Ethylene Production by Autoclaved Rubber Injection Caps Used in Biological Systems

While determining the ethylene production of plant tissue incubated in 30-ml flasks stoppered with rubber injection caps, we found high and erratic concentrations of the gas. It became obvious that the source of ethylene was not the tissue but the caps which had been autoclaved. To investigate this more fully, caps which had not been used previously were wrapped in aluminium foil and autoclaved at 15 pounds pressure for 20 min. They were then allowed to cool, unwrapped, and immediately fitted to empty flasks which had not been autoclaved; Caps which had not been autoclaved were fitted to flasks at the same time. The vessels were allowed to stand for 16 hr, and the contents of the flasks were assayed for ethylene by gas chromatography (4). Control flasks were found to contain 0.01 ,u/liter ethylene, which was about the same as for normal room air. The ethylene produced by autoclaved caps varied with the cap type and with the batch of caps. Type 11 produced 0.08 ,ul/liter ethylene (10 replicates, two experiments) while one batch of type 2 produced 1.5 ,ul/liter (8 replicates, two experiments) and another batch produced 0.4 Ml/liter (9 replicates, two experiments). Most of the ethylene was evolved in the first 12 hr, but it was still being produced slowly after 24 hr, so that values higher than those given could occur after longer periods of time. Caps were routinely autoclaved for 20 min at 15 pounds pressure, but ethylene production varied with the time of autoclaving and the severity of the conditions. Both types of caps were made from compounds based on natural rubber, and one might have expected a variety of gases to be produced and to increase in proportional amounts in increasingly severe conditions. In fact, ethane, ethylene, propane, and propylene were produced, but, while increases in ethane and ethylene were parallel, increases in propane and propylene were proportionately small. These data are important because it is common practice to autoclave these caps for use in work where sterile conditions are required and because the concentrations of ethylene which accumulated in the flasks are sufficient to cause a variety of effects in plant tissues. For example, 0.02 to 1 Asl/liter can cause morphogenic effects in leaves and stems, fruit ripening, and leaf abscission