*Microwave or Boiling Water Bath

Experiment 1 Oxidase Assay for Respiration
Materials
Nutrient Agar
Candle
Matches
*Distilled Water
*Bleach
*Microwave or Boiling Water Bath
*Permanent Marker
*Isopropyl Alcohol
Experiment Inventory
Labware
Disposable Gloves
Parafilm®
4 Sterile Cotton Swabs
Reusable Metal Inoculation Loop
DrySlide Oxidase Assay Cassette (contains the
substrate tetramethyl-p-phenylenediamine)
(2) 5 cm Petri Dishes
*Hot Pad
*Lab Notebook (optional)
*Scissors Note: You must provide the materials listed in *red.
EXPERIMENT 1: OXIDASE ASSAY FOR RESPIRATION
The electron transport chain (ETC) performs the final series of reactions in both aerobic and anaerobic respiration, ultimately
creating an electrochemical gradient that helps drive the production of ATP. In the ETC, electrons are transported from an electron
donor to an electron acceptor through redox reactions. These redox reactions are facilitated by enzymes, and the enzyme used
depends on the type of respiration being performed: aerobic respiration uses an oxidase, and anaerobic respiration uses a
reductase. In prokaryotes, the electron donors and acceptors can be a range of organic or inorganic molecules, depending on the
species.
In eukaryotic cells and some aerobic prokaryotic cells, the terminal oxidase in the ETC is cytochrome oxidase. This enzyme
transfers electrons from the cytochrome c molecules (making up the terminal complex of the ETC) to an oxygen molecule, fulfilling
the last step of respiration and allowing the cycle to start anew. Though many aerobic bacteria have an ETC with cytochrome c
oxidase, some prokaryotes have a different oxidase that makes up the terminal complex or are obligate anaerobes and utilize a
reductase. Microbiologists can distinguish between microorganisms that have cytochrome c oxidase and those that do not by using
an oxidase reagent.
Application of a cell culture containing cytochrome c oxidase to an oxidase reagent such as tetramethyl-p-phenylenediamine
results in oxidation of the substrate, turning it from clear to a purple/blue color. If the cell culture does not contain cytochrome c
oxidase, the cells cannot oxidize the tetramethyl-p-phenylenediamine, and the substrate will remain clear. Exposing cell cultures to
tetramethyl-p-phenylenediamine and observing the resulting color of the substrate is therefore a convenient method for determining
whether a microorganism is cytochrome oxidase positive or negative. It is important to remember, however, that although a
positive result means a microorganism is definitely aerobic, a negative result does NOT necessarily indicate the sample is an
obligate anaerobe. In this experiment, you will use the tetramethyl-p-phenylenediamine contained within the filter reaction areas
of the DrySlide Oxidase Assay Cassette to determine whether or not your cell cultures are oxidase positive or oxidase negative.
PROCEDURE
Note: If you are keeping a lab notebook, record the date, time, and experiment title on a fresh page before
you begin.
Prepare Agar Plates
1. Loosen or remove the cap on the nutrient agar bottle.
2. Place the bottle in a microwave. You will need to remove the bottle from the microwave and swirl the contents every 10
seconds to evenly distribute the heat. If you do not have a microwave, place the bottle in a heat-safe bowl, create a water
bath by pouring boiling water into the bowl (around the bottle), and heat until the entire bottle of agar is liquefied.
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. Once boiling has
stopped, use a hot pad to protect your hands and remove the bottle from the microwave. Use caution when
removing the bottle from the microwave as it will be HOT!
3. With a hot pad protecting your hands, remove the agar bottle from the microwave. Use caution when removing the bottle
from the microwave as it will be HOT!
4. Gently swirl the bottle a final time to mix the solution.
5. Pour the agar solution into the bottom half of two Petri dishes. USE THE AGAR SPARINGLY. YOU ONLY NEED ENOUGH
TO COAT THE BOTTOM OF THE PETRI DISH.
Note: Approximately 5 mL should be enough agar to cover the entire bottom of the dish. If not, continue to
add agar in approximately 1 mL increments until the bottom of the petri dish is completely covered. The agar
should be only 1 or 2 millimeters deep. If too much agar is poured, there will be no space under the cover for
microbial growth.
6. Place the lids onto the dishes slightly off-center to create a small space to allow condensation to evaporate and allow the
agar to solidify undisturbed. This should take approximately 30 – 60 minutes. If you will not be using the dishes immediately,
you can store them upside down in the refrigerator after they have fully gelled. Remove them from the refrigerator, and allow
them to sit at room temperature for at least 1 hour prior to use.
7. After the agar has gelled, use a permanent marker to draw a line on the back of the bottom of each plate, dividing them in
half.
8. Label each half as either “Sink,” “Shoe,” “Phone,” and “Control.”
9. Put on a pair of gloves, open a sterile cotton swab package, and quickly moisten the tip of the swab with distilled water.
10.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.
11. 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.
12.Carefully streak the cotton swab onto the agar. To do this, start at one end and work across the plate in a zig-zag motion.
Ensure that you do not press too hard when streaking the swab on the agar to prevent cutting into the agar surface.
13.Place the lid back onto the agar plate and set aside.
14.Repeat Steps 8 – 12 to obtain the “Shoe” and “Phone” samples. Use a new, sterile cotton swab for each sample. You may
use either a cell phone or landline for the phone sample. Be sure to spread each sample on the corresponding plate half.
15.Open a final sterile cotton swab, and quickly moisten the tip with distilled water. Without touching it to anything, streak the tip
of the swab onto the “Control” half of the plate.
16.Seal the plates with Parafilm® and allow them to incubate upside-down for approximately three days.
17.After your plates have developed sufficient isolated growth, remove the Parafilm® and open the DrySlide assay.
18.Sterilize your inoculation loop with your candle.
a. Pour 70% isopropyl alcohol into a 100 mL beaker to a depth of 2 – 4 cm. Place the cap back on the bottle, and position it
far out of the way.
b. Light your candle, and set it aside. Be very cautious that your candle is safely positioned away from the beaker of
isopropyl alcohol.
c. Dip the inoculation loop into the isopropyl alcohol for 10 seconds. Once you’ve put the inoculation loop in the alcohol,
keep it angled down so that no alcohol drips back onto your hand.
d. Without touching the inoculation loop to anything, carefully pass the end of the inoculation loop through the flame several
times.
e. Extinguish the flame when complete.
19.Use a sterile inoculating loop to remove a colony from the “Sink” section of the Petri dish, and gently transfer it onto one of
the four squares on the DrySlide.
20.Observe for a color change (colorless to purple/blue) within 20 – 30 seconds. Record your results immediately in Table 1.
21.Repeat Steps 18 – 20 with colonies from the “Shoe” and “Phone” halves on two other squares of the DrySlide. If keeping a
lab notebook, print out Table 1 and tape it into your lab notebook or re-create it by hand.
22.Pour approximately 5 mL of bleach onto the agar plates, allowing it to cover the entire surface. Pour enough bleach on the
DrySlide to completely cover the microbe samples. Incubate them for at least 20 minutes, and then pour the bleach down
the sink with running water.
23.Seal the Petri dishes and the DrySlide with Parafilm®, and dispose of them in the trash.
Data Sheet Experiment 1 Data Sheet
Table 1: Experiment 1 Assay Results
Colony Sample Color Change? (Yes or No) Oxidase Positive or Negative?
Sink
Shoe
Phone