The most critical aspect of microbial growth in microtiter plates is the closure system of the individual wells, which has to prevent cross-contamination (even during vigorous shaking), permit the exchange of headspace air, and limit evaporation.
Equal physical conditions in all wells is a further requirement: the wells in the corners should have exactly the same characteristics as the wells in the middle of the microtiter plate. This is especially important in the area of medium optimization and mutant screening where productivity improvements as low as 5% should be detectable.
Gas permeable membrane filters (sealing tapes) are available from many suppliers, and are well-suited for qualitative work, but have a number of distinct disadvantages for quantitative work. First, during vigorous shaking, droplets may cover smaller or larger parts of the membrane, leading to random well-to-well variations in evaporation and supply of oxygen. Second, the partial dissolving of potentially toxic glue material may harm growth.
An additional drawback of plastic membranes is their relatively low diffusion coefficient for oxygen and high diffusing coefficient for water. The result is substantial water loss by evaporation while the oxygen demand of the culture is often not met.
A solution that overcomes these problems with sealing tapes has been developed by Enzyscreen (), is manufactured by Kuhner (www.kuhner.com), and distributed in the U.S. by Biopro International (www.biopro.com). The solution consists of a sandwich cover with three layers: a silicone layer with 96 small holes in the middle of each well, a layer of cotton wool, and a stainless steel lid with 96 holes (Figures 1 and 2). The sandwich cover and microtiter plate are clamped together tightly in order to prevent spillage of the culture fluid during high G-force orbital shaking and to ensure that exchange of headspace air occurs solely through the center holes.
In this system, the rate of exchange of headspace air and evaporation is controlled by the diameter of the holes in the silicone layer; holes of 0.5 mm result in water evaporation rates of 10 µL per well per day at 50% humidity and 30C.
A thin stainless steel foil with 200-µm holes inserted between the silicone layer and the cotton wool layer results in even lower evaporation rates (4 µL per well per day) and is advisable for 100-µL cultures in low-well plates. Such evaporation rates correspond to 2% of the culture volume per day, which is acceptable for most purposes. Therefore, no air humidification is required, which avoids the nuisance of fungal wall growth, often seen in humidified incubators.
Since the diffusion coefficients of oxygen and water in air are practically equal (0.21 cm2/s at 20C), it can be readily calculated that an evaporation rate of 10 µl per day at a relative humidity of 50% correlates with an exchange rate of headspace air of 450 µl (containing 3.6 µmol O2) per minute, provided a mechanical diffusion barrier such as cotton wool is used. This guarantees a sufficient oxygen concentration (above 18%) in the headspace even at oxygen consumption rates in the culture as high as 40 mmol/L/h. Air supply rates in this range correlate well with the general rule of thumb for lab-scale stirred tank bioreactors: one vessel volume of air per minute.
A sufficiently high and universal exchange rate of headspace air is a prerequisite, but not a guarantee, that the cells growing in the wells are supplied with enough oxygen. Gas-liquid transfer is another limiting factor.