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Evaporation from Cell Culture Plates

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Evaporation from Cell Culture Plates No. 2 Evaporation from Cell Culture Plates When cells are cultured in Nunc Lab uses Evaporation Culture plates with loose-fitting lids, Nunc Plate index area/well Medium vol. Medium depth the medium will always lose cm/cm2 cm2 well/ml mm water to the plate sur round - Petri Dish 35 1.2 9 3 3.4 ings by evaporation, i.e. by Petri Dish 60 0.8 21 5 2.4 water vapour escape. Petri Dish 100 0.5 57 12.5 2.2 This may cause culture Petri Dish 140 0.3 146 35 2.4 problems since the medium Square Dish 235 0.2 520 135 2.6 components, especially the Multidish 4 rectang. 0.6 16 5 3.1 s a l t s , m a y r e a c h c o n c e n t r a - Multidish 8 rectang. 0.6 8.5 3 3.5 Multidish 4 SonicSeal 3.4 1.5 1 6.7 tions which are harmful Multidish 4* 2.1 2 1 5.0 to the cells. Multidish 6* 0.7 9.5 3 3.2 The evaporation is due to the Multidish 12* 1.0 3.5 2 5.7 fact that the air just above the Multidish 24* 0.9 2 1 5.0 surface of the medium is Multidish 48 0.8 1 0.5 5.0 always saturated with water MicroWell 96F 0.7 0.33 0.2 6.0 vapour, whereas the surround - *Plates having a reservoir for additional liquid. ings under realistic circum - Table 1 stan ces will never be com- Evaporation indices and other figures for Nunc cell culture plates with loose-fitting lids. The indices, which are the circumferences of the plates divided by the respective pletely saturated. Thus, water surface areas of medium, can be regarded as the relative speeds by which the water molecules will constantly losses become »critical« provided that the opening-slit widths and the depths of escape along a concentration medium are equal for all the plates. Thus, to obtain actual, relative figures, the indices should be multiplied by the respective actual slit widths and divided by the actual gradient from the surface of medium depths. See text for further explanation. the medium to the surround- Slit Area ings, by diffusion. Further, the time it takes before the The rate of evaporation may be water loss becomes critical is propor - In the following text, the assumed to be proportional to the tional to the volume of medium in the area of the slit between the lid and plate. If the depth of medium is con - various parameters the plate edge separating the plate stant, the volume of medium is in turn influencing evaporation lumen from the surroundings. Hence, pro portional to the surface area of the are discussed. if the slit width is constant, the speed medium in the plate. of water loss is directly proportional Therefore, the various Nunc cell to the plate's total edge length, i.e. its culture plates with loose-fitting lids circumference. can be characterized as more or less prone to critical water loss by eva po ra- given a ringshaped rib above each well, more exposed to the surround ings. tion according to their specific circum - as any narrowing in the vapour path In MicroWell Plates, the evaporation fe rence/medium surface area ratio, from the medium to the surroundings from the four corner wells has been which may be denoted »evaporation will add to the evaporation delay. shown to account for fully 15% of the index« (Table 1). Fig. 1 shows the results obtained from a total water loss regardless of the kind comparison of the evaporation from of lid used. In comparative studies, this In principle, the larger the index is for a MicroWell 96F Plates with new lids ver- figure was also found for corresponding product, the more prone it is to critical sus old lids, i.e. lids with edge rib and »low-evaporation« plates of another water loss by evaporation. rings versus lids without edge rib and brand. Due to this principle, the tendency rings. should be to use greater depths of As seen, water loss has been reduced Surrounding Humidity medium (i.e. larger volumes of medium by approx. 50% due to the edge rib and To avoid significant evaporaton from per square cm2), the larger the ratios are. rings. culture plates with loose-fitting lids, the Also, a critical water loss from evapora- plates are placed in an incubator with t i o n c a n b e p o s t p o n e d b y a d d i n g l i q u i d Edge Effect humidity control aimed at 100% relative to the reservoir between the wells, if In the multiwell plates, where all the humidity. However, the highest present (see Table 1). wells are not positioned identically obtainable humidity in practice would Because of the relatively high evapora- in relation to the edge of the plate, normally be about 95%. A small water tion index for MicroWell Plates, the i.e. Multidish 6, Multidish 24, and vapour gradient is therefore still MicroWell Plate Lid has been equipped MicroWell Plates, a larger water loss is operating causing a water loss which with an edge-rib for narrowing the slit seen in the peripheral wells than in the may become critical during long term width and thus diminishing the total central ones. This is espe ci ally so culture. One reason for the ina bi lity to slit area. For the same reason it has been in the corner wells because they are keep the humidity at 100% is that the Fig. 1 Evaporation curves for MicroWell Plates incubated at 37°C with 200 µl growth medium per well. Closed circles (red curve): Incubation at 95% relative humidity, lids with edge rib and rings. This curve represents the most realistic evaporation from a Nunc MicroWell Plate. Open circles: Incubation at 95% relative humidity, lids without edge rib and rings (out of production). Triangles: Incubation at 80% relative humidity, lids with edge rib and rings. Each of these three curves represents the average evaporation from four plates, which were removed at intervals, weighed, and returned to the incubator. Squares: Incubation at 95% relative humidity, lids with edge rib and rings. As opposed to the method described above, these data points represent the average evaporation from separate pairs of plates which were removed at intervals from an PERCENT LOSS BY WATER EVAPORATION incubated series, weighed, and discarded. A regression line is fi tted to these data points. The axis-intercept represents the evaporation that occurs between removal and weighing of the plates. The dotted line is a parallel displacement of the regression line through the zero point representing the (theoretical) evaporation curve when plates are left undisturbed in the incubator. See text for discussion. DAYS OF INCUBATION 2 incubator is normally opened frequently indicated by the levelling off tendency more water at 37°C than at 20°C, some during incubation. of the evaporation curves for plates that of the water vapor will be trapped by Fig. 1 shows the results from a compari- are returned to the incubator (see Fig. condensation. This is observed by drop son between evaporation from Micro- 1): the longer the interval is between formation on the inside of the lids soon Well Plates incubated at 95% and at the measurements, the less steep the after removal of the plates from the 80% relative humidity. As seen, evapo- evaporation curve is. This can be incubator. ration is almost four times higher at explained by the occurence of a rela- Obviously, plates should not be outside 80% humidity than at 95%. tively large additional evaporation the incubator for longer than absolutely A critical factor affecting evaporation every time the plates have been out of necessary. seems to be the frequency with which the incubator. Therefore, the number of plates are removed from the incuba- outside inspec tions should be kept at a Air Circulation tor for microscopic examination of the minimum. If the plates are exposed to »draught«, cells. the evaporation will be more rapid than The effect of this was investigated by Temperature if only pure diffusion forces are operat- monitoring the evaporaton (by weigh- Animal cell cultures are normally ing. ing) from plates which were removed at i n c u b a t e d a t 3 7 ° C , a t w h i c h t e m p e r a t u r e There is always some draught even intervals from the incubator and com- the pressure of saturated water vapour inside a closed incubator due to its paring the results with those obtained is about 2.5 times higher than at 20°C air circulating system, but when the from plates which had been incubated (room temperature). In addition, the incubator is opened, or when the plates undisturbed for the same length of surrounding humidity is far lower are moved outside the incubator, a sig- time. outside the incubator than inside. nificant draught may be experienced. As shown in Fig. 1, evaporation is Therefore, the evaporation gradient is Therefore, the incubator should only be almost halved if the plates are left greatly increased when the plates are opened when absolutely necessary, and undisturbed in the incubator. removed from the incubator. plates should be moved slowly and only Thus, it seems that the measured However, the evaporation will decrease taken the shortest possible distance evaporation is to a large extent due to with the falling temperature, and from the incubator.
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