Project Protocol Worksheet 2 – Materials & Methods
By Dana Krempels, Ph.D.
Title:
Section and Instructor:
Team# & Members:
INSTRUCTIONS: Use this PPW2 form to write your final experimental protocol. The document has
been formatted to display your work in BLUE to distinguish it from the assigned question. Use as
much space as necessary.
I. Materials
A. How many runs, each, of your treatment and control groups will you perform?
Treatment:
Control:
B. You will be supplied with the following standard reagents:
 Stock yeast suspension (70g yeast/L pH 7 sodium phosphate buffer)
 Stock hydrogen peroxide (33mL of 9.1% H2O2/L pH 7 sodium phosphate buffer)
 Stock sodium phosphate buffer (0.05M, pH 7)
 Deionized (DI) water
Remember that each control reaction is run with 10mL of standard yeast suspension and 20mL
of H2O2 in pH 7 buffer. With this information, calculate the amount of each of the stock solutions
of yeast, peroxide, buffer, and water your team will need.
Description (e.g., molarity, pH, etc.)
Name
Quantity/volume
Yeast suspension
70g/L in pH 7 sodium phosphate buffer
Hydrogen peroxide
9.1% H2O2/L in pH 7 sodium phosphate
buffer
NAPO4 buffer
pH 7.0, 0.05M
water
deionized
In addition to the stock solutions from #2, what reagents will you need, and how much of each?
List them here.
Description (e.g., molarity, pH, etc.)
Name
Quantity/volume
C. If your protocol requires that you prepare reagents not supplied as stock solutions or
solids, calculate how you will prepare each of your reagents. Use these formulas to
calculate concentrations and dilutions.
1. To calculate the mass (g) of a solid chemical needed to make a solution of a desired volume
and concentration of Chemical X, use this equation:
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Project Protocol Worksheet 2 – Materials & Methods
By Dana Krempels, Ph.D.
(desired concentration in moles/L) x (desired volume in L) x (molecular weight of Chemical X in g/mole)
= g of Chemical X needed
2. If you have a stock solution of known molarity and you want your final yeast suspension to be a
different molarity, use this equation:
(concentration of stock solution in moles/L)(volume of stock solution in L)
= (desired final concentration of NaCl in your yeast suspension)(desired final volume)
Remember: If the amount of yeast suspension and the value of x do not add up to your desired final
volume, you should add deionized water to your suspension to achieve the desired final volume so
that treatment and control are the same.
Create a step-by-step “recipe” for each reagent you plan to prepare. Anyone should be able to
follow your directions to replicate your work. Use this template, or create one of your own. But each
team member should have a copy to take home at the end of lab.
Reagent #1:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Reagent #2:
1.
2.
3.
4.
5.
6.
7.
If you are examining the effect on catalase of an environmental variable (e.g., temperature, pH,
concentrations, you will not need chemical reagents beyond the stock solutions in #2. However,
you still must create a protocol.
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Project Protocol Worksheet 2 – Materials & Methods
By Dana Krempels, Ph.D.
II. Methods
Below, you will find the Sample Experimental Protocol you used in a previous lab session.
Modify it to reflect YOUR team’s protocol (it’s in MS Word), and submit the completed protocol to
your instructor. Wherever you modify the protocol, change the color of the font to BLUE if it
does not automatically show up in blue. This will allow your instructor to easily note the
methods you are modifying for your own experiment.
Modify this Protocol as necessary to create your own Research Protocol
Most of this protocol can be used “as is.” Modify only those segments of the protocol necessary for
your team’s original experiments.
1. Check workstation and equipment. Clean any dirty items with deionized water and drain on a
paper towel. (Check the list of supplies, above, that should be at your station. These items must be
clean and back at your station when you check out for the day or your grade will be docked. When
you are ready to leave, ask your Undergraduate Learning Intern (UGLI) to check your station and
approve it before you go.)
2. Using labeling tape and a marker, re-label syringes and other supplies, as necessary.
Remember: If an item is clean, it doesn’t need a label until you have contaminated it with a
reagent. If any item (syringe, graduated cylinder, beaker, etc.) touches a solution, it MUST
be labeled with the type of solution (concentration and chemical) and your team name (in
case items may get mixed up between neighboring groups). Clean and re-label items
throughout the lab as necessary.
3. Open the LoggerLite program on the laptop your team will use for data collection. (The
download link is on page 3 of this chapter.)
4. Keeping the O2 sensor probe UPRIGHT in its box, connect it to the laptop using the adaptor
cable. Keep the probe dry and upright in its labeled beaker at all times when not in use.
5. To ensure that the sensor is working correctly, click “Collect” on the toolbar. The %O2 should be
recorded once per second in the table to the left and on the graph. Try breathing into the dry
plastic respiration chamber and putting the sensor in the top. The oxygen readings should go
down. Now click “Stop.” You can click “store” on the toolbar to save one data run and “save” to
save an entire batch. To open a fresh data table and graph, click “New.”
6. Before beginning the experiment, delegate duties to everyone in the team. Every person must
know how to do every task, but it will be easiest to assign duties such as manning the computer,
cleaning glassware, recording times, and so on amongst the team members.
A. Preparing your Treatment Samples
If treatment reagent needs time to induce a response in the yeast, those solutions should be set
up first.
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Project Protocol Worksheet 2 – Materials & Methods
By Dana Krempels, Ph.D.
1. Label (insert number of your variable’s values) beakers Treatment
(Example: If you are running trials with catalase concentrations of 0.1M, 0.2M, and 0.3M, you will have
FIVE beakers, labeled Treatment 1, Treatment 2, Treatment 3, Treatment 4, and Treatment 5)
2. Find the stock yeast suspension on a tray at the center of the lab table. Mix the contents to
ensure that yeast are evenly distributed and then decant 70mL into a labeled beaker. Stir the
suspension with a stirring rod often to keep yeast from settling.
3. Add 10 mL of yeast suspension to each Treatment beaker, using the labeled syringe.
4. Insert your protocol step here.
5. Insert your protocol step here.
6. Insert your protocol step here.
7. Insert your protocol step here.
8. Insert your protocol step here.
9. Insert your protocol step here.
…etc. for as many steps as you require
B. Preparing your Control Samples
After the treatment groups are set up, the control groups can be prepared. Because you are not
manipulating any variable in the controls, you may place each control sample directly into the
Vernier plastic respiration chamber and run each trial immediately, without placing your samples
in separate beakers. Use the following procedure for your control runs.
1. Mix your remaining yeast suspension to ensure that yeast are evenly distributed.
2. Add 10 mL of yeast suspension to the plastic Vernier respiration chamber.
3. Add 5 mL of deionized water to ensure equal volumes between the treatment and control.
4. Find the stock hydrogen peroxide solution on the tray at the center of your lab table. Decant
about 135 mL into a labeled beaker.
5. Back at your station, draw 20mL of your H2O2 solution into a labeled syringe.
6. Prepare everything for an experimental reading, making sure your LoggerLite software is
ready, that everyone is in position and ready to do his/her job.
7. When everyone is ready to start the experiment, quickly and carefully squirt 20 mL of H2O2
solution into the plastic Vernier chamber already containing your yeast suspension.
8. Quickly and carefully insert the O2 probe to seal the chamber.
9. Gently swirl the container (hold it by the neck, not the body) to stir. Do not rub the bottom of
the chamber on the tabletop, to avoid friction. Swirl just enough to keep the mixture moving,
but do not wet the probe!
10. The reaction begins very shortly after the introduction of hydrogen peroxide, so click
“Collect” in the LoggerLite program once you have begun swirling your suspension. Be very
careful to keep the probe dry or your readings will become erratic. If this happens, use
one of the ringstand/fan assemblies set up along the back table to dry your probe. Do not
clamp the probe tightly, to avoid damaging it.
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Project Protocol Worksheet 2 – Materials & Methods
By Dana Krempels, Ph.D.
11. When the %O2 readings begin to plateau, click “Stop.” This will happen automatically after 300
seconds.
12. Click “Store” to save the readings from that sample.
13. Gently remove the O2 sensor and place it upright in its dry, labeled beaker.
14. DISCARD ALL USED REAGENTS IN THE LABELED “WASTE” BEAKER AT YOUR STATION.
NOTHING SHOULD BE POURED DOWN ANY SINKS!
15. Repeat Steps 10-14 for the remaining control runs. Work quickly and carefully in
order to be ready for the treatment groups when their incubation time is up, if your samples
are incubating.
16. Follow Steps 1-13 for each of your treatment runs, substituting your treatment variable for
the control reagent appropriately. All steps in the protocol should be the same for treatment
and control groups except for the single variable in your treatment runs.
C. Data Analysis
What statistical test will you use to analyze your results?
D. Before you Leave…
When your team has completely finished all its preparations and has protocol sheets ready to use
for data collection next week, do the following:
1. Provide each team member with electronic copies of your team’s completed PPW1
and PPW2 documents.
2. Provide your lab instructor with electronic copies of your team’s completed PPW1
and PPW2 documents.
Any team leaving without instructor approval will lose 5 points per team member.
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