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Canonical Carol Canon O

Canon O: Assembly of Canonical Carol Canons on the Oroboros Ecosystem
Supplement to: Β»X-mass CarolΒ«

Canon O1: A Canonical O2k-Manual for high-resolution respirometry: why the living cells died and dyed

Communicated by Gnaiger Erich 2020-06-25
Q1: The question is, who you are and how you are, when entering the Canoncal O2k-Manual in the jukebox of the Canonical Carol.
Pop up to Β»Canon P: Are you Practical&Technical-Theoretical&Canonical with a bite of PTC-mood?

Is DatLab part of the O2k?

Q2: Is the DatLab software of the high-resolution respirometer HRR (which is installed on my PC [that is connected to my O2k]) part of the O2k?
In the O2k-Catalogue, DatLab is listed as part of the O2k, which was shipped in a Hardware box H, that contained the hardware O2k H(O2k), and a separate USB-flashDrive, which is the Software box D. After assembly of the O2k, H is unfortunately empty, since there is no instruction on how to copy the O2k before taking it out of the box β€” this would be a most economical approach to the Power-O2k. This is different with the Software box D, since there is an instruction to copy DatLab rather than taking it out of the box D, such that DatLab stays inside the box D containing DatLab, represented as D(atLab).
Before opening box H, it is instructive to clarify the canonical representation of H, to address question Q2, if DatLab is part of the O2k.
  1. YES, then H(O2k[D{atlab}])
  2. NO, then H(O2k, D[atlab])
  3. Before deciding too quickly, consider the important higher-level question in this nested network: Is H part of the O2k? β€” O2k(H[O2k{D<atLab>}])
  4. Postpone the decision and think of your living-dying cells on the bench - now we are a bit practical even in Theoretical&Canonical TC-mode.

Where is O2k-Manual F?

Q3: Is the instruction 'Add cells' in the O2k-Manual F(Add cells) part of the O2k-Hardware or Software, and where is the F.. manual?
We should add the cells into the O2k for HRR measurements. If I proceed with the perception that DatLab is part of the O2k-system, then the part of the O2k-Manual that instructs me to add the cells into the O2k should explicitly (canonically) define, into which part the cells are to be addded, else a TC-user may try to add the cells into the USB-flashDrive D(atlab). It goes without saying that the proper part where to add the sample are the O2k-Chambers 2K (for the German 'Kammer' for chamber, and there are NK = 2 x K chambers used in the O2k). The O2k-Manual says, that the O2k should be taken out of the Hardware-box H, DatLab should be copied from the Software-box D(atlab) to be installed on my PC, and β€” BINGO! Here is the answer to Q1(Q2) in Canon O: DatLab is not part of my O2k, the O2k-Catalogue should get this right for all TC-users (neither PT-realists nor TT-nerds would care).
The O2k-Manual shows in a video, how two O2k-Chambers are taken out of the Assembly box A(3K) β†’ A(1K) + 2K, pushing down from system A to entity K, leaving one spare O2k-Chamber in box A(1K). This is followed by popping up 2K to the O2k-System S. In my TC-mode there is a Theoretical voice that says: Think of normalization, count the cells first. The Technical side transfers a subsample of cells to the cell counter. And here comes the Canonical again.
Do the instructions of the cell counter include the unit 'counting-unit' [x] in their system of units? Does the cell counter manual introduce properly the quantity 'count' with symbol NX, meaning 'number of unit-entities of unit-type X, expressed in the unit 'counting-unit' with symbol [x], where in the cell counter X = ce? If I do not re-write the cell counter manual at this stage, since the living cells are dying on the bench, I should nevertheless update the 'Add cells' instructions in the O2k-Manual.
Where is the O2k-Manual F (pronounced as 'the F.. manual')? The instruction says: H(O2k, D[adtlab, F{DatLab, Assembly, Operation, Disassembly}]) ↓ push down to find the USB-FlashDrive D(adtlab, F[DatLab, Assembly, Operation, Disassembly]) in box H and copy the Manual F(Assembly, Operation, DatLab). But since the living cells are dying on the bench, there is no time to read the F.. manual. Just look for 'Add cells'. ↓ push down to find 'Add cells'. There are several options for pushing, with failures and problems:
  1. Manual F(DatLab, Assembly, Operation[Add cells], Disassembly) β€” failed, take an alternative thread, mining and grinding on the same level without pushing:
  2. Manual F(DatLab[Add cells], Assembly, Operation) β€” failed, give up grinding and pop up
  3. D(adtlab, F[DatLab, Assembly, Operation, Disassembly]) β€” ?? β€” switch from system D to system PC, where you have installed DatLab and DatLab is running
  4. PC(DatLab[DL-Protocols{F<How to Find instructions in F, Add cells>}]) β€” Found !!
The great success to find the instruction 'Add cells' is a great testimony to the fact, that the O2k-Manual is the most simple, logical, practical, easy-to-use, userfriendly instrumental manual of type F in the world. There exists no simpler, logical, easy-to-use structure of F:
  1. HHH: push down taking all you need out of the box or copy the software.
  2. O2k↔PC(DatLab): pop up (set up) the instrumental system with your PC and DatLab connected to the O2k.
  3. 2K: pop up (insert) the instrumental chambers into the O2k
  4. H: get started with High-resolution respirometry: switch on the O2k, connect DatLab, select a DL-Protocol for 'cells', define the experimental temperature of your experimental system, add mitochondrial respiration medium.
  5. DatLab(DL-Protocols[F{How to Find instructions in F, Add cells}]): In DatLab, the selected DL-Protocols designed for 'cells' as a sample type, include within the DL-Protocol the F-Manual, which provides instructions how to Find help, and where you find the instruction on 'Add cells'.
There is no way to make this structure of the nested system simpler, more logical, easy-to-use. So how do you find this structure? You may find it to be useful, but in the first place you do not find the structure at all. When trying to Find the instruction 'Add cells', we are not trying to find information on how to Find, but without any choice we have to use the structure of F to Find 'Add cells'. We cannot best use a structure of a nested system, if we are not pushing down into the instructions to find instructions, without forgetting to pop up back to the current task. If you have time to play pushing and popping, even complex structures will precipitate. This is how we learn using a new software when we like to play with it, and the more PTC- one adds to a PT-approach, the more sophisticated and error-free applications are possible. Any sophistication of TC-mode does not help in using or designing a nested structure, if the practical side is missing. Therefore, diversity of operation- and cognition-modes is the key to innovation of design and success of application, based on gender balance, cultural diversity, Extroductory Canons in the team β€” if only they can communicate.

The big improvement of the O2k-Manual that makes it worse

Of course, there is lots of scope for simplifying the structure of O2k-Manual F and making it even more logical and canonical. Oroboros, please stack all Operation instructions into DatLab, such that failures and problems 1-3 above will not happen, even without thinking about the structure of F. Change from:
  1. D(adtlab[DL-Protocols{F<How to Find instructions in F, Add cells> F[DatLab, Assembly, Operation, Disassembly]), to let all external F disappear altogether, devoured by DatLab itself, to obtain the linear thread,
  2. D(adtlab[DL-Protocols{F<How to Find instructions in F, Assembly, Operations<How to operate DatLab, How to operate the O2kΒ»Add cellsΒ«>, Disassembly}])
No more frustration in Finding everything on the O2k in DatLab and everything on DatLab in DatLab. With its self-referencing, retrograde structure this linear thread gets fuzzy on recursive nodes, and canonical perfection can never be achieved in a perfectly linear structure: If all instructions on the O2k and DatLab are nested in DatLab, where do you find the instruction how to find DatLab? The instruction on how to open the box must not be popped into the box. F on F nested in the box implies infinite regress without bottoming out, and all you need is falling in love with the Extroduction that contemplates about the system in the box and out of the box.

When the living cells have died

With too much obsession by the Extroduction, the living cells have died on the bench and their nuclei dyed in the cell counter. At this stage, exclusively continued TC-mode of the Unrealist provides escape. I have a living cell preparation on the bench, but all are dying, without application of any mild detergent to permeabilize the plasma membranes enabling the dying of all nuclei in a cell viability test. Being aware of this theoretical question, I prepared a parallel subsample as a negative control for the cell viability test: experimental sample 'ce' = living cells; negative control sample 'pce' = experimentally permeabilized cells using digitonin. In the TC-approach all living cells were dying, such that sample 'ce' is not intact any more when it should be popped into the 2K of the O2k. The cell count Nce of the experimental sample is identical to the cell count Npce of the negative control. The cell viability index IVce = NvceΒ·Nce-1 of the experimental group and the cell viability index IVpce = NvceΒ·Npce-1 of the negative control group are identical, both are exactly zero, since subscript 'vce' indicates 'viable cells'. This is fine for the pce-control cells. But an appropriate term is required for the ce-experimental cells. These living cells ce are not intact any longer β€” they are certainly not intact cells. But these living cells are not viable cells at a viability = 0. But this experimental sample ce should have a viability >0.95 in a more Practical and less Canonical approach. This is where Mitochondrial physiology bottoms out BEC 2020.1.

Canon O2: The experimental chamber in the instrumental O2k-Chamber

Communicated by Gnaiger Erich (2020-06-04) last update 2020-06-19
Glass chamber.jpg
The O2k-chamber of the Oroboros O2k is taken as an example, but the concept is entirely general.

The volume of the experimental chamber

The instrumental chambers of the O2k consist of Duran glass with an inner diameter of either 16 mm of the '2 mL chamber', or a smaller diameter for the '0.5 mL chamber' of the O2k-sV-Module. 2 mL and 0.5 mL are the 'operation volumes', which do not include the volume of the glass wall. The operation volumes, therefore, are not the instrumental volumes but the experimental volumes, including the liquid volume and sample enclosed in the chamber with a volume-calibrated stopper inserted, to obtain a closed system. It is useful, therefore, to distinguish the experimental chamber as the closed or open system operated under experimental conditions of temperature and pressure, with a particular stirrer inserted, but without including the volume of the stirrer in the volume V of the experimental system. For the experimental chamber it may be entirely irrelevant, if the wall is composed of Duran or quartz glass. But an instrumental chamber of Duran glass is a different product than an instrumental chamber of quartz glass. You may have a number of instrumental spare chambers, which are stored in the accessory box. An experimental chamber, however, is by definition inserted into the O2k, containing a defined medium (usually aqueous, but it might be gaseous) and operated under experimental conditions of temperature, pressure, stirring speed and optionally containing a sample. Thus the experimental chamber is defined as the thermodynamic system including the sample if a sample is inserted, whereas the instrumental chamber is a particular product described in the catalogue. The 2 mL and 0.5 mL volumes V refer to the experimental chamber volumes of the instrumental chambers.
  • The O2k-Chamber is the instrumental chamber of the O2k with product ID 23100-01. The instrumental chamber may be a physical object inserted in the O2k or placed in the accessory box or storage room, or it may even be a non-physical concept displayed on the website. The instrumental chamber is effectively the chamber wall (product ID 23100-01 does not include the stirrer bar).
  • The O2k-chamber is the experimental chamber in the O2k operated under experimental conditions. The experimental chamber does not include the chamber wall, but the system boundary is the area separating the wall from the contents β€” an area of zero thickness and hence the boundary has zero volume. Yet the experimental chamber is a physical object, characterized by the experimental contents of the chamber. The stirrer bar is inserted into the instrumental chamber, but the volume of the stirrer bar is excluded from the experimental chamber volume.
Whereas the instrumental chamber lasts over time until it breaks (the O2k does not have disposable chambers), the experimental chamber exists for the duration of an experiment. The instrumental chamber is the container (made of glass), the experimental chamber is defined by the experimental contents and experimental conditions.

The volume of the study object, the sample, the medium, and the experimental chamber

In a sample consisting of a pure substance S, the sample volume VS equals the experimental chamber volume V β€” the sample is the system:
Sample S for a pure substance B: VB = V
Take a sample s of freeze-dried yeast cells as countable objects or entities X. The pure sample of yeast cells is added into the experimental chamber containing mitochondrial respiration medium β€” the sample is not the system, the sample is not the object.
Take a sample s of a tissue homogenate of volume Vthom and inject this volume into the experimental chamber containing mitochondrial respiration medium. In this mixture the sample does not contain countable objects but units of sample volume, occupying a volume Vthom that is smaller than V β€” the sample is not the system, the sample is the object:
Sample s for a mixture: Vs < V
For cells suspended at high dilution, the volume Vce is small to the extent that it can be ignored relative to the volume VMiR of the mitochondrial respiration medium β€” the medium is the system:
Cells at high dilution: VMiR ~ V

The experimental chamber is not an ideally closed system

The polarographic oxygen sensor is neither part of the instrumental O2k-Chamber nor experimental O2k-chamber. Oxygen diffuses across the system boundary from the experimental medium to the cathode of the sensor, where 4 electrons reduce O2 to H2O, and the corresponding electron flow is measured as a current Iel which is the primary signal of the polarographic oxygen sensor (polarized at 800 mV to push the reduction of oxygen). In an ideally closed system (= experimental chamber), oxygen would be prevented from escaping to the oxygen sensor. Therefore, this external flow of oxygen (a negative flow has the direction out of the system) is accounted for as a component of the instrumental background oxygen flux JΒ°O2. The experimental background oxygen flux includes additionally the chemical background oxygen flux due to processes of autooxidation, if any unstable components are added to the incubation medium (correction for chemical O2 background).

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