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 grow in the presence or absence of oxygen. Molecular oxygen (O₂) is the major 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.

I. In the most general 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 (less appropriately called "facultatives" or "facultative aerobes") – those which can grow in the presence or absence of O₂.

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

• 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.
  
  
• 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.
  

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 use in our Bacteriology laboratory courses – is a "standard" medium often used for the determination of oxygen relationships of bacteria.

One must consider the following limitations of Thioglycollate Medium:

• The medium will support the growth of common, easily-grown chemoheterotrophic bacteria, and the pattern of growth of an inoculated organism will indicate whether it can respire (using O₂) and/or ferment. Secondarily, the oxygen relationship designations derived from the growth observations (strict aerobe, facultative anaerobe, etc.) can be helpful in comparative studies of the physiologies of these organisms.
  
• 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 in this medium.
  

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.

The results we see in Thioglycollate Medium are shown below. The accompanying table gives related information. (A photo of a microaerophile will be added eventually.)

IV. In conclusion: 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
  

A couple items forming part of our learning experience in Bacteriology 102: (1) A procedure similar to the use of Thioglycollate Medium which tests purple non-sulfur bacteria for "obligate or facultative phototrophy" is shown here. (2) A relevant quiz/exam question is shown here.