A critical assessment of the occurrence and extend of oxygen contamination during anaerobic incubations utilizing commercially available vials

For rate determinations of anaerobic metabolism it is essential to maintain strictly anoxic conditions throughout the experiment. However, even if oxygen contamination can be avoided while preparing the incubation containers, it is still possible that the incubation containers themselves contaminate the samples by oxygen diffusing from or through their plastic or rubber components. In this study, we investigated the sources and extent of oxygen contamination during anoxic incubations, and present solutions to minimize oxygen contamination. In particular, we investigated oxygen contamination in Labco® Exetainers, glass vials with a butyl rubber septum in the screw cap, which are frequently used in microbiological experiments. Our results show that significant oxygen contamination occurred at different stages during the incubation. Contamination occurred when Exetainers were either filled or incubated for more than 16 h under oxic atmosphere, but also under an oxygen-free atmosphere due to diffusion of oxygen out of the butyl rubber septum. Therefore, to avoid oxygen contamination during incubations, we suggest (1) filling and incubating the incubation containers under anoxic atmosphere (glove bag) and (2) deoxygenating all elastomers in sample processing and incubation equipment. If initial oxygen contamination cannot be avoided, introduction of an anoxic headspace might help extract oxygen from the incubated sample and present a buffer against oxygen diffusing out of the septum. We modeled the amount of oxygen diffusing out of butyl rubber septa under different conditions, and results fitted well with the observed oxygen contamination. Thus, the model can be used to predict oxygen contamination under varying conditions and for differently sized septa.

► Oxygen leaking out of rubber seals contaminates anoxic samples during incubations.
► The degree of oxygen contamination was modeled.
► The model was verified with experiments.
► Solutions are provided to diminish oxygen contamination from rubber seals.