Fabrication of an autoclavable one-way valve for venting fermentation gases.

Culturing fermentative microorganisms often requires that accumulating carbon dioxide be vented during growth. Preventing the release of such by-products usually causes inhibition of the organism, and this can then lead to a decreased yield of fermentation products such as organic acids and ethanol. It is common to release pressure with a needle placed through a septum covering the fermentation vessel. Unfortunately, this design will only keep the system anaerobic if fermentative gases are being produced rapidly enough to result in a positive displacement of air within the fermentor. Thus, the system may become aerobic as oxygen enters through the vent during the initial and late stages of the fermentation. One-way valves can be used for this purpose, but they are typically quite expensive for routine growth involving numerous culture tubes or flasks. We present a very simple and rapid method to construct an inexpensive, autoclavable one-way valve using a 1-cc syringe and a few miscellaneous items that can be found in most laboratories. First, the plunger is removed from a 1-mL syringe (Figure 1). The rubber nub is pulled off the plunger and cut in half as shown in the diagram, with either scissors or a razor blade. The very tip portion (2a) is discarded, and the remaining half of the rubber nub is reinserted back into the syringe in the same orientation as before. A 4-mm glass bead creates the valve by sitting against the fabricated rubber washer in the syringe. To keep the bead snugly against the opening, a spring is inserted. Small springs may be purchased from most hardware stores, but we have found that those used in disposable pens work excellent. If desired, a small amount of glass wool can be added at this point to prevent contamination from entering through the valve. Finally, the apparatus is completed by reinserting the plunger back into the syringe until only a slight amount of pressure is against the spring. It is sealed into place by poking a needle heated with a Bunsen burner through the syringe and into the plunger at the location shown by the arrows (Figure inset). This will melt the plastic and essentially “weld” the plunger in place. The valve may be made more compact by cutting the top portion of the syringe and plunger off by using wire cutters. Two critical points should be made regarding the construction of these valves. Occasionally, it may be necessary to fasten the fabricated rubber washer (2b) to the syringe if it does not fit tightly. We find that a couple of drops of cyanoacrylate (e.g. Superglue) will secure it to the bottom of the syringe. Also, it is important not to put too much pressure against the spring since it takes very little force to keep the glass bead seated against the rubber seal. Exceeding this will only cause extra pressure to accumulate in the fermentor before the valve is opened. We use these valves by connecting a hypodermic needle to the end, which can then be inserted through a septum on the culture vessel. Very little oxygen was able to get into the closed system as demonstrated by using resazurin in an uninoculated tube of medium. These valves have been shown to stand up well even with rapid and abundant carbon dioxide production with Saccharomyces cerevisiae in a high sugar medium. This one-way valve can also be autoclaved numerous times without any problems.