Instrumental background oxygen flux

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Instrumental background oxygen flux


Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and intact or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibres and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.

Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. If no experimental background test has been performed, the system default values are used, which are a°=-2.0 pmol/(s·ml) for the intercept at zero oxygen concentration, and b°=0.025 for the slope of background flux as a function of oxygen concentration.

Automatic correction for the instrumental background oxygen flux is an essential standard in high resolution respirometry. At the same time an instrumental background experiment is the ultimate test for instrumental performance, evaluating chamber performance after completion of all elements of the Oxygen sensor test. The instrumental background oxygen flux measured at air saturation should reflect the theoretically predicted volume-specific oxygen consumption by the oxygen sensor. The actual agreement using experimental respiration medium provides at the same time a test that excludes microbial contamination of the medium or serves to evaluate any autoxidation processes in newly tested experimental media.

Abbreviation: J°O2

Reference: MiPNet14.06 Instrumental O2 background; Gnaiger_2008_POS; Gnaiger_2001_RespPhysiol

MitoPedia concepts: MiP concept 

MitoPedia methods: Respirometry 

MitoPedia O2k and high-resolution respirometry: DatLab, Oroboros QM 

» See MiPNet14.06 Instrumental O2 background for experimental details of the instrumental background test, and for downloading the Excel template for analyzing instrumental background experiments.
» Compare Chemical background correction of oxygen flux.

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O2k-technical support

This information is part of O2k-technical support and open innovation.

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O2 slope in the closed and open O2k-Chamber

In an open chamber of the O2k the liquid phase in the chamber (aqueous medium) is in equilibrium with the atmosphere. All oxygen consumed by the polarographic oxygen sensor (POS) is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the (negative) time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab, has to be zero. The background corrected oxygen flux is meaningless for the open chamber situation. This is because the background correction at air saturation subtracts the consumption of oxygen by the sensor from the negative slope, when diffusion into and out of the chamber is zero at air saturation. Therefore, the background-corrected oxygen flux in the open chamber at air saturation is shown as a negative value. To avoid this apparent artefact, the "O2 slope uncorr." is selected to be shown while the chamber is open. Only Graph Layouts that display "O2 slope uncorr." are suitable for assessing the stability of the oxygen signal when the chamber is open. Such Layouts are:
  • 01 Calibration show Temp
  • 02 Calibration - Background
The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability and equilibrium of oxygen between the gas and aqueous phases have been reached. Therefore no experiment should be started before a zero "O2 slope uncorr." has been reached with an open chamber. The suggested criterion for signal stability is a "O2 slope uncorr." between -1 pmol/(s mL) and + 1 pmol/(s mL).


  • Second step - O2k Quality Control 2
Instrumental background experiment, measuring oxygen flux without biological sample at four oxygen levels (left), and linear relation between instrumental background oxygen flux and oxygen concentration (right). Modified after: Gnaiger E (2001).

Standard operating procedure

If all quality control criteria of the O2 sensor test are met, the operator can be assured that the quality of the sensor signal is acceptable. Next, the quality of the O2k-Chamber assembly has to be tested, described in detail as an O2k-SOP:
The O2k-chamber test provides quality control at an instrumental level beyond the O2 sensor test:
  1. O2k-Chamber not properly assembled or broken.
    1. OroboPOS-Holder not properly positioned.
    2. O2k-Chamber Holder not properly positioned; V- and O-rings not properly mounted (V-ring\30-35-4.5 mm, O-ring\Viton\18x2 mm).
  2. Volume-Calibration Ring not properly positioned by chamber volume calibration.
  3. O-ring\Viton\12x1 mm injured and must be replaced on the stopper.
  4. Stopper\black PEEK\conical Shaft\central Port broken conical edge or O-ring not properly applied.
  5. OroboPOS-Seal Tip leaky.
  6. Experimental medium consumes oxygen due to microbial contamination.
  • Next step - when measuring cytochrome c oxidase activity: Autoxidation of ascorbate and TMPD causes a chemical background oxygen flux. DatLab provides real-time correction for instrumental and chemical background.

Trouble shooting

If specifications given in the Instrumental O2 background are not obtained

Check components for locating the problem.
  1. Check the stirring bars for any contamination.
  2. Check the stoppers for the quality of the O-rings and the conical edges.
  3. If no indications of a defect are observed, disassemble the O2k-chamber.
  4. Check the glass chamber for contamination or for broken edges.
  5. Clean the copper block of the O2k and reassemble the O2k-chamber.
  6. Reassemble and clean the chambers: MiPNet19.03 O2k-cleaning and ISS.
  7. Perform an O2 sensor test and - if successful - an O2k-chamber test, using fresh incubation medium.
  8. If the problem with the instrumental O2 background remains in one chamber, switch stoppers between chambers A and B.
  9. Perform an O2k-chamber test (the sensor test is not necessary).
  10. If the problem with the instrumental O2 background remains in the same chamber, switch glass chambers between the left and right side of the O2k.
  11. Perform an O2 sensor test and - if successful - proceed with the O2k-chamber test.
  12. If the problem with the instrumental O2 background remains in the same chamber, switch sensors between the left and right chamber.

Instrumental oxygen background test for permeabilized muscle fibers

  1. While biopsy sampling and fiber preparation proceed: Perform air calibration in MiR06Cr, then close the chamber to evaluate instrumental background at air saturation (c. 10 min): This is a quality control of the medium, important under field conditions, where medium preservation (sterility) may be less controlled than in the lab.
  2. Elevate oxygen concentration to 450-500 µM with oxygen gas (Syringe\60 ml\Gas-Injection), close and after two to three min perform a stirrer test (using the automatic stirrer test function of DatLab). This is important, since the OroboPOS may have a different response time at elevated oxygen concentration. If the response time increases dramatically, then the sensor may even show a non-linear response to oxygen concentration at high oxygen levels.
  3. Instrumental background: After 20 min, open the chamber and allow O2 to drop to c. 350 µM, close for 20 min, open and drop O2 to c. 250 µM (this should be the lowest experimental O2 concentration).
  4. Increase O2 with H2O2 injection (c. 2 µl) to 400 µM, measure for 15-20 min instrumental background, simulating a re-oxygenation during the experiment.
  5. Increase O2 with H2O2 injection (c. 1 µl) to 450-500 µM, until the fibers are added, for equilibrating the instrument at high O2.
  6. Addition of permeabilized fibers into the O2k-Chamber: » Permeabilized muscle fibers.

Apparent oxygen flux in closed chamber near air saturation with pure medium

The uncorrected slope of the oxygen signal over time in a closed chamber at air-calibrated oxygen concentration is an important control parameter. It reflects the consumption of oxygen by the polarographic oxygen sensor (POS). The theoretical value is calculated by DatLab in the O2 Calibration window (Supplement C in MiPNet06.03 POS-calibration-SOP). The theoretical value at 37 °C (O2 slope uncorrected) is usually between 2 and 3 pmol·s-1·ml-1. The actual values should correspond very closely to the expected slope, i.e. within ± 1 pmol·s-1·ml-1.
  • Values higher than 4-5 pmol·s-1·ml-1 at 37 °C may therefore indicate a biological contamination in the chamber or in the medium.
  • Lower values may indicate:
  1. Air bubbles in the closed chamber: switch on the internal illumination of the O2k and inspect the chamber through the front window. Remove any air bubbles.
  2. A large volume of medium collected in the receptacle of the stopper: siphon off any medium.
  3. A larger chamber volume: check O2k-Chamber volume calibration.
  4. If an O2k-MultiSensor stopper with multiple ports is used, it is particularly important to siphon off excess liquid from top of stopper. Any convection of liquid must be avoided, which otherwise results in an apparently leaky chamber.

Instrumental O2 DLP

Instrumental O2 DL-Protocols (DLP) are used for calibrations and instrumental quality control, typically without experimental sample in the incubation medium. All Instrumental DL-Protocols available can be found in C:\DatLab\DL-Protocols\Instrumental\ (default directory after installation of DatLab).

A general description of DL-Protocols (Instrumental DL-Protocols and SUIT DL-Protocols) is displayed in DL-Protocols.

DatLab-Analysis templates and DatLab Demo Files

2017-05-17 P2-01 Instrumental O2 background TIP2k.jpg

DatLab 7: Instrumental O2 background.xlsx
DatLab 6: O2k-Background.xlsx
DatLab 5: O2k-Background.xlsx

Demo file:
MiPNet14.06_2014-07-24_P4-02_Instr-background.DLD (norm. oxygen)
MiPNet10.04_2014-02-20_P4-02_ O2-calib high-O2.DLD (high oxygen)


  • Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - »Bioblast link«
  • Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - »Bioblast link«
  • Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - »Bioblast link«
  • Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«