Differential Media:
"Oxygen Relationships" and the Use of Thioglycollate Medium

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In Bergey's Manual and other bacteriological texts, reference is made to the oxygen relationships of various bacteria – that is, how bacteria metabolize and replicate (if at all) in the presence or absence of oxygen.

Molecular oxygen (O₂) is the electron acceptor utilized by organisms which obtain energy from respiration (i.e., aerobic respiration). However, it does not function as such for organisms which obtain energy from fermentation, photosynthesis or anaerobic respiration.

For the following discussion, it may be helpful to review basic catabolic processes such as what is covered on this page. Also, a review of respiration vs. fermentation is given here.

I. In the most elementary sense, living organisms can be classified according to "oxygen relationships" as follows: (1) strict (or obligate) aerobes – those which require O₂, (2) strict (or obligate) anaerobes – those which can only grow in the absence of O₂, and (3) facultative anaerobes – those which can grow in the presence or absence of O₂. Practicing bacteriologists do not settle for this oversimplification as we see in the following two sections.

II. Bergey's Manual applies oxygen relationship categories to the chemotrophic bacteria, and the definitions which follow are taken verbatim from those in Bergey's Manual of Determinative Bacteriology (9th ed., 1994) except for the items in brackets:

• [Strict or Obligate] Aerobe: An organism that is capable of using oxygen as a terminal electron acceptor [i.e., aerobic respiration], can tolerate a level of oxygen equivalent to or higher than that present in an air atmosphere (21% oxygen), and has a strictly respiratory type of metabolism. Some aerobes may also be capable of growing anaerobically with electron acceptors other than oxygen [i.e., anaerobic respiration].
  
  
• Facultative anaerobe: An organism that can grow well both in the absence of oxygen and in the presence of a level of oxygen equivalent to that in an air atmosphere (21% oxygen). Some are capable of growing aerobically by respiring with oxygen and anaerobically by fermentation [anaerobic respiration is also possible]; others have a strictly fermentative type of metabolism and do not respire with oxygen. [We form the "aerotolerant anaerobe" category with the latter type; see below.]
  
  
• Microaerophile: An organism that is capable of oxygen-dependent growth but cannot grow in the presence of a level of oxygen equivalent to that present in an air atmosphere (21% oxygen). Oxygen-dependant growth [i.e., aerobic respiration] occurs only at low oxygen levels. In addition to being able to respire with oxygen, some microaerophiles may be capable of respiring anaerobically with electron acceptors other than oxygen.
  
  
• [Strict or Obligate] Anaerobe: An organism that is incapable of oxygen-dependent growth and cannot grow in the presence of an oxygen concentration equivalent to that present in an air atmosphere (21% oxygen). Some anaerobes may have a fermentative type of metabolism; others may carry out anaerobic respiration in which a terminal electron acceptor other than oxygen is used. [The primary consideration for defining an organism as a strict anaerobe is its total intolerance of oxygen.]

With these Bergey's Manual definitions, phototrophs would be categorized with difficulty if at all. As one example, the purple non-sulfur photosynthetic bacteria can respire and can also grow anaerobically, but anaerobic growth is associated with the organisms' use of energy derived from light, not (except for certain exceptional strains and species) from fermentation or anaerobic respiration.

III. Thioglycollate Medium – which we utilize in our Bacteriology laboratory courses – is a "standard" medium for the determination of oxygen relationships, and it will support the growth of common, easily-grown chemoheterotrophic bacteria. The observed growth patterns of organisms in this medium determine their oxygen relationship designations (strict aerobe, facultative anaerobe, etc.) which correlate with such physiological abilities as respiration, fermentation and the catalase reaction and also whether there is an inhibitory effect on the organism in the presence of air. See the table under the photo below. Thus, a description of a chemoheterotrophic organism as a "strict aerobe" can imply a number of associated characteristics that may be unnecessary to specify separately (able to respire, unable to ferment, catalase-positive, azide-sensitive, etc.).

The amino acids and glucose in the medium can be respired, and glucose is the only fermentable energy source in the medium except for those exceptional organisms such as certain species of Clostridium which can ferment amino acids.

With Thioglycollate Medium, we are able to differentiate two distinct patterns of growth for those classified in the Bergey's Manual definitions (above) as "facultative anaerobes":

• Those which are indifferent to oxygen and have a strictly fermentative type of metabolism grow evenly throughout the medium. We term such an organism an aerotolerant anaerobe and set that off as an additional category of oxygen relationship (added to the list of four above).
  
  
• Those left in the facultative anaerobe category show greater concentration of growth at the top of the medium where oxygen is present and aerobic respiration is then possible. Comparing the degree of growth under aerobic vs. anaerobic conditions can be a good demonstration of the relative efficiencies of aerobic respiration and fermentation when it comes to generation of cell mass.
  
  
• The terms "facultative" and "aerotolerant" are always meant to modify another term such as "anaerobe" and they should not be used by themselves. Describing an organism as simply "facultative" may mean "facultative anaerobe," "facultative phototroph," or a variety of other things.

One must consider the following limitations of Thioglycollate Medium:

• Many organisms (including a lot of chemoheterotrophs) cannot grow in this medium for one reason or another.
  
  
• No allowance is given in the medium or method for anaerobic growth (1) with alternate electron acceptors (such as nitrate) or (2) in light (such as what is seen with the anoxygenic photosynthetic bacteria). Thus, an organism which may be termed a "strict anaerobe" in the more general sense – i.e., one which cannot tolerate oxygen and can only obtain energy by reactions which do not involve O₂ – would only show anaerobic growth in this test if it were capable of fermentation of the glucose in this medium.
  
  
• The results in Thioglycollate Medium can be difficult to read. As shown in the table below, an organism's oxygen relationship designation can be determined by a combination of other methods which can be used as a check to see if the medium is showing the correct results – i.e., (1) testing for fermentation in Glucose Fermentation Broth, (2) performing the catalase test, and (3) testing if the organism can grow in the presence of oxygen. These methods tend to be quite reliable and can be utilized if Thioglycollate Medium is not available or even specified for use in the identification process. With that in mind, Thioglycollate Medium could be considered redundant.

The results we see in Thioglycollate Medium are shown below. (Note that microaerophiles are not included.) The accompanying table gives related information.

*  These are the basic things tested for in this medium. Whether or not any organism can obtain energy by anaerobic respiration or phototrophy is not relevant to these designations of oxygen relationships.

So, in becoming a practicing bacteriologist, one will see that there is more to this concept than whether bacteria simply "like" or "don't like" oxygen – which, unfortunately, is the extent to which oxygen relationships are too-often and unconscionably taught.

IV. Rather than (or in addition to) using "oxygen relationships" as descriptive terms – however they may be determined or defined – we can characterize and classify bacteria more consistently and comprehensively by applying the method(s) of energy generation of which an organism is capable:

• aerobic respiration
  
• anaerobic respiration
  
• fermentation
  
• anoxygenic phototrophy
  
• oxygenic phototrophy

Remember that Thioglycollate Medium tests for an organism's ability to perform aerobic respiration and/or fermentation – the results of which give us the "oxygen relationship" categories for those organisms which can grow in the medium under the incubation conditions provided. Anaerobic growth in this medium is only associated with fermentation.

V. The following summary may help to explain how media formulations can allow anaerobic growth for organisms capable of doing so for one reason or another. The same organism – a typical strain of E. coli – was inoculated into tubes 1, 2 and 3, and a "facultative phototroph" was inoculated into tube 4.

In Tube #1, we have a medium containing peptone and agar plus other nutrients a "typical Bacteriology 102 organism" (i.e., a commonly-found, easy-to-grow chemo- or photoheterotroph) might require for metabolism and replication – except that nothing is included which would support anaerobic growth such as glucose (or something else that could be fermented) or nitrate (or some other electron acceptor/"oxygen substitute" that could be used in anaerobic respiration). After inoculation of this medium and incubation in the dark, any growth would be due to aerobic respiration with the growth only at the top of the medium. There would be no anaerobic growth except for some rare, exceptional organisms which can ferment amino acids; these we do not consider in Bact. 102.

Tube #2 is the same medium as in #1, but glucose has been added. After incubation (in the dark), any anaerobic growth would be due to fermentation of the glucose. Thus the medium can be used to detect whether or not an organism can respire (aerobically) or ferment. An example of such a medium is the Thioglycollate Medium we use to test common chemoheterotrophs for "oxygen relationships" (discussed above).

Tube #3 is the same medium as in #1, but potassium nitrate has been added. After incubation (in the dark), any anaerobic growth would be due to anaerobic respiration where the organism is using nitrate as the electron acceptor. In Bact. 102 (Exp. 7), we do a test in a broth medium for nitrate reduction; with reagents we can detect nitrite formation, and with the Durham tube we can detect N₂ gas formation. One can probably see why we would not want to include nitrate in the Thioglycollate Medium above.

Tube #4 is the same medium as in #1, but we have incubated the tube in the presence of light. With light as the ultimate energy source, anaerobic growth would be due to anoxygenic phototrophy. This is the basis for the test we do in Bact. 102 (Exp. 11.1) to see if our isolates of purple non-sulfur bacteria are either "strict phototrophs" (just anaerobic growth in the light) or "facultative phototrophs" (anaerobic growth in the light, plus aerobic growth due to aerobic respiration whether in the dark or the light). Click here for a summary of this test.

A relevant quiz/exam question which we repeatedly give in Bact. 102 is shown here.

A special page on Thioglycollate Medium that may be suitable as a handout can be found here.
A page on the Bact. 102 site summarizing catabolism and the oxygen relationship test is here.

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Page was last modified on 10/18/08 at 1:00 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison