. After the plates have been sufficiently incubated, use a sterile disposable inoculating needle to pick up an individual colony from the “Skin” plate.
17. Inoculate the skin FTM tube by inserting the needle straight to the bottom of the tube and slightly mixing the needle in the medium (by sliding the needle up and down, not side to side). Pull the needle straight back out through the initial needle track to limit introducing oxygen to the medium. Replace the lid on the tube.
18. Repeat steps 16-17 with the remaining samples and the other FTM tubes. Remember to use a new inoculating needle each time.
19. Inoculate the Control FTM tube with a new, sterile inoculating needle. Do not expose the needle to any surfaces!!
20. Incubate all tubes in a warm location (not to exceed 37.7 °C or 100 °F) for 2-3 days. Examine the tubes for growth and record your results in Table 2.
21. Seal your bacterial plates with Parafilm™ and store them in a warm place for later experiments in this lab. Use bleach to disinfect the control plate, seal it with Parafilm™ , and dispose of the plate in the trash.
22. Cover your FTM tubes with bleach and allow them to sit for 20 minutes. Rinse the bleach down the drain with running water, seal the tubes with Parafilm™, and dispose of them in the trash.