User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Anaerobic Cell Culture

Allison M. Speers¹, Dena L. Cologgi¹, Gemma Reguera¹

¹Michigan State University, East Lansing, Michigan

Publication Name:

Current Protocols in Microbiology

Unit Number:

Appendix 4F

DOI:

10.1002/9780471729259.mca04fs12

Online Posting Date:

February, 2009

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Abstract

Although the ability of some microorganisms to grow without O₂ has long been recognized, the application of new methodologies has greatly expanded the known diversity and potential of anaerobic microorganisms and processes. In particular, anaerobic techniques that permit the successful cultivation of microorganisms on solid media have opened new avenues for the study of the physiology and metabolic potential of many new microorganisms using molecular, genomic, and proteomic tools. One technique above all has proven instrumental for anaerobic studies over the years: the use of the anaerobic chamber. This unit gives a brief description of the methods used for the cultivation of anaerobic microorganisms, and describes in detail the principles and applications of anaerobic chambers, with special emphasis on vinyl glove boxes. The methodologies described in this unit should provide the interested but inexperienced investigator with the basic tools to successfully cultivate anaerobic microorganisms and study anaerobic processes. Curr. Protoc. Microbiol. 12:A.4F.1-A.4F.16. © 2009 by John Wiley & Sons, Inc.

Keywords: anaerobes; gassing station; anaerobic chamber; anaerobic techniques; glove box

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Introduction
Overview and Principles
Anaerobic Chambers: Operation and Maintenance
Preparing Anaerobic Liquid Medium
Preparing Anaerobic Solid Medium
Cultivation of Hydrogen Sulfide–Producing Microorganisms
Acknowledgements
Literature Cited
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure A.4F.1

Prototype of gassing station for anaerobic culturing. (A) Control panel board with manifold and syringe ports. A 3-way ball valve with vacuum and gas positions is also pictured. The “vacuum/gas” valve interrupts the gas flow and opens the connection to a vacuum pump to aspirate gases when in the “vacuum” position. When in the “gas” position, this valve directs the gas flow to the pressure gauge and manifold and syringe ports. Three metering valves (black round valves) are pictured that control H₂ gas flow to the H₂ flow meter, as well as gas exiting the control panel through the manifold and syringe outlet ports. (B) Back of the panel showing the copper tubing connections. (C) Glass copper column wrapped in heating tape.

View Image
Figure A.4F.2

Vinyl anaerobic chamber. (A) COY vinyl glove box with two pairs of glove ports (type B chamber), two fan boxes, an incubator, shelves, a gas analyzer, and an electrical outlet. The airlock cabinet is attached to the right side of the chamber and the entry cap to the left. Close-ups of a heated fan box (B), catalyst Stak-Pak (C), and O₂-H₂ gas analyzer (D) also are shown.

View Image
Figure A.4F.3

Anaerobic media preparation using a gassing station and manifold ports. Reusable needles are attached to the manifold hoses to bubble the medium without (A) and with (B) the stoppers in place.

View Image

Literature Cited

Literature Cited
	Aranki, A., Syed, S.A., Kenney, E.B., and Freter, R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17:568-576.
	Brewer, J.H. and Allgeier, D.L. 1966. Safe self-contained carbon dioxide-hydrogen anaerobic system. App. Microbiol. 14:985-988.
	Drasar, B.S. 1967. Cultivation of anaerobic intestinal bacteria. J. Pathol. Bacteriol. 94:417-427.
	Hall, I.C. 1929. A review of the development and application of physical and chemical principles in the cultivation of obligately anaerobic bacteria. J. Bacteriol. 17:255-301.
	Hosoya, S. and Kishino, S. 1925. The influence of the sulfur-containing substances upon the growth of anaerobic bacilli. Sci. Rept. Govt. Inst. Dis. Tokyo Imp. Univ. 4:123-127.
	Hungate, R.E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bateriol. Rev. 14:1-49.
	Kashefi, K., Holmes, D.E., Reysenbach, A.L., and Lovley, D.R. 2002. Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: Isolation and characterization of Geothermobacterium ferrireducens gen. nov., sp. nov. Appl. Environ. Microbiol. 68:1735-1742.
	Killgore, G.E., Starr, S.E., Del Bene, V.E., Whaley, D.N., and Dowell, V.R. 1972. Comparison of three anaerobic systems for the isolation of anaerobic bacteria from clinical specimens. Abstr. Ann. Meeting Am. Soc. Microbiol.: (abstr. M86).
	Leach, P.A., Bullen, J.J., and Grant, I.D. 1971. Anaerobic CO₂ cabinet for the cultivation of strict anaerobes. Appl. Microbiol. 22:824-827.
	McIntosh, J. and Fildes, P. 1916. A new apparatus for the isolation and cultivation of anaerobic micro-organisms. Lancet 1:768-770.
	Nottingham, P.M. and Hungate, R.E. 1969. Methanogenic fermentation of benzoate. J. Bacteriol. 98:1170-1172.
	Pouliquen, F., Blanc, C., Arretz, E., Labat, I., Tournier-Lasserve, J., Ladousse, A., Nougayrede, J., Savin, G., Ivaldi, R., Nicolas, M., Fialaire, J., Millischer, R., Azema, C., Espagno, L., Hemmer, H., and Perrot, J. 2005. "Hydrogen sulfide". Ullman's Encyclopedia of Industrial Chemistry, 7th ed. Wiley-VCH, Weinheim, Germany.
	Rosebury, T. and Reynold, J.B. 1964. Continuous anaerobiosis for cultivation of spirochetes. Proc. Soc. Expetl. Biol. Med. 117:813-815.