Oroboros Chamber

Oroboros Chamber

Description

Oroboros Chamber: Duran® glass polished, with experimental volumes (V) of 2.0 mL (classic volume cV, 16 mm inner diameter) or 0.5 mL (small volume sV, sV-Module, 12 mm inner diameter). The optical properties of Duran® allow application of fluorometric sensors (Duran® optical properties).

Oroboros Chamber models: cV and sV

Classic-volume Chamber cV: 16 mm inner diameter, Duran® glass polished, with standard operation volume V of 2.0 mL.

Product ID: 23100-01 Product details and purchase information

Small-volume Chamber sV: 12 mm inner diameter, Duran® glass polished, with standard operation volume V of 0.5 mL.

Product ID: 33100-01 Product details and purchase information

Paradigm shift in HRR from mimimum to optimum chamber volume

Small experimental chamber volumes of 20-50 µL generate problems rather than providing resolution with small amounts of tissue samples, low numbers of cultured cells or few isolated mitochondria. The surface to volume ratio increases with decreasing chamber volume, thus various boundary effects entail larger errors at smaller volume, in particular oxygen diffusion. While the rate of oxygen depletion per unit amount of sample increases linearly with decreasing chamber volume, side effects may increase to a larger degree. Then, sensitivity is lost with decreasing chamber size. We recommend a minimum chamber volume of 0.5 mL of the sV-Module or 2.0 mL for the classic volume of the Oroboros Chamber supporting all Oroboros Modules.

1. Minimization of chamber volume represents a past paradigm, aming at high rates of oxygen consumption per volume. The advantage appears to be obvious, whereas the drawbacks are frequently overlooked — see below.

2. Advancements of electronics, data acquisition and analysis, polarographic oxygen sensor specifications and chamber design made possible a superior approach, allowing for respirometric measurements at high dilution, as reviewed by Gnaiger E (2001). In specifically designed mitochondrial respiration media, respiration is stable at high dilution, multiple substrate/inhibitor titrations are possible without oxygen depletion, and a low-oxygen regime may be chosen to prevent elevation of oxidative stress at air-level oxygen saturation. In contrast, micro-chambers are characterized by a high surface-to-volume ratio which hinders optimum stirring, increases unfavourable surface effects and oxygen-backdiffusion, and poses problems with accurate titrations and dilution effects of the sample. These potential - and mostly hidden - artefacts are avoided in high-resolution respirometry (HRR), using glass chambers, titanium stoppers, and avoiding teflon-coated stirrers or perspex (yielding high back-diffusion of oxygen).

Assume you have 0.1 mg mitochondrial protein for a respirometric assay. Approach (1) would lead you to search for a 100 µL volume respirometer, to maintain a classical 1 mg/mL protein concentration. In contrast, HRR allows for dilution of mitochondria to 0.02 mg/mL protein concentration. Dilution of 0.1 mg mitochondrial protein in a 2-mL chamber yields an optimum concentration for multiple substrate/inhibitor titrations and kinetic measurements. The high-resolution approach of the Oroboros offers the unique advantages of a versatile and ready-to-use system for studies in mitochondrial physiology and pathology.

Compare small 0.5 mL and classic 2.0 mL Oroboros Chambers

experimental volume V	sV 0.5 mL	cV 2.0 mL
Oroboros Manual	MiPNet24.14 Oroboros sV-Module Manual	MiPNet22.11 O2k-FluoRespirometer manual
Oroboros Chamber	Chamber sV - exclusively for HRR: • O₂ consumption	Chamber cV - Glass chamber (cV) and PVDF stirrer of the Oroboros for • O₂ consumption, fluorespirometry • H₂O₂, • mt-membrane potential • ATP production • Ca²⁺, TPP⁺-Module • mt-membrane potential, pH-Module, NO, Q-Module, NADH-Module, PB-Module
inner diameter of glass cylinder	12 mm	16 mm
outer diameter of glass cylinder	24 mm	24 mm
thickness of glass wall	6 mm	4 mm
stirrer bar	Stirrer-Bar sV\white PVDF\11.5x6.2 mm	Stirrer-Bar cV\white PVDF\15x6 mm Stirrer-Bar\black PVDF\15×6 mm
Stopper	Stopper sV\black PEEK\conical Shaft\central Port	Stopper cV\black PEEK\conical Shaft\central Port
O-ring	O-ring sV\Viton\9.5x1 mm	O-ring\Viton\12.5x1 mm
volume-calibration ring A/B	Volume-Calibration Ring sV	Volume-Calibration Ring cV
stopper capillary diameter	1.00 mm	1.30 mm
stopper capillary crossectional area	0.79 mm²	1.33 mm²
stopper capillary length	50.64 mm	48.86 mm
stopper capillary volume	39.8 µL	64.9 µL
stopper capillary dead volume V_stc including small meniscus	0.04 mL	0.07 mL
volume V´ for volume calibration	0.54 mL	2.07 mL
Stopper-Spacer		Stopper-Spacer
Chamber-Holder	Chamber-Holder sV	Chamber-Holder cV
POS-Holder	POS-Holder sV	POS-Holder cV

Oxygen calibration and experimental volume

During air calibration (Oroboros quality control 1), in the open chamber, the raw oxygen signal is independent of the volume of the aqueous phase in the chamber and should be between 1 and 3 μA (volt [V] in DatLab 7.4 and older) from sea level up to 1000 m altitude (p_b 90 - 101 kPa) and temperature between 25 °C and 37 °C in both 0.5-mL and 2-mL chambers. However, the instrumental oxygen background flux (Oroboros quality control 2) is volume-dependent in the closed chamber and higher values of J°₁ are obtained in the 0.5-mL compared with the 2-mL chamber. The standard oxygen calibration procedure is described in detail here: MiPNet06.03 POS-calibration-SOP.

Titrations into the chambers

In application of various SUIT protocols available in DatLab, the standard Oroboros microsyringes are used for titrations. There is no apparent retention of chemicals injected through the stopper. Despite the slight recess of the needles in the sV-stopper (Stopper sV) depending on the particular microsyringe, the entire injected volume is mixed into the effective 0.5 mL experimental volume. In contrast to the stoppers of the 2-mL chamber (Stopper sV), the needles fit the stopper capillary diameter of 1.00 mm rather tightly, such that upon removal of the syringe the residual dead volume is mixed into the experimental chamber volume, since the small space between needle and capillary ensures laminar downwards flow (in contrast to turbulent flow) with a jet effect directed into the mixed experimental compartment. Our experimental evidence does not show any delay of mixing after removal of the microsyringe. Accurate kinetic titrations, however, require application of the TIP2k, with accurately placed needles extending slightly into the mixed volume without touching the stirrer.

Cleaning the Oroboros chamber

There are different kinds of contamination that can accumulate in the experimental chamber of the Oroboros and may cause problems. All of them have to be treated in different ways:

Biological contamination: The ideal counter agent is 70 % ethanol with 30 % water (NOT 100 % ethanol). If this does not help, the biological contamination may be embedded in a protein contamination and the glass chamber should be disassembled and cleaned as described below.
Protein contamination and other macroscopic deposits: After long-term use, a whitish deposit can form on the glass walls of the chamber. Additionally, small glass splinters (difficult to see) may stick to such deposits. A sign of this is a jumping or stuck stirrer bar. In this case, remove the glass chamber from the Oroboros. Immerse the Oroboros Chamber (without the stirrer and without stopper) into a glass beaker with 10 mol/L hydrochloric acid (HCl) under a hood for at least an overnight period. The glass beaker should be covered to avoid evaporation of the HCl solution.
Contamination by hydrophobic inhibitors: The ideal counter agent for this case is pure ethanol.
Carry-over of inhibitors or uncouplers: It is recommended to wash the chambers with cells, thom or imt. Specific inhibitors bind to the mitochondria and are thus removed with high affinity to the mitochondria. Excess amounts of experimental samples of cells, tissue homogenate are isolated mitochondria can be used after storage at -20 °C for washing the chambers. If available, living cells are preferred. Without disassembling the Oroboros, fill the chambers to the rim of the Chamber-Holder with the suspension, insert the stoppers loosely (sliding down without engaging the O-ring at the glass chamber), and stirr for at least 30 min.

DL-Protocols (DLP) should be used for cleaning the chambers, stirrers and stoppers before and after experimental use. A list of Instrumental DL-Protocols for cleaning is displayed in the 'Protocols' menu ('Run DL-Protocol / Set O2 limit' window). See [Instrumental:_Browse_DL-Protocols_and_templates Browse Instrumental DL-Protocols for cleaning]

» Further details: Oroboros cleaning and ISS

Inhibitors may be introduced accidentally, one example being 70 % ethanol used in hospital settings containing antiseptics. Such inhibitors may accumulate in plastic parts and inhibit subsequent experiments.

» Further details: Discussion.

In specific cases (e.g. working with bacteria), the chamber can be sterilized with 3% formaldehyde for 10 min before the experiment, followed by three washing steps with ddH₂O.(https://wiki.oroboros.at/index.php/Steininger_2002_J_Antimicrob_Chemother)

Oroboros Chamber assembly - possible problems

A properly assembled Oroboros Chamber can remain in the Oroboros for extended periods of time.

It is recommended to disassemble, clean and reassemble the chamber at least every 1 - 2 years, to avoid the chamber from getting stuck, to clean the glass properly, and to detect any possible damages.

After assembly, medium is leaking out of the chamber

Siphon off medium and disassemble the chamber.
Check for apparent damages of the glass chamber, the POS-Seal Tip and O-rings on Chamber-Holders and stoppers. If any damage is visible, replace the part.
Reassemble the chamber and proceed with an QC1: Oxygen sensor test.

Oxygen leak of the chamber (strongly negative intercept in the instrumental background test)
Unstable O₂ signal
Unexpected high or low O₂ signal

The possible contribution of chamber assembly is checked by removing the OroboPOS from the chamber and observing, if the signal changes to the expected values.
If the Oroboros signal responds as expected, reassemble the Oroboros Chamber and proceed with an QC1: Oxygen sensor test.

Leaky chamber due to damaged POS-Holder

» [[POS-Holder#A_damaged_POS-Holder_may_cause_liquid_leak_from_the_Oroboros_chamber |POS-Holder]

Oroboros chamber disassembly

After siphoning off any medium from the chamber, remove the SmartPOS or OroboPOS with OroboPOS-Connector and Chamber-Holder from the Oroboros.
Insert your finger (with gloves on) into the chamber and then gently pull the glass chamber up.
Problem: Glass chamber stuck in the Oroboros, and the Oroboros Chamber cannot be removed. It remains stuck even after washing with water.

This may be caused by spilled medium acting as an adhesive between glass chamber and copper block. Wash with plenty of warm water and try to remove the chamber while the Oroboros is switched on and heated up to 37 °C. Then GENTLY screw in the POS-Holder to push the glass chamber upwards (as performed during the Oroboros chamber assembly).
If this does not work, increase the temperature to 40 °C (or slightly higher) for washing and continue with removing attempts.