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Difference between revisions of "Talk:O2k-chamber"

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Previous Product ID 23100-21


== Different fluxes in left and right chamber ==
:Version 01
 
=== How to check the O2k-chamber for gas leakage if you do not have time to run an instrumental O<sub>2</sub> background test?===
'''Question:''' In our experiments we get consistently higher fluxes in chamber A as compared to chamber B.
:Version 02
 
=== How to test the airtightness of the O2k-chamber if you do not have time to run an instrumental O<sub>2</sub> background test? ===
'''Answer:''' To exclude instrumental causes please follow the procedure described in the section
:::* When a chamber is newly assembled or reassembled due to previous medium leakage, but there is not enough time to start an instrumental O<sub>2</sub> background test to check whether the chamber is tight, run the following test overnight:
'''Calibration and quality control of the OroboPOS (O2k-SOP)''' in [[MiPNet06.03 POS-Calibration-SOP]].
::::# Add MiR05 to the O2k-chamber and close it by gently pushing the stopper downwards. Siphon off excess medium from the top of the stopper using the [[ISS]]. Check that any air bubbles are removed from the chamber.
::::# Decrease the oxygen concentration down to ~ 50 μM (using [[Dithionite]] or N<sub>2</sub> injection); see: [[Setting the oxygen concentration]].
::::# Let the O2k recording overnight with the [[closed chamber|chamber closed]] and illumination switched off.
::::# The oxygen concentration should decrease over time due to the O<sub>2</sub> consumed by [[OroboPOS]] as it is shown in the figure. In this case, the chamber is assembled properly.
::::# If the oxygen concentration increases over time and/or air bubbles are observed in the chamber, a reassemble of the chamber is needed. For details: [[MiPNet22.11 O2k-FluoRespirometer manual]]- 5.2 O2k-chamber assembly
[[File:Leaking_chamber_2020-02-03 P2-03.png|1500px]]


Possible instrumental and protocol related causes for fluxes differing between chambers:
=== A story about gas bubbles appearing in the closed chamber ===
[[User:Gnaiger Erich|Gnaiger Erich]] ([[User talk:Gnaiger Erich|talk]]) 16:27, 19 March 2020 (CET)
::::* You are an O2k user who assembles the O2k-chamber carefully following the SOP (manual & video). Usually this works very well - but not always ..
::::# You are stressed because you have a persistent problem with a leak after chamber assembly, loosing medium from the chamber (observed by medium dropping out from the OroboPOS holder and connector, or a gas bubble appearing slowly in the closed chamber).
::::# ‘Leaky chamber’, therefore, has to be defined: Here it is not just the oxygen leak (as observed in an instrumental O2-background test), it is actual medium leak – two entirely different processes, although medium leak causes simultaneously an O2 leak, but not the other way around.
::::# You re-assemble again and again, until the chamber appears to be tight. You have no clue, what you did differently. And you are not 100 % sure, if the chamber is now really tight. (This is meant by “When the leakage is so little that cannot be noticed”).
::::# You could test this easily with an instrumental O<sub>2</sub>-background test - SOP! You can do this even in a short amount of time, if you have other tasks to complete meanwhile. But unfortunately it is already evening, and the lab will be closed. (This is meant by “in a short amount of time”). Well, then you may quickly reduce the oxygen level .. the overnight ‘leaky chamber test’ (we will find a better title).
::::# You may use dithionite quickly. But probably you performed your tests of loosing medium after chamber assembly with H<sub>2</sub>O, to avoid any sticky medium clogging your sensor connector when medium leaks from the chamber. Do you now titrate dithionite solution into the closed chamber containing H<sub>2</sub>O? YOU WOULD BE IN TROUBLE! This is well explained in our SOPs, but it is a good idea to clarify, that you should use MiR05 (or a highly concentrated phosphate buffer). (Marco: did you actually use MiR05 – I guess absolutely YES).
::::# You come back to the lab in the morning – BINGO. The chamber is tight. Does this replace the instrumental O<sub>2</sub>-background test?


* wrong O2 calibration in one chamber, see [[MiPNet06.03 POS-Calibration-SOP]]
* wrong chamber volume calibration: The total amount of oxygen in the chamber with the smaller volume will be smaller. Therefore, the O2 concentration will drop faster resulting in a higher flux, see [[MiPNet19.18A O2k-Start]].
* wrong background parameters: wrong background parameters will cause a O2 concentration dependent offset to the flux, the value of the offset will be independent of the absolute value of the flux. E.g. a constant offset of 10 pmol /ml s at ca 100 µM O2 concentration and 20 pmol/ml s at 300 µM O2 concentration could be caused by wrong background parameters, but  a difference of always e.g. 20% at very different absolute values of the flux can not be caused by wrong background parameters. In any case, it is advisable to check the background parameters (perform an instrumental O2 background experiment). See [[MiPNet14.06 InstrumentalBackground]].
* hydrophobic inhibitors, see [[MiPNet19.03 O2k-cleaning and ISS]]
* biological contamination, see [[Biological contamination]]
Hardware defects: While hardware defects (sensors, electronics,..) can obviously have many negative effects (noise signal, slow response, no signal,..) it is difficult to see how a real hardware defect can cause a systematic error in flux calculation once it was possible to do a correct calibration of the O2 sensor (at air saturation and at zero oxygen). [[User:Fasching Mario|Fasching Mario]] 11:04, 6 November 2014 (CET)
Previous Product ID 23100-21


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Latest revision as of 10:24, 9 November 2020

Previous Product ID 23100-21

Version 01

How to check the O2k-chamber for gas leakage if you do not have time to run an instrumental O2 background test?

Version 02

How to test the airtightness of the O2k-chamber if you do not have time to run an instrumental O2 background test?

  • When a chamber is newly assembled or reassembled due to previous medium leakage, but there is not enough time to start an instrumental O2 background test to check whether the chamber is tight, run the following test overnight:
  1. Add MiR05 to the O2k-chamber and close it by gently pushing the stopper downwards. Siphon off excess medium from the top of the stopper using the ISS. Check that any air bubbles are removed from the chamber.
  2. Decrease the oxygen concentration down to ~ 50 μM (using Dithionite or N2 injection); see: Setting the oxygen concentration.
  3. Let the O2k recording overnight with the chamber closed and illumination switched off.
  4. The oxygen concentration should decrease over time due to the O2 consumed by OroboPOS as it is shown in the figure. In this case, the chamber is assembled properly.
  5. If the oxygen concentration increases over time and/or air bubbles are observed in the chamber, a reassemble of the chamber is needed. For details: MiPNet22.11 O2k-FluoRespirometer manual- 5.2 O2k-chamber assembly

Leaking chamber 2020-02-03 P2-03.png

A story about gas bubbles appearing in the closed chamber

Gnaiger Erich (talk) 16:27, 19 March 2020 (CET)
  • You are an O2k user who assembles the O2k-chamber carefully following the SOP (manual & video). Usually this works very well - but not always ..
  1. You are stressed because you have a persistent problem with a leak after chamber assembly, loosing medium from the chamber (observed by medium dropping out from the OroboPOS holder and connector, or a gas bubble appearing slowly in the closed chamber).
  2. ‘Leaky chamber’, therefore, has to be defined: Here it is not just the oxygen leak (as observed in an instrumental O2-background test), it is actual medium leak – two entirely different processes, although medium leak causes simultaneously an O2 leak, but not the other way around.
  3. You re-assemble again and again, until the chamber appears to be tight. You have no clue, what you did differently. And you are not 100 % sure, if the chamber is now really tight. (This is meant by “When the leakage is so little that cannot be noticed”).
  4. You could test this easily with an instrumental O2-background test - SOP! You can do this even in a short amount of time, if you have other tasks to complete meanwhile. But unfortunately it is already evening, and the lab will be closed. (This is meant by “in a short amount of time”). Well, then you may quickly reduce the oxygen level .. the overnight ‘leaky chamber test’ (we will find a better title).
  5. You may use dithionite quickly. But probably you performed your tests of loosing medium after chamber assembly with H2O, to avoid any sticky medium clogging your sensor connector when medium leaks from the chamber. Do you now titrate dithionite solution into the closed chamber containing H2O? YOU WOULD BE IN TROUBLE! This is well explained in our SOPs, but it is a good idea to clarify, that you should use MiR05 (or a highly concentrated phosphate buffer). (Marco: did you actually use MiR05 – I guess absolutely YES).
  6. You come back to the lab in the morning – BINGO. The chamber is tight. Does this replace the instrumental O2-background test?


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