14-CO-2 trapping assembly for monitoring anaerobic microbial cultures.

This note describes a "4CO2 trapping assembly for anaerobic microbial cultures which is easily operated, cleaned, and assembled, and requires no specialized glassware. The assembly in operation has the advantages of providing a means of: purging an anaerobic microbial culture with a gas containing C02; monitoring of '4CO2 produced while the assembly is operating; trapping 14Cvolatilized non-1"CO2 materials prior to the CO2 traps; and reproducing gas flow rates from one experiment to another. The assembly in operation is shown in Fig. 1. High-purity, water-pumped 5% C02-95% N2 (a) is passed through a two-stage regulator (b; Matheson Co., Inc., Joliet, Ill.) set at a pressure of 6 lb per square inch on the second-stage gauge. The gas then flows through a column of reduced copper filings (d) heated by a combustion chamber (c; P. P. Williams, Appl. Microbiol. 14:1045, 1966). The gas is then metered with a type 2M Nupro valve (e; Nuclear Products Co., Cleveland, Ohio) at a flow rate of 28 ml/min, as measured with a soap-bubble meter (t; Beckman Instrument Co., Fullerton, Calif.). From the valve (e) via the tubing (f), the gas flows into a 20-ml syringe barrel (i) containing 10 ml of culture medium, and then into a cartridge of anhydrous magnesium perchlorate (q; Frederick Smith Chemical Co., Columbus, Ohio) where water vapors and organic constituents can be removed. The gas then passes through 5 ft (1.52 m) of Teflon tubing (r) coiled in a container filled with dry ice and methanol, where additional organic materials can be removed from the gas. The gas is then bulbled through two 14CO2 traps (u) for entrapment of 14CO2 and CO2. Each of these traps contains 15.0 ml of monoethanolamine and ethylene glycol monomethyl ether (1:2; Fisher Scientific Co., Fairlawn, N.J.). When sampling these traps, a 1.0-ml sample is removed through n and 1 and dispensed in a scintillation vial containing 10.0 ml of scintillator solution and 5.0 ml of ethylene glycol monomethyl ether. One milliliter of trapping solution is then added to the 14CO2 trap through the Teflon connector (n) and a twoway valve (1) to maintain a volume of 15.0 ml. Each of these traps has the capacity to trap approximately 40 mmoles ofCO2. The culture vessel (i) test solution is maintained at 38 C by means of 0.25-inch (0.64-cm) Tygon tubing (h) wrapped around the vessel, which is filled with water being circulated via a water bath and an immersible pump (Fisher Scientific Co., Chicago, Ill.). At the termination of an experiment, the culture vessel medium is released of CO2 by the addition of 2.0 ml of 80% (w/v) citric acid solution through the connector (n) and the valve (k). If foaming of the test material is a problem, 0.02 to 0.1 ml of caprylic alcohol is added prior to acidification. The scintillator solution used contains 0.5% (w/v) PPO (2,5-diphenyl-oxazole) and 0.03% (w/v) POPOP [p-bis-2-(5-phenyloxazole) Pilot Chemicals, Inc., Watertown, Mass.] in toluene (Fisher Scientific Co., Fairlawn, N. J.). The solution is counted at 14 C in a liquid scintillation spectrometer. Disintegrations per minute are calculated by the channels ratio method for quench correction. The syringe barrels, Multifits, used in this assembly were purchased from Becton, Dickinson and Co., Rutherford, N.J. Thetwoand three-way valves with Teflon plugs and 18-gauge stainless steel needles, Teflon tubing, and connectors were purchased from Analabs Incorporated, Hamden, Conn. The neoprene stoppers were obtained from A. H. Thomas Co., Philadelphia, Pa. Laboratory results with this assembly are shown in Table 1. Na2"C03 is efficiently trapped as 14CO2. Trifluralin (2,6-dinitro-di-n-propyl-114C-a,a,a-trifluoro-p-toluidine), which is rather volatile, does not occur in the 14CO2 traps after 18 hr of incubation. The starch experiment shows the ability of an obligate anerobic protozoan, Isotricha sp., to function in this assembly with the production of 14CO2 from radioactive starch.