! When the orange indicator floats and the red indicator is on the bottom of the jar (EtOH between 50% and 60%), the ethanol concentration of the remaining fluid can be brought back between 70% and 80% by the use of three different stock solutions. There is no need to perform additional density measurements. The topping up procedure can be carried out in the most efficient way.
||discard fluid until volume is 50-60%
top up with EtOH 95%
||top up with EtOH 90%
||top up with EtOH 80%
! The indicators will only function accurately when put in ethanol-water solutions without added substances like salts (buffers) or heavy alcohols.
! The addition of methanol (as in methylated spirit) will not alter the specified shifting range provided no other additions such as salts are present in the fluid.
! Lipids migrating from the specimen into the fluid will not seriously affect the specified shifting range of the indicators and therefore will not affect the reliability of the read-out.
! In case of freshly mixed ethanol, dissolved air in the fluid can attach in the form of air-bubbles to the indicators and can cause an incorrect read-out (it will take a few weeks before the bubbles disappear and a proper read-out is possible).
Therefore, it is advised to add the indicators to the fluid al least one week after mixing has taken place (the dissolved air through mixing should have dissipated by then).
! To prevent indicators getting trapped underneath specimens, the indicators can be put into small glass vials or tubes closed with a perforated cap or a piece of cotton/polyester wool and placed (visible from the outside) on the bottom of the specimen jar.
! Although the indicators are extremely stable in ethanol-water solutions, it is recommended that these be checked every 10 years for accuracy (shifting-point).
This can be done by randomly selecting a small amount of indicators and testing them in calibrated ethanol-water solutions which concentrations lie just beyond the specified shifting range.