Gas distribution manifold

The system provides a facility to blow anoxic gases aseptically through labware, media and containers. If necessary, a Widdel flask may be attached. The system provides six regulated outputs, delivers two different gasses and features a flow-meter. We have successfully developed a protocol for anaerobic media preparation that does not require a Widdel flask.

See the videos below for a walkthrough of a model two Buddy Engineers designed and built for a client at the University of Warwick. Or click here for some photos we took immediately prior to shipping.

Here is latest model featuring a lightweight aluminum framework, ready for shipping to the client:

manifold_crate

Pressure vessel

One of our Buddy Engineers describes a pressure vessel manufactured for a client growing Methanococcus maripaludis.

Pressure gauge

The pressure gauge features a diaphragm to protect the sensitive electronics from harmful gasses.

Preparation of Anaerobic Culture Media

This is a brief overview of our method for making media to grow anaerobic bacteria. The essential first step is removing oxygen from the media. This is an efficient way to do it without a lot of fuss, and can be done without using a Widdel flask. Our gassing station and anaerobic chamber are already set up.

Place the media in a 70 C water bath, the heat helps displace the oxygen. The hot water bath in this arrangement is on the counter, directly under the gas panel. The custom gas panel has 2 inputs and 6 outputs.

Next take a 0.2 micrometer filter, on the minimal chance that there are any bacteria in the lines of the gas delivery system. The end of the line in our set up is a derived from a tuberculin syringe; these are inexpensive and easy to use. Most filters will set on the end of them quite easily.

Before handling sterile or autoclaved parts, it’s a good idea to wash your gloved hands with ethanol . Next, take a tube that has been autoclaved and connect it to the line through the filter. Then place the other end of the tube into the prepared media that has been in the hot water bath, wrap foil around the juncture, and turn on the gas. Allow the gas to bubble through the media while it sits in the water bath. You can process more than one flask of media at a time because of the design of the gas manifold.

The color of this media is purple, in about 20 minutes the color will change to transparent, indicating an anoxic state. Once again it is a good idea to sterilize your gloved hands with ethanol before removing the tubes and placing autoclaved caps on the media. Turn off the gas, unwrap the caps. Place the cap over the flask after removing the foil. Then gently pull out the tube and tighten the cap. You may notice that some of the liquid media has traveled up into the tube and saturated the filter. It sometimes happens that when the filter gets wet at the end of the bubbling process it will not let any more gas through. However, you can see that it did not harm the anoxic treatment because the media is already clear.

Occasionally the media is pink at the end of the bubbling step. We then put some Cystein in the media to further reduce it. At the end of the day or the next day when we come into the lab, we find it perfectly clear. Do not forget the heat step, you really need the heat to remove the oxygen and without heat Cystein cannot reduce it. After this process, you can return the media to room temperature and you can use it at any time.