Sandwich covers (background, working principles)The most critical aspect of microbial and cell growth in microtiter plates is the closure system of the individual wells. The closure system must: •prevent (cross)-contamination even during vigorous shaking•permit a reproducible and defined exchange rate of headspace air•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 centre of the microtiter plate.Inspired by the general principle used for multi-litre bioreactors (an average aerobic microbial culture needs an air supply of one working volume per minute), we designed the standard sandwich covers (for fast growing bacteria) to have a headspace refreshment rate of 1-2 culture volumes per minute. Such a headspace refreshment rates secures oxygen concentrations in the headspace of at least 18% (v/v), even at an oxygen consumption rate by the culture of 40 mmol O2/L/ minute. Such a headspace refreshment rate assures a sufficient oxygen supply, but limits the evaporation to about 2% of the culture volume per day (at an ambient humidity of 50% and 30ºC, see hydrodynamics, oxygen transfer rates): this degree of water loss is acceptable for most projects, and so avoids the need for air humidification in the incubator for most applications. The latter additionally prevents fungal growth in the incubator and so contributes to a safe working environment. To increase the gas-liquid transfer, orbital shaking is our method of choice since it is proven to be effective (see literature), and gives rise to exactly the same hydrodynamic conditions in each well (see hydrodynamics, oxygen transfer rates). For slow growing strains and (animal) cell cultures, we supply sandwich covers with slower headspace exchange rates (in the order of 0.2 culture volumes per minute). This allows cultivation times of 5-14 days without excessive evaporation.We supply sandwich covers with either:•“Spongy” silicone, which is relatively soft, and easy to compress, and so closes off the wells already at relatively low forces. Please note that this kind of silicone is not “ spongy” is the sense that it take up water: it has closed cells, filled with air, and the surface is a solid film, with no pores.•Solid silicone layers, for these applications where the permeability of the silicone has to be absolutely minimized (e.g. for low culture volumes, such as in the covers for 96-round shallow well plates CR1596), but also for certain type of fungi that may tend to grow to inside the spony silicone.
Sandwich covers have 4 or 5 layers (all re-usable and autoclavable): A: a stainless steel lid for rigidity B: a microfiber filter (blue) C: an ePTFE filter (0.3 micron) laminated between two polyester/polyamide fabrics (green) D: a stainless steel foil with pinholes to control the headspace refreshment rate and evaporation (only in covers CR1596 and CR1524a) E: a silicone layer with 96 holes (to hermetically seal the "mini-reactors")