Suitability of butyl rubber stoppers for closing anaerobic roll culture tubes.

Stringently anaerobic bacteria have been successfully cultured in rubber-stoppered roll tubes. Stoppers of certain types of rubber are so permeable to oxygen that they are unsuitable (Hungate, J. Bacteriol. 86:848, 1963). Butyl rubber is less permeable to gases than are many other types. The permeabilities of butyl stoppers and a common laboratory type to gases concerned in anaerobic culture work have been compared, under conditions comparable to those commonly employed for anaerobic cultures. Both sets of stoppers were obtained from Arthur H. Thomas Co., Philadelphia, Pa. (HR108 #00 recessed stoppers as supplied under catalog no. 8806); similar stoppers of butyl rubber were generously provided especially for this test by the company, and are now stocked as catalog item 8820-B. Twenty-four glass culture tubes (16 by 150 mm; Beilco Glass, Inc., Vineland, N.J.) with narrow necks were freed from air by passing carbon dioxide gas through each tube and then seating the stopper without access of air. Eight additional tubes were filled with H2. Half of the tubes in each group were closed with HR-108 stoppers, the other half with butyl rubber. The carbon dioxide and H2 were freed from oxygen by passing the gases over reduced copper heated to 350 C. The gas in the tubes was initially at atmospheric pressure plus a slight additional pressure due to 1 to 2 ml of gas pushed in as the stopper was inserted. Samples of the gas in tubes 1-4 (for both sets of stoppers) were removed immediately with a 1-ml syringe (the dead space filled with C02), and were analyzed for oxygen by use of a Perkin-Elmer gas chromatograph with thermistor detectors and a silica gel column. None was found, though the method was sensitive to 0.025 ,uliter of oxygen per ml of sample. Tubes 5-8 were incubated in an atmosphere of oxygen at a temperature of 39 C for 10 days. The tubes were removed to room temperature, and the amount of water required to bring the pressure to atmospheric was measured by introducing water from a 5-ml syringe (fitted with a 1x-inch 21-gauge needle) until atmospheric pressure was reached. The volume of water required to bring each tube to atmospheric pressure is recorded in Table 1. In two tubes, little carbon dioxide had diffused out and the pressure was above atmospheric. Tubes 9-12, after incubation in oxygen, were removed and the pressure in each was increased to 5 psi above atmospheric by introducing 02free carbon dioxide through the stopper with a syringe needle connected by a rubber tube to