Changes in ethanol concentration during incubation in multiwell tissue culture trays

Ethanol can have direct effects in tissue culture and is often used as a solvent. Analysis of these effects will require a precise knowledge of the concentration over time in the particular system employed. We measured the disappearance rate of ethanol from multiwell culture trays (open system) containing single or multiple concentrations of ethanol over a 48-hr time period. The ethanol concentration was also measured at 72 hr in stoppered Erlenmeyer flasks (closed system). In multiwell culture trays, 1 and 0.3% ethanol (V/V) evaporated with a t1/2 of 6 to 12 hr. Media containing no ethanol but adjacent to wells with 1% ethanol accumulated ethanol with a peak of 0.2% at 12 hr. The evaporation of 0.3% ethanol was slower from wells adjacent to those containing 1% ethanol. At 72 hr, stoppered Erlenmeyer flasks, which originally contained 1% ethanol, still had a concentration of 0.85%. Since both evaporation and transfer can occur in an open system, it is necessary to specify precise conditions or measure concentrations in such systems. Alternatively, a closed system in which the concentration of ethanol is maintained can be employed.