2. General Microbiology Laboratory Anaerobic Bacteria

3. Categories Based Upon Oxygen Requirements Aerobic bacteria Require oxygen as electron acceptor Microaerophilic bacteria Require oxygen in reduced quantity (2-10%) Facultative bacteria Grow either with or without oxygen Anaerobic bacteria Both obligate and aerotolerant (Aerotolerant do not use aerobic metabolism but have some enzymes that detoxify toxic forms of oxygen)

4. Methods for excluding oxygen Fluid Thioglycollate Media (FTM) 1. Fluid Thioglycollate Media (FTM) supports aerobic & anaerobic bacteria. Sodium thioglycollate:Sodium thioglycollate: removes O2 from the medium ResazurinResazurin as an oxidation reduction indicator. dye that turns pink in presence of O2

6. 2- Anaerobic Jar Inoculate FTM tube : B. subtilis or P. aeruginosa (strict aerobes) S. pyogenes (microaerophile) E. coli (facultative anaerobe) C. sporogenes (strict anaerobe) Place in anaerobic jar. Incubate overnight at 37C.

7. Anaerobic Jar

8.  For this exercise, we use the GasPak anaerobic system. This system consists of a polycarbonate jar, a lid with a gasket to prevent air flow, a strip infused with methylene blue, and a pouch containing sodium borohydride, sodium bicarbonate, citric acid, and a palladium catalyst.  When water is added to the pouch, the sodium borohydride, sodium bicarbonate, and citric acid react to form hydrogen and carbon dioxide. The palladium catalyzes a reaction between the hydrogen and the oxygen within the jar; this reaction creates water, which forms as condensation on the inside of the jar.  Methylene blue is blue in the presence of oxygen but is colorless in an anaerobic environment. When the oxygen is converted to water and condensation forms on the side of the jar, the indicator strip will turn from blue to white.

9. Candle jars  The candle flame will consume most of the oxygen in the jar.  Produce an elevated level of carbon dioxide.  These conditions are ideal for the growth of microaerophilic organisms.

10. Growing Microaerophilic Microbes CO2 Generating Packet

11. Anaerobic Chambers  The Vinyl Anaerobic Chambers provide a strict anaerobic atmosphere of 0-5 parts per million (ppm) using a palladium catalyst and hydrogen gas mix of 5%.

12. End of lecture