Experiment 2: Catalase Assay

The ETC contains many different types of electron carrier molecules, including the cytochrome proteins that were discussed in Experiment 1. Flavoproteins are another important electron carrier protein and are derived from riboflavin (which is vitamin B₂). Flavoproteins occur early in the ETC and can either pass electrons to the next carrier in the chain or can bypass the ETC and directly reduce oxygen. This latter path generates hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^- ), both of which are very toxic to the bacteria. Bacteria that utilize this alternative pathway have developed mechanisms to degrade these toxic molecules to harmless ones. Superoxide dismutase converts O₂ ^- to H₂O₂ then catalase converts H₂O₂to water and oxygen (O₂). Catalase is typically present in aerobic and facultatively anaerobic bacteria. Since catalase plus H₂O₂ generates O₂ (in gaseous form), bubbles are released and can be used as an indicator of the presence of catalase in a bacterial sample.

Figure 6: Eukaryotic energy production is performed in mitochondria. This mitochondria has an ETC very similar to the ETC found in Oxidase positive bacteria. The mitochondria also includes bacterial ribosomes and a bacterial-like genome. In fact, mitochondria are thought to have evolved from a primitive bacteria (that used aerobic respiration) proximately 2 billion years ago after it was eaten by a larger cell (that likely used anaerobic respiration). This symbiotic relationship would have provided increased energy for the larger cell and was an important step towards developing more advanced cell types with high metabolic needs.

Procedure

1. Loosen or remove the cap on the nutrient agar bottle.
2. 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 from the microwave 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 to remove the bottle from the microwave. Remember, this will be HOT!
3. Gently swirl the bottle to mix the solution.
4. Slowly pour the liquefied agar solution into the bottom half of two 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.
5. Place the lids onto the dishes and allow the agar to gel undisturbed. If you will not be using the dishes immediately, store them upside down in the refrigerator after they have fully gelled. Remove from the refrigerator and allow them to sit at room temperature for at least one hour prior to use.
6. After the agar has gelled, remove the Parafilm™ and use a permanent marker to draw a line down the diameter on the back of the plates.
7. Label each of the four halves as either “Sink”, “Shoe”, “Phone”, and “Control”.
8. Put on a pair of gloves, open a sterile cotton swab package, and quickly moisten the tip of the swab.
9. Without touching anything else, place the tip of the cotton swab in a sink. Roll the swab around on the sink to ensure good contact is made. A kitchen sink is ideal for this, but a bathroom or other sink is also sufficient.
10. Remove the lid from the agar plate that has one half labeled as “Sink” and hold it closely over the top of the plate to shield the plate from airborne contaminants.
11. Carefully streak the cotton swab onto the sink half of the 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.
12. Place the lid back onto the agar plate and set aside.
13. Repeat Steps 8 - 12 this to obtain the shoe and phone microbe samples. Use new, sterile cotton swabs for each sample. Also note you may use either a cell phone or landline for the phone sample. Be sure to spread each sample on the corresponding plate half.
14. Open a final sterile cotton swab and, without touching it to anything, streak the tip of the swab onto the final control half.
15. Seal the plates with Parafilm™ and allow them to incubate upside-down for approximately three days.
16. After your plates have developed good, isolated growth, remove the Parafilm™.
17. Turn the third petri dish (the last dish; without agar in it) upside down so that the bottom of the dish is facing you. Use a permanent marker to divide the plate into three areas. Label each area as “Shoe”, “Sink”, or “Phone”.

Figure 8: Set-up reference for Step 6.

18. Turn the dish back over, remove the lid, and use the wax pencil to draw two circles in each of the three sections. See Figure 8.
19. Use an inoculating loop to transfer a microbial colony from the “Sink” half of the nutrient agar dish to one of the circles in the “Sink” third on the dish.
20. Use the transfer pipette to add 1 - 2 drops of H₂O₂ to the same circle you just transferred the microbe to. Immediately look for bubble formation (effervescence). Record your results in Table 2.
21. Repeat Steps 19 - 20 in the second “Sink” circle for a second trial with the “Sink” growth.
22. Repeat Steps 19 - 21 for the “Shoe” and “Phone” growth. Remember to look for effervescence immediately and record your results in Table 2.
23. Pour approximately five mL of the 10% bleach solution onto each surface of the three 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.
24. Seal the petri dishes with Parafilm™ and dispose of them in the trash.