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

2.  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.

4.  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.

5. GROWTH INDICATED BY GRAY AREAS

6.  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.

7.  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.

8.  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