Oxygen and Growth of Bacteria
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1.
2.
3.
4.
5.
6.
Explain what is meant by aerotolerance.
Explain what is meant by oxic and anoxic.
Explain what is meant by aerobes and anaerobes.
Explain what is meant by obligate and facultative.
Explain why the Fluid Thioglycollate Broth allows for a range of aerotolerance.
Demonstrate an understanding in the interpretation of the thioglycollate tubes.
Microorganisms exhibit great diversity as to their oxygen requirements for cellular respiration.
Many bacteria will grow in the presence of oxygen but there are also many bacteria that find
oxygen to be toxic or at least inhibitory to their existence. An environment in which oxygen is
present is known as an oxic environment; where as an environment that is devoid of oxygen is
known as an anoxic environment. There are five major groups in which microorganisms can be
classified as to their aerotolerance or ability to exist in either of these two environments.
1. Obligate Aerobes: Require the presence of oxygen for growth thus they are restricted to growth
in an oxic environment.
2. Obligate Anaerobes: Require the absence of oxygen for growth thus they are restricted to
growth in an anoxic environment.
3. Facultative Anaerobes: Prefer the presence of oxygen for growth but will grow in an anoxic
environment as well.
4. Microaerophiles: Prefer the presence of only a small amount of oxygen for growth.
5. Aerotolerant Anaerobes: Do not require the presence of oxygen for growth but will grow in oxic
and anoxic environments equally well.
Culture methods have been developed to assist in the aerotolerance classification of
microorganisms. This laboratory exercise utilizes a reducing medium that is suitable for a broad
variety of aerobic and anaerobic bacteria known as Fluid Thioglycollate Broth. It contains sodium
thioglycollate, which reacts with molecular oxygen (O2) keeping free oxygen levels low. The
sodium thioglycollate in the broth creates a redox potential in the tube, with higher levels of
oxygen at the top of the tube (an oxic environment), and a complete absence of oxygen at the
bottom of the tube (an anoxic environment). Fluid Thioglycollate Broth also typically contains a
redox potential indicator such as resazurin, which produces a pink color in an oxidized
environment. Microorganisms will only be able to grow where their oxygen requirements are met,
and will localize to the area(s) of their oxygen requirements in the fluid thioglycollate broth, see
Figure 1.
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Figure 1. Fluid Thioglycollate. (http://people.rit.edu)
Tube a: Obligate Aerobe -- note the growth is only in the top portion of the tube where oxygen is
present.
Tube b: Obligate Anaerobe -- note the absence of growth in the top portion of the broth where
oxygen is present.
Tube c: Facultative Anaerobe -- note the uneven distribution of growth from top to bottom (more
growth at the top).
Tube d: Microaerophile -- note the growth is only in the top portion of the tube where some
oxygen is present.
Tube e: Aerotolerant Anaerobe -- note the uniform growth from top to bottom.
In this laboratory exercise you will be using Fluid Thioglycollate Broth to determine the
aerotolerance categories of bacteria.
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Materials (per student)
1 Pure Culture Broth of Pseudomonas fluorescens or Staphylococcus epidermidis
1 Pure Culture Broth of Escherichia coli or Proteus vulgaris
2 Fluid Thioglycollate Broth Tubes
1 Inoculating/Transfer Loop
China Marker or Sharpie
Bunsen Burner
Procedure 1
Inoculation of Fluid Thioglycollate Broth Tubes
Acquire a pure culture broth tube of Pseudomonas fluorescens or Staphylococcus epidermidis and
a pure culture broth tube of Escherichia coli or Proteus vulgaris. You will inoculate an individual
Fluid Thioglycollate Broth tube for each of the cultures.
Note: Use aseptic technique throughout all inoculations.
1. Setup and light a Bunsen burner.
2. Label the uninoculated Fluid Thioglycollate Broth tubes to be inoculated with the name of the
organism, your initials and the date.
3. Inoculate the Fluid Thioglycollate Broth tube by transferring one loop-full of inoculum from
the pure culture broth tube using aseptic technique.
4. Incubate the inoculated tubes at 37°C for 24 to 48 hours.
5. Observe the growth patterns in the tubes and record your results.
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Results and Observations
For the inoculated Fluid Thioglycollate Broth tubes record your observations below:
Tube
Pseudomonas fluorescens or
Staphylococcus epidermidis
Escherichia coli or Proteus
vulgaris
Growth (+) or (-)
Aerotolerance Classification
Laboratory Review
1. Explain why the Fluid Thioglycollate Broth allows for a range of aerotolerance:
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2. Explain the difference between oxic and anoxic environments:
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3. Explain the difference between aerobes and anaerobes:
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4. Explain is meant by an obligate anaerobe and a facultative anaerobe:
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5. In your own words, define aero tolerance:
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6. What category of aerotolerance is noted by growth only in the top portion of the tube where
some oxygen is present?
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______________________________________________________________________________
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