Standard Operating Procedure
Title: Oxytherm Respirometer Calibration and Operating Instructions
Department: Agronomy
Created by: Whitney Bouma
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Allen Knapp
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
Oxygen uptake measurements of mitochondrial and cellular respiration can be achieved with
the Oxytherm respirometer and associated Oxygraph Plus software. The following are
instructions necessary for operating this equipment, including electrode preparation, system
calibration, and electrode cleaning/maintenance.
Equipment and reagents necessary:
Electrode preparation:
Equipment:
Clark electrode
Beaker
Sharpie marker
Weight boat
Spatula
Analytical balance
Stir bar
Stir plate
Thermometer
PTFE membrane
Cigarette paper
Membrane applicator
Dropper
O ring
Squeeze bottle with DI water
Plastic waste container
Reagents:
Potassium chloride
Sodium hydrosulfite
DI H2O at 25 C
Oxytherm system:
Equipment:
Oxytherm control unit and power cord
Oxytherm peltier electrode chamber w/ reaction vessel and plunger
Oxygraph Plus software
RS-232 cable fitted with USB adaptor
Electrode connection cable
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Calibration:
Equipment:
Vacuum aspirator fitted with rubber pipette tip
Erlenmeyer flask
Mini stir bars (flea)
Spatula
Reagents:
Sodium dithionite
DI H2O at 25 C
Clean up & Maintenance:
Cleaning kit (polish and cotton buds)
Procedure:
The set up procedure for operating the Oxytherm system can be broken into five parts: 1)
electrode preparation, 2) system setup, 3) system calibration, 4) system operation, 5)
storage/preventative maintenance. Please follow the instructions carefully and see Whitney
Bouma if you have any questions (rm. 1523 or 294-3886).
1) Electrode Preparation: The high precision Clark type electrode sensors are very sensitive
and require proper preparation for accurate measurement. Please refer to the manual under
the help menu in the Oxygraph Plus software for more information on the electrode
operating principles and references for the oxygen electrode theory. You will also use this
manual for detailed instruction on how to properly prepare the electrode and calibrate the
system.
The electrode disc is prepared so that an electrolyte bridge is established between the
anode and cathode. The will allow a current to flow in the presence of oxygen. You will first
need to prepare the electrolyte which is a 50% saturate Potassium chloride solution. If there
isn’t any fresh electrolyte already prepared, complete the following procedure:

Using a spatula, weigh boat and analytical scale, weigh 17.5 g of Potassium chloride
and set aside.

In a glass beaker with a minimum of 100 ml capacity, measure out 100 ml of de-ionized
H2O. Using a thermometer, check the temperature. You will want the H2O to be close
to room temperature (25 C), so it may be necessary to slightly warm the water.

Add a stir bar to the beaker containing the water and mix in the 17.5 g of KCl by placing
the beaker on a stir plate and gently mixing the solution. You will want to make sure
that the KCl is fully dissolved so continue mixing until this is achieved.

Place the solution in a glass bottle with a lid and label appropriately with contents and
date prepared (remember no short hand). This solution should be good to use for up to
a month.
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You are now ready to prepare the electrode for measurements. Please read the help
manual on “Preparing the Electrode Disc” thoroughly before beginning. It may take multiple
tried to prepare the electrode properly if this is your first time. It is vital that there are no air
bubbles or creases directly over the platinum cathode on the top of the electrode dome.
Creases along the sides of the dome however are sometimes unavoidable and will not
interfere with the electrode signal.
The electrode preparation is completed in 4 stages (visual aid is available in the help menu
under “Preparing the Electrode Disc” of the online help manual). When applying the paper
spacer and membrane, it is best to use foreceps as oils from your fingers may interfere with
the electrode signal.

Place a small drop of electrolyte on top of the dome of the electrode disc. This will be
directly on top of the platinum cathode.

Place a 1.5 sq. cm paper spacer (cigarette paper) over the electrolyte ensuring that at
least one corner of the spacer is in the electrode well to act as a wick. Cover this with a
slightly larger, 2.5 sq. cm, piece of PTFE membrane.

Place the small electrode disc O-ring over the end of the applicator tool. Hold the
applicator vertically over the dome and slide the applicator shaft down to push the Oring over the dome.

Check that the membrane preparation is smooth and that there are no trapped air
bubbles. Top the reservoir well up with several drops of electrolyte.
It is vital to make sure the larger O-ring is placed in position on the recess around the
electrolyte well. If this second O-ring is not in place when the disc is installed into
the electrode chamber, the silver anode will not be sealed from ambient air and
measurements may be affected.

You will want to test the response of the electrode before putting the electrode disc into
the chamber.

To do this you will connect the electrode disc to the rear of the control unit at the
electrode input.

Open the Oxygraph Plus software and start a recording by clicking the GO button on the
toolbar. The signal will take up to 20 minutes to stabilize but the disc should read
between 2000mV to 1800mV in air. Make sure that the electrode has properly stabilize
and doesn’t drift downward over time.

Once this reading is stable, breathe or exhale across the disc and observe the reaction
the screen. The signal should drop due to the decrease oxygen in the exhaled breathe.

The signal should soon begin to return to the original level as ambient oxygen begins to
equilibrate around the electrode disc.
Once this test has been completed satisfactorily you can stop the recording, set up the rest
of the equipment, and then mount the disc into the base of the Peltier chamber.
2) System Set Up: This step covers how to connect all the components of the Oxytherm
system. On the back of the control unit from left to right are the following connections and
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inputs: 25 A fuse, 12 V Power supply input, RS232 input and output, electrode disc
connection, and the auxillary input connection.
Before making any measurements, it is important that the system is set up
correctly. The diagrams in the online manual show the various connections that
must be made to the control units. Please ensure the following are done before
continuing on with system calibration.
Ensure that the 12V power supply is connected to the 12V input socket on the control unit.
Confirmation that this connection has been made is shown by an illuminated green LED
on the front panel of the control unit.
Connect the RS232 serial cable to an available serial port on the PC. You will need to
make sure to use the USB converter attachment for the PC input. Connect the other end
of the RS232 cable to the INPUT RS232 connection on the rear of the control unit.
Mount the electrode into the Peltier chamber using the following directions:

Remove the black base ring from the Peltier electrode chamber and mount the prepared
electrode disc into the recess at the top. The electrode cable connection slots into a
keyway in the chamber base meaning that the disc can only fit one way.

Fit the chamber base and the disc assembly onto the Peltier chamber itself so that the
electrode cable connection fits with the keyway on the chamber base thread. Gently
hold the base in position while threading the base ring on to the Peltier chamber. Just
slightly more that finger tight will suffice.
Connect the Peltier Electrode Chamber and the prepared electrode disc, if necessary, to
the rear of the base unit. You are now ready to start the calibration part of the set up.
3) System Calibration – Before any measurements can take place, the electrode disc must
be calibrated so that the electrical signal received from the disc can be presented as
actual calibrated units (nmol/ml). The calibration procedure is performed so that the signal
from the oxygen electrode can be referenced to two known oxygen concentrations (air line
and zero air) to derive an offset and a calibration factor.
Temperature (°C) [Oxygen (ppm)] [Oxygen (nmol/ml)]
0
14.16
442.5
5
12.37
386.6
10
10.92
341.3
15
9.76
305
20
8.84
276.3
25
8.11
253.4
30
7.52
235
35
7.02
219.4
At a given temperature and atmospheric pressure, air saturated deionized water contains
a known concentration of dissolved oxygen which may be calculated mathematically. The
following information in the figure above is used by the Oxygraph Plus software in order to
accurately reference the electrical signal from the electrode for the air line stage of
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calibration. The data was published by Truesdale & Downing (Nature 173: 1236, 1954).
Additional formulas and information on these values can be referenced in the Oxygraph
Plus Online help manual under “System Calibration – liquid phase”.
Using the information derived from the figure 1 below we can begin the calibration
process. The Oxygraph software will guide you through most of the process with easy to
follow prompts. Please follow these guidelines:

Ensure that the electrode disc is properly installed into the base of the Peltier Chamber.

Using a P1000 pipette, pipette 2 ml of air saturated deionized water into the reaction
vessel of the chamber. Air saturated water is obtained by vigorously shaking a small
quantity of deionized water (~50 ml) in a large Erlenmeyer flask (~ 1L). Then drop one
of the small magnetic stir bars (fleas) into the vessel.

Next is to set the assay temperature through the Oxygraph software, by selecting
HARDWARE > TEMPERATURE CONTROL from the menu bar. This will display the
actual temperature and then the setup temperature you desire. Set the temperature you
wish to run your assays, for example 25 C.

It is important to remember that the sample of air saturated H2O should be equilibrated
to the assay temperature before the calibration procedure actually begins. This may take
several minutes to allow the H2O to reach temperature once added to the reaction
vessel.

Once the H2O temperature and assay temperatures have equilibrated, initiate the
calibration sequence from the CALIBRATE > LIQUID PHASE CALIBRATION menu
option. Enter the appropriate temperature for your assay. The pressure should be
standard atmospheric, but make sure to check the weather to see what it is and make
conversions accordingly. You will already have H2O in the chamber so proceed by
pressing OK.

The next window prompts you to enter the stir bar settings. This operation can be
accessed by selecting HARDWARE>STIRRER SPEED. Enter a speed of 75. This is
usually sufficient to ensure that the dissolved oxygen within the samples is kept evenly
distributed. Then press OK.

The next window will show the signal as it changes and stabilizes. Once it has reached
a plateau the window will prompt you to press OK to continue on to the second stage of
the calibration procedure, establishing zero oxygen line.

To perform this stage, we will use the chemical Sodium Dithionite.

Add a few crystals to the reaction vessel. It really only requires a small pinch of crystals
to deplete the oxygen in the vessel so the tip of a spatula worth of crystals should be
sufficient. Once they are added, press OK to continue.

Watch the signal drop. You should expect to see a signal of less that 1% of the air line
signal. For example, if your previous signal was 1802 mV, you would expect to see a
signal less than 18.02 mV for the zero line. If you are satisfied with the signal, press OK
to continue.

The next window will give you the calibration factor and the calibration offset. If these
values are adequate, click the save button to store the calibration details.

You will need to remove all traces of sodium dithionite from the reaction vessel. Using
the aspirator, fit with the rubber topped pipette tip, suck out the waste. Be mindful of the
stir bar and the electrode. Fill with dionized H2O for rinsing and remove. Repeat the
rinsing several times to ensure the vessel is clean.
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
For more details about the calibration process refer to the online help portion of the
Oxygraph software manual

You are now ready to begin measurements.
4) System Operation/Data Acquisition – Full details on the Oxygraph software functions
and system configuration can be viewed on the online help manual in the Oxygraph
software. You will need to thoroughly read this section before commencing with your
measurements. This information will help you determine the appropriate software settings
and actions depending on the type of experiment you will be running. The specific details
of conduction your experiment should be included in the experiment SOP.
5) Cleaning& Storing Electrode/Preventative Maintenance - To ensure accurate
measurement, it is imperative that proper electrode cleaning and preventative
maintenance measure be taken. Deposits of silver chloride and/or silver oxide will build
up on the silver ring of the disc. Again, detailed instruction and illustrations of well and
poorly managed electrodes are available in the online help manual of the Oxygraph
software.
It is important that the electrode is never left to dry out while prepared with the KCl
electrolyte. You will need to complete the electrode cleaning procedure at the end of each
day and ensure it is properly stored. Please follow these instructions for cleaning the
electrode:

Using the polishing paste, provide by Hansatech, you will want to polish both the silver
anode and platinum cathode.

Take a cotton bud moistened with a bit of DI H2O. Apply a small amount of polishing
paste on the tip. Insert the bud into the well of the electrode and gripping the bud just
above the tip, apply a moderate amount of pressure and gentry rotate the bud around
the electrode well in a circular motion, 6-10 times.

Using the other end of the cotton bud, again moistened with a bit of DI H2O, apply a
small amount of polishing paste. Paying attention to restrict the polishing to the small
platinum cathode, use a circular motion to gently polish the cathode. Make sure to avoid
as much of the epoxy resin dome as possible.

Once adequately cleaned, rinse the electrode several times with DI H2O to ensure all
the polishing paste has been removed. If necessary, gently brush the area with a soft
bristled toothbrush to remove any extra paste.

Dry the electrode with a kimwipe and store the electrode in the designated, dessicated
box.
Personal Protective Equipment / Engineering Controls:
Lab coat, eye protection and hand protection must be selected as required by Section D of the EH&S
Laboratory Safety Manual. Check the box(es) next to the item(s) which are appropriate for your
procedure.
x
Nitrile gloves
x
Safety glasses
Dust mask
Lab coat
Fume hood
Latex gloves
Splash goggles
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Eye wash station
Safety shower
Respirator
Insulated gloves
x
x
Face shield
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Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
.
Handling & Storage Precautions:
Potassium chloride: May cause skin, eye and respiratory tract irritation. Use appropriate PPE as listed
above. Keep containers tightly closed in a dry, cool and well ventilated place. Avoid dust formation.
Sodium hydrosulfite: May cause skin, eye and respiratory tract irritation. Use appropriate PPE as listed
above. Avoid dust formation Is.unstable and can be reactive, self-heating, may catch fire. Keep cool,
dry, well ventilated area and protect from sunlight. Do not store near acids or combustible materials.
Refer to MSDSs for full details.
Waste Disposal Procedures:
With the implementation of the new Lab Safety Manual, EH&S eliminated the ability to dispose
of non-hazardous waste by flushing it down a drain. Instead, all waste needs to be collected
for EH&S disposal regardless of what the MSDS indicates. The following should be all you
need in this section unless you have been specifically instructed otherwise.
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
Potassium Chloride (and all reagents made with Potassium chloride): will need to be collected
by EH&S. Please place them in the satellite accumulation area with proper labeling.
Sodium hydrosulfite: Do not let this product enter drains. Collect in a suitable, properly
labeled container for collection by EH&S.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Potassium Chloride – collect any chemical that has spilled and keep contained in a suitable
container or with a proper spill kit for collection by EH&S.
Sodium hydrosulfite: collect any chemical that has spilled and keep contained in a suitable
container or with a proper spill kit for collection by EH&S.
Remember, all waste needs to be collected for EH&S disposal – including that from a
spill clean up.
Health & Safety Summary for Required Reagents:
Potassium Chloride:
Eye Contact: Rinse immediately with plenty of water, also under the eyelids for at least 15
minutes. Obtain medical attention.
Skin Contact: Wash off immediately with plenty of water for at least 15 minutes. Get
medical attention immediately if symptoms occur.
Inhalation: Move to fresh air. If breathing becomes difficult, give oxygen. Get medical
attention immediately if symptoms occur.
Ingestion: Do not induce vomiting. Obtain medical attention.
Sodium Hydrosulfite:
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Eye Contact: May cause eye irritation. Rinse immediately with plenty of water, also under the
eyelids for at least 15 minutes.
Skin Contact: May be harmful if absorbed through skin. May cause irritation. Wash off
immediately with plenty of water for at least 15 minutes.
Inhalation: May be harmful if inhaled. May cause respiratory tract irritation. Move to fresh
air.
Ingestion: Toxic if swallowed. Rinse mouth with water and consult a physician.
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Incompatibilities
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 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the contents
of the entire MSDS(s) before starting this procedure.
References:
Oxygraph Plus software online help manual.
http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do
http://www.fishersci.com/msds?productName=BP366500&productDescription=POTASSIUM+
CHLORIDE+500G&catNo=BP366-500&vendorId=VN00033897&storeId=10652
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