MiPNet14.06 Instrumental O2 background

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MiPNet14.06 Instrumental O2 background

Publications in the MiPMap
O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux.

» Bioblast pdf » Versions

Oroboros (2018-08-07) Mitochondr Physiol Network

Abstract: Fasching M, Gnaiger E (2018) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(07):1-14.

O2k-Protocols SOP: Correction for instrumental background oxygen flux is a standard in high-resolution respirometry, automatically performed by DatLab. Background measurements provide a test of instrument function. In the Oroboros O2k, background corrections are usually within a few % of experimental flux over the entire experimental oxygen range. At minimum activities, however, even the small background effects become highly significant and require compliance to standard operating procedures described in this chapter as part of the MitoFit Quality Control System. This is part 2 of O2k Quality Control.

» O2k Quality Control 1 »MiPNet06.03 POS-calibration-SOP«
» Product: O2k-FluoRespirometer, O2k-Catalogue

Keywords: Instrumental O2 background

O2k-Network Lab: AT_Innsbruck_Oroboros

Labels: MiParea: Respiration, Instruments;methods 

HRR: Oxygraph-2k, O2k-Manual 

DatLab, O2k-SOP 

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O2k-technical support

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


  • 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.

A full list 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«