Clinical MicrobiologyA vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes
Introduction
Common procedures for removal of oxygen from aqueous solutions include boiling, sparging with oxygen-free gas, or exposing the solution to negative pressure. Boiling and sparging are methods of choice for large volumes of liquid. The Hungate technique [1] and its modification by Balch et al. [2] have been standard procedures for cultivating strict anaerobes that require a low reducing potential. By use of these procedures large volumes of media may be prepared and dispensed into culture tubes, bottles, or vials in an anoxic chamber. A procedure is described below in which small amounts of anoxic media may be prepared on the lab bench and dispensed into tubes or vials without use of heat or an anoxic chamber. For example, an aerobic solution in a sealed culture tube may be rendered anoxic in 90 s.
Section snippets
Equipment
The gassing station shown in Fig. 1, is a modification of that published by Balch et al. [2], the basic components being an oxygen scrubber for gasses, a 3-way valve, a pressure gauge for measuring positive and negative pressures, a vacuum pump, a moisture trap, and a gas dispensing attachment. Fig. 2 shows the construction of the gassing attachment through which gasses are added to and withdrawn from sealed tubes or vials.
Preparation of an anoxic solution in a culture tube
This procedure involves use of alternate cycles of evacuating the atmosphere over a solution in a sealed culture tube, followed by vortexing the solution under negative pressure, followed by addition of anoxic gas to the atmosphere. The following steps are recommended in preparation of anoxic solutions (When negative or positive gas pressures are being used in glass containers, safety goggles should be worn with use of a safety shield).
- 3.1
Turn the 3-way valve on the gassing station to the “GAS”
Preparation of a sterile, anoxic solution of a heat-labile compound in a culture tube
In this procedure the atmosphere in a sealed non-sterile culture tube is evacuated prior to sterilization, leaving a negative pressure in the sterilized tube. An anoxic solution of the heat-labile compound is then injected through a sterile membrane filter into the sterile tube. The following steps are recommended:
- 4.1
Add a drop of distilled water to a culture tube.
- 4.2
Add a Balch stopper to the tube and crimp it in place.
- 4.3
Insert the needle of the gassing attachment through the Balch stopper and
Vacuum-vortex procedure for liquids in serum vials
Small serum vials may be used instead of culture tubes, procedure 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 (Fig. 3), the bottom of the vial contacting the vortex cup or platform. Small vials pose no safety risk, but the tip of the syringe needle inside the vial must be maintained at a maximum distance from the liquid-surface; the liquid volume should not exceed one third of the total volume of the vial to avoid contamination of the membrane filter and gas line with liquid and to ensure efficient
Procedure for preparation of sterile, anoxic culture medium in culture tubes
In this procedure a 50 ml volume of anoxic culture medium is prepared and then dispensed into culture tubes.
- 6.1
Add 50 ml of a basal culture medium to each 158 ml serum vial, omitting reducing agents such as sulfide or cysteine, or volatile substrates such as methanol, and crimp a Balch stopper in place. Resazurin is an ideal O/R indicator to use in anoxic culture media for anaerobes requiring a low reducing potential of −330 mv or lower [1].
- 6.2
Expose the medium in the vial to three vacuum-vortex
Discussion
The Hungate procedure [1] introduced a new concept in the cultivation of strict non-sporeforming anaerobes by providing a prereduced medium with an O/R potential below −330 mv, and a technique for maintaining this reducing environment during aseptic cultivation of anaerobes. In preparation of medium oxygen was removed from the liquid medium by boiling under a stream of anoxic gas from a gassing probe. Resazurin was added as an O/R indicator. The reduced medium was transferred at near boiling
Acknowledgments
Supported by a grant from the Department of Energy DE-FG02-02ER 15296
We thank Gargi Kulkarni for providing medium prepared by the conventional procedure.
References (4)
Commentary on the Hungate technique for culture of anaerobic bacteria
Am J Clin Nutr
(1972)- et al.
Methanogens: reevaluation of a unique biological group
Microbiol Rev
(1979)
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