Hungate technique

Miller TL, MJ Wolin (1974) A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl Microbiol 27 985-987. 

Strain K4 was tested in the absence of oxygen at various concentrations of glycerol (10,20,30,40 g L ).Two serum bottles were used with a modified Hungate technique one ofthe bottles contained resazurin and a reducing agent, namely cysteine-H Cl, and the other did not contain resazurin. The gas was analyzed by GC with TCD. 

In a separate experiment, various concentrations of glycerol were used in the modified Hungate technique (see above). The bacterial strain K4 was also used in a reactor attached to a gas meter in order to evaluate the maximum amount of dihydrogen produced. The same strain was used in a reactor with periodic withdrawal of gas in order to evaluate the effect of pressure. The same strain was also used in a high-pressure autoclave equipped with a pressure gauge and the maximum pressure of dihydrogen produced was monitored. 

Ruminococcus albus strain 7, (1), was grown on cellulose roll tubes according to the Hungate technique (2). Either Avicel (FMC Corporation, Marcus Hook, Pa.) or balled filter paper was used as the cellulose source. Three milliliters of melted cellulose-medium at 45 °C. in 16 X 150 mm. tubes were inoculated with the bacteria under a gas phase of 95% C02 and 5% H2. The tubes were subsequently stoppered and rolled in a tray of ice in order to form a film of agar medium around the... 

The methods required to create and maintain conditions that are suitable for growing anaerobic cultures are more difficult than those required for culturing aerobic cultures. Nonetheless, use of methods such as the serum bottle modification of the Hungate technique [24] is now routine in many laboratories. As discussed in the previous section, the formulation of the medium depends on whether the fiber or fabric is to serve as the sole source of carbon, nitrogen or sulfur. In addition, the formulation of the anaerobic culture medium depends upon which terminal electron acceptor is to be considered in the study. As shown in Table 1.2, the list of terminal electron acceptors includes Mn(IV), nitrate, Fe(III), and sulfate. In addition, bicarbonate (carbon dioxide) serves as the terminal electron acceptor for methanogenesis (equation 1.1). Supplementing the medium with an abundant supply of one of the terminal electron acceptors prescribes the microbial process that occurs in the cultures. Fermentation, in which some oxidized organic compound serves as the terminal electron acceptor, also occurs under anaerobic conditions. 

Research with anaerobic cultures can be in batch or continuous culture. Fed-batch anaerobic reactors are not known to us but may very well be feasible. A reliable technique for batch cultures uses serum bottles sealed with butyl rubber stoppers and crimp sealed with an aluminum cap. Anaerobic microorganisms in batch flasks are mainly cultured by Hungate s methods [7] that are widely accepted in the research community. We will not describe these techniques but will focus on the different continuous culture apparatuses that have been developed over the years. 

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