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Chapter 2.1.2 : Anaerobic Cultivation

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Abstract:

Anaerobes are key players in global cycles of elements and nutrition in natural and anthropologic ecosystems and are also causative agents of human and veterinary diseases. Numerous studies have sought to develop culturing techniques for anaerobes to enable the elucidation of their basic physiology, pathogenic mechanisms, and ecological functions. This chapter will describe a brief history of the development of anaerobic culturing techniques from the historical Hungate technique to techniques and apparatuses commonly used in modern laboratories. In addition, recent progress in anaerobic culturing techniques (e.g., single-cell manipulation and isolation, the six-well plating method, the co-culture method, bioreactor-based enrichment, and in situ/in vivo cultivation) will be described, with several examples of the application of these techniques for the isolation of anaerobes from natural and artificial ecosystems.

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2
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FIGURE 1

(a) Gas cylinders for replacing head space (e.g., N, N/CO, and H/CO). (b) Gassing manifold in safety cabinet. All operations can be done aseptically. (c) Gassing manifold in draft chamber (Deoxygenized Gas Pressure Injector types IP-8, Sanshin Industrial, Yokohama, Japan). (d) The medium is dispensed to serum vials and replaced the head space with anoxic gas. (e) Plugging the tube with butyl rubber stoppers. (f), (g) sealing with crimp-closure aluminum seals. (h) Reducing agents (e.g., NaS and -cysteine hydrochloride) can be prepared (the head space has to be replaced with N) and autoclaved in advance. (i) Incubation in thermostatic chamber. (j) Vinyl-type anaerobic chamber (COY Laboratory Products, Grass Lake, MI, USA). doi:10.1128/9781555818821.ch2.1.2.f1

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2
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Image of FIGURE 2
FIGURE 2

Schematic illustration of brief procedure of six-well plate method with aerobic inoculation. This figure is a reprint with slight modifications from Nakamura et al. ( ) with the permission of the . doi:10.1128/9781555818821.ch2.1.2.f2

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2
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Image of FIGURE 3
FIGURE 3

(a) Colonies of strain KNH grown in the gellum gum medium in a well of six-well plate for 3 weeks of incubation at 45°C. (b) Phase contrast microphotograph of strain KNH. Bar indicates 10 µm. This figure is a reprint from the Nakamura et al. ( ) with the permission of . doi:10.1128/9781555818821.ch2.1.2.f3

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2
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FIGURE 4

(a) Appearance of colonization system (ISCS) after 5 days of incubation in a hydrothermal vent emission. Inner diameter and inner length of the ISCS were 45 and 250 mm, respectively. Filamentous white microbial mat formations were observed. (b) Pumiceous stuffing settled in the ISCS after the 5 days of incubations. Outer diameter and length of the white rings were 6 and 9 mm, respectively. This figure is a reprint from the Nunoura et al. ( ) with the permission of . doi:10.1128/9781555818821.ch2.1.2.f4

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2
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Tables

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TABLE 1

Reducing agents used for anaerobic culturing

Citation: Narihiro T, Kamagata Y. 2016. Anaerobic Cultivation, p 2.1.2-1-2.1.2-12. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.1.2

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