Strict anaerobic conditions

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Strict anaerobic conditions,hungate technique microbes involved and cultured in anaerobic chamber,4 stages involved acidogenesis,methanogenesis,

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<ul><li> 1. Aerotolerants are anaerobes that can grow inthe presence of O2 (compared to the strictanaerobes which would likely die), but theydo not use it. And last, but very common, arethefacultative anaerobes which prefer to use O2when present but will grow without it.</li></ul> <p> 2. Another way to culture and grow anaerobes is the use ofreduced media--- media without oxygen. Thioglycollatebroth has a reducing agent in it---the chemicalthioglycollate---which binds any free oxygen within themedium. You will also notice that these tubes have screwcaps, allowing a tight closure, to reduce oxygen entry.However, some oxygen will be in the tube between the capand the broth and there is no way to get rid of it. So therewill be some diffusion of oxygen into the top portion ofthe broth, and that is where any aerobic bacteria maygrow. An indicator, resazurin, in the medium will be a lightpink in the area of higher oxygen. You can determinewhether the bacterium is an anaerobe, facultativeanaerobe, or an aerobe by checking out where theorganism grows in the column of media. 3. GROWTH INDICATED BY GRAY AREAS 4. OBJECTIVES: Identify the 3 major categories of microbes based on oxygenrequirements. Learn different ways to culture anaerobic bacteria. MATERIALS NEEDED: per table 1 thioglycollate broth per table 3 TSA plates (divide into pie-shaped sections) GasPak container for entire lab + GasPak sachet for the jar +methylene blue indicator strip candle jar for entire lab cultures: your tables unknown bacterium a strict anaerobe + a strict anaerobe used as controls (your instructor will give you the names at beginning of lab) Your instructor will set up the strict aerobe and the strictanaerobe cultures in thioglycollates for the class to view. 5. THE PROCEDURES: Thioglycollate broth 1. The thioglycollate broth should be either boiledfirst before inoculation OR recently made so that theoxygen content is very low. (Your instructor will tellyou if it needs to be boiled). 2. Inoculate a tube of thioglycollate broth with yourunknown bacterium: make sure that the loop orneedle goes down to the BOTTOM of the broth (donot get metal holder in the sterile broth). 3. Incubate at 25 or 37 degrees C as directed. 6. TSA plates in 3 different oxygen environments 1. Label 3 plates for the table---candle jar, ambient air, andGasPak anerobic jar. 2. Divide the 3 plates into sections, one for each organismyourunknown, the strict aerobe, the strict anaerobe. 3. Inoculate the section by streaking a straight line or a zig-zag(as seen below). HOWEVER, be sure that you inoculate all 3 platesusing the same technique. 4. Be sure that the jar has a methylene blue indicator strip (seenabove) inside. The methylene blue is blue when oxidized, butcolorless when reduced. Before the jar is opened, the stripshould be checked to make sure that it is COLORLESS.5. Incubate at 30 or 37 degrees C</p>

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