Technical Documents
Nunc Tech Support Index
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Evaporation from Tissue
Culture Plates
When cells are cultured in plates
with loose-fitting lids, the medium will always lose water to the plate surroundings by
evaporation, i.e. by water vapor escape.
This may cause culture problems since the medium components, especially the
salts, may reach concentrations which are harmful to the cells.
The evaporation is due to the fact that the air just above the surface of the
medium is always saturated with water vapor, whereas the surroundings under realistic
circumstances will never be completely saturated. Thus, water molecules will constantly
escape along a concentration gradient from the surface of the medium to the surroundings,
by diffusion.
In the following text, the various parameters influencing evaporation are
discussed. |
Slit Area
The speed of evaporation may be assumed to be proportional to the area of the slit between
the lid and the plate edge separating the plate lumen from the surroundings. Hence, if the
slit width is constant, the speed of water loss is directly proportional to the
plate’s total edge length, i.e. its circumference.
Further, the time it takes before the water loss gets critical is proportional
to the volume of medium in the plate. If the depth of medium is constant, the volume of
medium is in turn proportional to the surface area of the medium in the plate.
Therefore, the various Nunc cell culture plates with loose-fitting lids can be
characterized as more or less prone to critical water loss by evaporation according to
their specific circumference/medium surface area ratio, which may be denoted »evaporation
index« (Table 1). In principle, the larger the index is for a product, the more prone it
is to critical water loss by evaporation.
Due to this principle, the tendency should be to use greater depths of medium
(i.e. larger volumes of medium per square cm2), the larger the ratios are.
Also, as seen from the index figures in Table 1, critical water losses through
evaporation can be postponed by adding medium (or water) to the spaces between the wells
in Multidishes and MicroWell™ Plates (F).
Because of the relatively high evaporation index for MicroWell Plates, the MicroWell Plate
Lid has been equipped with an edge-rib for narrowing the slit width and thus diminishing
the total slit area. For the same reason it has been given a ring-shaped rib above each
well, as any narrowing in the vapor path from the medium to the surroundings will add to
the evaporation delay.
Fig. 1 shows the results obtained from a comparison of the evaporation from
MicroWell 96F Plates with new lids versus old lids, i.e. lids with edge rib and rings
versus lids without edge rib and rings. As seen, water loss has been reduced by approx.
50% due to the edge rib and rings.
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|
| Nunc Plate |
Evaporation
index
cm/cm2 |
Culture
area/well cm2 |
Nunc Lab uses
 |
| Medium vol./well ml |
Medium depth
mm |
|
Petri Dish 35
Petri Dish 60
Petri Dish 100
Petri Dish 140
Square Dish 235
Multidish 4
Multidish 4*
Multidish 24
Multidish 24*
Multidish 6
Multidish 6*
MicroWell 96F;U
MicroWell 96F* |
1.2
0.8
0.5
0.3
0.2
2.1
1.0
0.9
0.5
0.7
0.5
1.0
0.7 |
9
21
57
146
520
2
2
2
2
10
10
1/3
1/3 |
3
5
12.5
35
135
1
1
1.5
1.5
3
3
0.2
0.2 |
3.4
2.4
2.2
2.4
2.6
5.0
5.0
7.5
7.5
3.1
3.1
5.9;7.6
5.9 |
|
Table 1
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
surface areas of medium, can be regarded as the relative speeds by which the water losses
get »critical« provided that the opening-slit widths and the depths of 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 medium depths.
See text for further explanation.
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Edge Effect
In the multi-well plates, where all the wells are not positioned identically in
relation to the edge of the plate, i.e. Multidish 6, Multidish 24, and MicroWell Plates, a
larger water loss is seen in the peripheral wells than in the central ones. This is
especially so in the corner wells because they are more exposed to the surroundings.
In MicroWell Plates, the evaporation from the four corner wells has been shown
to account for fully 15% of the total water loss regardless of the kind of lid used. In
comparative studies, this figure was also found for corresponding »low-evaporation«
plates of another brand.Surrounding Humidity
To avoid significant evaporaton from culture plates with loose-fitting lids, the
plates are placed in an incubator with humidity control aimed at 100% relative humidity.
However, the highest obtainable humidity in practice would normally be about 95%. A small
water vapor gradient is therefore still operating causing a water loss which may become
critical during long term culture. One reason for the inability to keep the humidity at
100% is that the incubator is normally opened frequently during incubation.
Fig. 1 shows the results from a comparison between evaporation from MicroWell
Plates incubated at 95% and at 80% relative humidity. As seen, evaporation is almost four
times higher at 80% humidity than at 95%.
A critical factor affecting evaporation seems to be the frequency with which
plates are removed from the incubator for microscopic examination of the cells.
The effect of this was investigated by monitoring the evaporaton (by weighing)
from plates which were removed at intervals from the incubator and comparing the results
with those obtained from plates which had been incubated undisturbed for the same length
of time.
As shown in Fig. 1, evaporation is almost halved if the plates are left
undisturbed in the incubator.
Thus, it seems that the measured evaporation is to a large extent due to the
plates’ »outside trips«.
This is also indicated by the levelling off tendency of the evaporation
curves for plates that are returned to the incubator (see Fig. 1): the longer the interval
is between the measurements, the less steep the evaporation curve is. This can be
explained by the occurence of a relatively large additional evaporation every time the
plates have been out of the incubator. Therefore, the number of outside
inspections should be kept at a minimum.
Temperature
Animal cell cultures are normally incubated at 37°C, at which temperature the pressure of
saturated water vapor is about 2.5 times higher than at 20°C (room temperature). In
addition, the surrounding humidity is far lower outside the incubator than inside.
Therefore, the evaporation gradient is greatly increased when the plates are removed from
the incubator. However, the evaporation will decrease with the falling
temperature, and because air can contain about 2.5 times more water at 37°C than at
20°C, some of the water vapor will be trapped by condensation. This is observed by drop
formation on the inside of the lids soon after removal of the plates from the
incubator. Obviously, plates should not be outside the incubator for longer
than absolutely necessary.
Air Circulation
If the plates are exposed to »draught«, the evaporation will be more rapid than
if only pure diffusion forces are operating. There is always some draught even
inside a closed incubator due to its air circulating system, but when the incubator is
opened, or when the plates are moved outside the incubator, a significant draught may be
experienced.
Therefore, the incubator should only be opened when absolutely necessary, and
plates should be moved slowly and only taken the shortest possible distance from the
incubator. |
 |
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. These data points represent the average evaporation from separate pairs of
plates which were removed at intervals from an incubated series, weighed, and discarded. A
regression line is fitted 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. |
Summary
Generally, evaporation from cell culture plates only causes problems during long term
culture, e.g. two weeks or more. However, peripheral wells, especially in MicroWell
Plates, may lose »critical« amounts of water long before then‚ but the evaporation
very much depends upon the actual culture conditions.
The following precautions would help to postpone evaporation problems:
- Use an optimally performing humidity control.
- Limit the number of outside inspections.
- Keep the »outdoor« periods as short as possible.
- Take the plates the shortest possible distance from the
incubator.
- Move the plates slowly.
- Do not open the incubator unnecessarily.
- If possible, add water or medium to the spaces between the
wells.
- Use larger volumes of medium in corner wells, or in all
peripheral wells.
- Use greater depths of medium in the plates which have a high
evaporation index (see Table 1).
These precautions can to a large extent be substituted by
keeping the plates in a humidity chamber during incubation and transport. The Nunc Square
Dish equipped with a wet filter paper in the bottom is highly suitable for this purpose.
As illustrated in Fig. 2, the Nunc Square Dish will hold four MicroWell Plates.
Peter Esser
Nunc Laboratories |
 | Fig. 2
Placing pattern of four MicroWell Plates in a Nunc Square Dish. |
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