STRICT ANAEROBIC CONDITIONS

Aerotolerants are anaerobes that can grow in the presence of O2 (compared to the strict anaerobes which would likely die), but they do not use it. And last, but very common, are the

facultative anaerobes which prefer to use O2 when present but will grow without it.

Another way to culture and grow anaerobes is the use of reduced media--- media without oxygen. Thioglycollate broth has a reducing agent in it---the chemical thioglycollate---which binds any free oxygen within the medium. You will also notice that these tubes have screw caps, allowing a tight closure, to reduce oxygen entry. However, some oxygen will be in the tube between the cap and the broth and there is no way to get rid of it. So there will be some diffusion of oxygen into the top portion of the broth, and that is where any aerobic bacteria may grow. An indicator, resazurin, in the medium will be a light pink in the area of higher oxygen. You can determine whether the bacterium is an anaerobe, facultative anaerobe, or an aerobe by checking out where the organism grows in the column of media.

GROWTH INDICATED BY GRAY AREAS

OBJECTIVES: Identify the 3 major categories of microbes based on oxygen

requirements. 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 table’s 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 strict anaerobe

cultures in thioglycollates for the class to view.

THE PROCEDURES: Thioglycollate broth 1. The thioglycollate broth should be either

boiled first before inoculation OR recently made so that the oxygen content is very low. (Your instructor will tell you if it needs to be boiled).

2. Inoculate a tube of thioglycollate broth with your unknown bacterium: make sure that the loop or needle goes down to the BOTTOM of the broth (do not get metal holder in the sterile broth).

3. Incubate at 25 or 37 degrees C as directed.

TSA plates in 3 different oxygen environments 1. Label 3 plates for the table---candle jar, ambient air,

and GasPak anerobic jar. 2. Divide the 3 plates into sections, one for each organism

—your unknown, 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 plates using the same technique.

4. Be sure that the jar has a methylene blue indicator strip (seen above) inside. The methylene blue is blue when oxidized, but colorless when reduced. Before the jar is opened, the strip should be checked to make sure that it is COLORLESS.

5. Incubate at 30 or 37 degrees C