User manual

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10.2.5 Some solutions for reducing Dead Volume
Some solutions for reducing this dead volume could be:
1) Keep the dead volume as small as possible by using as small diameter tubing and as short a length
of tubing as possible for the tubing where the dead volume occurs. And whether the dead volume is
a problem also depends on how big the bath volume is.
2) Automatically pre-flush much of the dead volume tubing before switching (as with AutoMate
Scientific’s AutoPrime system).
Whether you need to use the pre-flush system that requires twice as many valves as the standard
system depends on your perfusion requirements. If you are using small ca 60 ml syringe reservoirs
without reservoir heating or with reservoir heating (for example using AutoMate Scientific’s
BubbleStop Syringe Heater) that maybe extends the dead volume from 10 to 15 cm in a standard
system, a pre-flush system would not be particularly advantageous. However, if you are using
larger 250 and 500 ml bottles as reservoirs (perfusing for hours at 2-5 ml/min) and with pre-heating
using a water bath that maybe extends the dead volume from 10 cm to 1 meter in a standard
system, a pre-flush system would be advantageous in this situation. Obviously, its up to you to
decide. WinLTP just tries to provide the tools.
3) Use clear Teflon tubing where the dead volume occurs.
Clear Teflon tubing is less permeable to oxygen diffusion of oxygen across the tubing wall than
polyethylene or tygon tubing, but we don’t know if this decreased permeability makes any real-world
difference in a standard system and if it would negate needing a pre-flush system.
4) Possibly re-bubble (re-oxygenate) the perfusion fluid between the reservoir and the chamber. Some
researchers at Bristol have a perfusion line break by dripping into a syringe near the chamber, and
some bubble the contents of this syringe.
5) Whatever you do, we would strongly suggest empirically testing it. After normal ACSF dead volume
less-oxygenated perfusion fluid has ‘sat’ in the tubing a ‘long’ time, try switching from normal ACSF
fully-oxygenated perfusion fluid to the dead volume less-oxygenated perfusion fluid. And then a) at
minimum, see if field EPSP amplitude or slope changes, and (hopefully) b) measure changes in
oxygen and/or changes in pH.
10.2.6 Alternatives to using Pre-Flush for Extracellular Slice Experiments
The necessity of using 95% O
2
/ 5% CO
2
prohibits running a perfusion line directly from solution reservoir
to chamber (as with the Single-Line (Section 10.3) and Double- or Triple-Line/Stepper (Section 10.4)
perfusion systems). In Sections 10.2.4 and 10.2.5 we have discussed using a Pre-Flush or AutoPrime
system. However, using 2 pinch-valves is a costly solution.
In certain situations, a single valve can be used to.keep the tubing between the reservoir and the pinch
valve filled with aerated solution of the correct pH. Note that with these systems, there is still a short
length of tubing between the pinch valve and the manifold that is not aerated.