Mini-Chamber for Regulating Gaseous Environment During Culture

1. The basic unit is constructed from a media filtration unit such as Corning ’s 115 ml filter system (catalog numbers 430944-430947).

2. Tygon tubing (3.2 mm i.d. x 6.4 mm o.d.) with a stopcock fastened to the end is attached to the hose connector.

3.  A 00 size rubber stopper is inserted into the pour spout of the filter system.

4. The membrane in the filter system is punctured in several places to facilitate movement of gases.

5. Culture plates (up to a 60 mm petri dish plate) are gently positioned on top of the membrane. If the plate is large, it is difficult to maneuver the plate into the chamber with fingers.  In this case, a cradle underneath the plate can be constructed with tape to facilitate placement of the dish.

6. The lid of the filter system is taped securely with scotch tape and parafilm is placed around the lid to make the chamber airtight.

7. Gases are injected for 3 min through the pour spout from a hose connected to the gas cylinder while the stopcock was in the open position.

8. After injection of gases is completed, the rubber stopper is fastened to the pour spout and the stopcock is moved to the closed position.

9. To prevent contamination during injection of gases, a filter (0.22 mm) is attached to the gas hose.

<center> <p> <font><img height="140" src="http://www.animal.ufl.edu/hansen/protocols/gaschamber.JPG" width="186" /><br /> Photograph of assembled chamber</font></p> </center>