Microbiology - Using the Anaerobic Chamber


Jeff Lawrence

Bringing Items into the Chamber

1. Verify that the oxygen level in the camber is below 100 ppm and that the hydrogen level is above 1%. Also verify that the N2 and the N2 /CO2 /H2 tanks are open and not empty.

2. Verify that the inner interchange door is sealed, and that the gas, catalyst, and vacuum systems are set to "Auto".

3. Open the outer interchange door and place items inside. Close and latch door.

4. Press the "Start" button. The following sequence should occur:
- Interchange chamber is evacuated
- Interchange chamber fills with N2
- Interchange chamber is evacuated
- Interchange chamber fills with N2
- Interchange chamber is evacuated
- Interchange chamber draw gas from the main chamber to equilibrate
5. Wait until the proper lights indicate that the chamber is anaerobic, and that it is safe to open the inner interchange door.

6. Open the inner interchange door by
(a) lifting the handle,
(b) rotating the handle counterclockwise, and
(c) pushing the door towards the back of the chamber.
7. Pull interchange tray into the main chamber and unload items. Return tray.

8. Reseal the inner interchange door. DO NOT work with the interchange compartment open to the chamber.

Bringing Items out of the Chamber

9. Verify that the interchange chamber is anaerobic. If it is aerobic, check to see if any supplies need to be moved into the chamber. Then, press the "Start" button. The interchange will cycle as described above. If the chamber is already anaerobic, there is no need to cycle the gases.

11. Open the inner interchange door and pull interchange tray into the main chamber.

12. Load items on the tray and return the tray to the interchange chamber.

13. Reseal the inner interchange door.

14. Open the outer interchange door and unload items.

15. Reseal the outer interchange door. DO NOT leave the interchange chamber open to the atmosphere.

Earlier Comments:

Anaerobic-Techniques
Charlotte Grabau

INSTRUCTIONS FOR USING ANAEROBIC CHAMBER AND GASSING STATION

Note: Please don't attempt to use the station or chamber without being shown by someone knowledgeable. These notes are not intended to be complete instructions, but merely helpful reminders. Incorrect use leads to ruined experiments, both yours and those of others using the equipment, as well as possible damage to the equipment.

To make anaerobic liquid culture tubes

Boil desired medium under a stream of N2. Seal flask tighly and bring into chamber.
Dispense (5ml) into anaerobic tubes.
Stopper with black rubber stoppers.
Bring out of chamber.
Cap with aluminium seals.
Autoclave.

All additions to tubes are made with sterile syringes at the gassing station.

To use gassing station

Turn oven on. Let warm up about an hour.
Exchange air in syringe with nitrogen.
Briefly flame top of bottle. Use syringe to withdraw solution.
Flame top of bottle. Dispense desired volume into tube (same syringe can be used for several tubes).
Innoculate with 0.1 ml cells (can be aerobic culture).

To use anaerobic chamber

Again, please have someone explain the operation and function of the chamber before attempting use. Be very gentle with the gloves and plastic front of chamber - they are very easy to puncture. Also, creases and rough handling can lead to O2 leaks.
1. To bring something into the chamber.

Make sure inner door is closed.
Open outer door, place object in outer chamber.
Close outer door.
Press start button. This starts the cycle - three evacuations (first and second are filled with N2, third with mixture of CO2, N2, H2).
When anaerobic light turns on, open inner door and bring objects inside.
Always close inner door when finished.

2. To bring something out of the chamber:

Make sure both doors are closed.
Press start button (to exchanged atmosphere in out chamber).
When anaerobic light turns on, open inner door and put plates in out chamber.
CLOSE INNER DOOR!
Open outer door and remove objects. Close outer door.

3. Upkeep of chamber:

READ INSTRUCTIONS in the manual which is kept in a drawer near the chamber.

The catalyst and dessicant wafers should be changed periodically - every 2 weeks, depending on use. To change, place in drying oven for about an hour. Let cool and bring into the chamber. Place dessicant wafer below catalyst. Bring out old wafers to be baked when needed.

CaCl2 is used to absorb moisture in the chamber. When bringing in a large number of plates, check and change CaCl2 every day.

Hydrogen levels inside the chamber have to be high in order to keep O2 levels low. To maintain a high hydrogen concentration, the atmosphere of the chamber should be exchanged periodically. To do so, READ MANUAL for instructions.



Last Update: Thursday, 19-Jun-2014 11:54:36 PDT
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Eric Kofoid eckofoid at ucdavis.edu