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FAQs - Environmental Control

Tissue Culture Incubators keep cells viable and stress free because three parameters are controlled; Temperature , CO2 Atmosphere and Humidity . Solent Scientific's CO2 Enrichment Accessory simply mimics this technology.

Temperature Control

Cells are typically cultured in a 35mm petri dish, 6 Well Plate or Incubation Flask. Such dishes are placed in the small inner chamber. When located on the microscope's stage inside the full enclosure incubator the first parameter, temperature is brought under control at a physiologically significant temperature (generally 37 Degrees).

How quickly will the incubator be at temperature?

The temperature sensor indicates that temperature is being controlled at 37 °C within 45 minutes of switching the heater on. For best results however the entire microscope and incubator must be brought into thermal equilibrium. This takes at least 3 hours but an overnight equilibration period is preferred.

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How stable is the temperature control?

Measurements taken at 10-second intervals over 24+ Hrs indicate that temperature is controlled to ± 0.2 °C (99% confidence limits).

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Will accessing the interior of my incubation chamber affect the temperature stability?

Every acrylic enclosure has several openings each of which has a specific function:

• The four doors in the front of the enclosure are used to exchange specimens on the stage or to make day to day adjustments to the microscope such as selection of the fluorescence cube for the next experiment. The minimum number of these doors should be opened for the shortest possible time to minimise heat loss from the enclosure.

• The Services Port allows cables and tubing to pass into the enclosure. This is considered to be a maintenance function and not a day to day operation of the microscope. Generally the Services Port will be open for a significant period of time which will disturb the thermal equilibrium inside the enclosure. As such the system should be allowed to return to equilibrium. In general terms for every 5 minutes that the Services Port is open you should leave the system for 1.5 hours to re-equilibrate. The minimum Recovery Period should be 1 hour and the maximum will be overnight (16 hours).

• Tilting of the illumination pillar is not recommended, the recovery time needs to be increased to 3 hours re-equilibration for every 5 minutes opening. The minimum Recovery Period in this case increases to 2 hours and the maximum will be overnight.

• Service Access Doors may have been specified in your chamber. They are to be used exactly as described, for Service Access only. By this we mean to allow easier access to set up a Perfusion Pump and Flow Apparatus. These Doors should not be used whilst undertaking temperature controlled experiments.

If they are in a vertical wall of the enclosure they should be treated in the same way as the Services Port above.

If these Service Access Doors are in the top horizontal face of the enclosure the recovery time needs to be increased to 3 hours re-equilibration for every 5 minutes opening. The minimum Recovery Period in this case increases to 2 hours and the maximum will be overnight.

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Must I position the temperature sensor in a specific location?

Yes. The position for the temperature sensor is shown in the installation manual. Any change from this position will affect the accuracy of the temperature control at the specimen.

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Will focus be stable?

It is important that focus remains stable for the duration of a time-lapse experiment, particularly when confocal microscopy is involved. Many laboratories turn off the heating at nights. This causes the microscope frame to cool and contract by as much as 40 microns. A full enclosure incubation chamber places the microscope into thermal equilibrium thereby stabilising focus.

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Can I monitor and record the temperature inside the chamber?

Yes. Temperature monitoring PC software is available. It records the signal from the platinum foil temperature sensor against time. Connection is made to the PC via the serial port.

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Will there be any condensation under the lid of my culture vessel?

No. Condensation is only possible when the lid of the culture vessel is cooler than the growth medium. This does not happen inside the incubation chamber.

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Will the incubator make my room hot?

No. The power requirement of the incubator, when at temperature, is less that a regular light bulb.

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CO2 Atmosphere Control

Pre-mixed 5% CO2 is fed into the inner chamber from a gas cylinder (tank) at a slow flow rate. This maintains a blanket of the gas over the surface of the growth medium that in turn regulates the pH of the growth medium.

What gas do I use with the CO2 Enrichment Accessory?

Generally 5% CO2 or whatever gas mixture was used in your tissue culture incubator.

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Is the entire chamber under CO2 & humidity control?

No only the small inner chamber or the interior of a multi well plate or incubation flask.

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What is the rate of gas consumption when using the CO2 Enrichment Accessory?

Typically 2 ml/min.

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I want a display of CO2 concentration. Is this possible?

This has not been included because we place complete faith in the Laboratory Gas supplier getting the mixture correct. If in doubt you should simply buy the more expensive Certified Gas Mixture.

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Does the CO2 Enrichment Accessory compromise the working distance of my objectives?

No. The base of the inner chamber is identical to the adaptor plate used with motorised X-Y stages.

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Humidity Control

The function of humidity control is to minimise evaporation from the growth medium. Humidity control to a specific RH value is not necessary. In an ideal world the gas will be at the saturated vapour pressure of water at 37 degrees. The gas heater/humidifier is installed inside the incubation chamber. The gas bubbles through the warm water to increase its humidity before it enters the inner chamber. This minimises evaporation of water from the growth medium.

Will the growth medium dry out during long term time lapse experiments?

This does not happen when the CO2 Enrichment Accessory is used.

Here a user reports "we found evaporation rates to be almost identical to that experienced in our tissue culture incubator"

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