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Relationship to Oxygen

Relationship to oxygen refers to the extent to which the presence or absence of oxygen influences bacterial growth. More often than not, closely related bacterial species share the same combination of relationship to oxygen and cell wall properties as revealed by the Gram stain. These properties are so informative that unless some other obvious feature gives away the identity of a bacterial isolate, our path to learning its identity starts there. The Gram stain method and rationale will be discussed in a separate document. Below we define several types of relationships to oxygen followed by our methods for determining what type of species we have.

Types of bacteria based upon their relationship to oxygen

  • Obligate aerobes rely on aerobic metabolism for growth and cannot grow in the absence of oxygen

  • Facultative anaerobes employ aerobic metabolism when oxygen is available but are capable of inducing alternative anaerobic pathways when oxygen is not available; they grow best in an oxygen atmosphere but demonstrate limited growth under anaerobic conditions

  • Obligate anaerobes cannot grow in the presence of oxygen; they are poisoned by oxygen and are unlikely to turn up in our samples or be cultivable in our laboratory without using anerobic chambers

  • Microaerophils rely on aerobic metabolism but their growth is suppressed in room air; they grow well when oxygen is present at relatively low partial pressures

Rationale behind the assay

Problems. How can we control the availability of oxygen to a culture in order to determine its influence on bacterial growth? How can we be sure that lack of oxygen and not simply lack of viability explains why we don't see growth in a culture?

Solutions. We incubate our cultures in sealed jars in which a chemical reaction removes oxygen, replacing it with carbon dioxide and water vapor. We incubate duplicate cultures aerobically to ensure that they were viable when placed in the jar. The aerobically grown cultures are our positive controls.

Applications. We use this assay as part of a plan to characterize bacterial species based upon phenotype. Analysis of the ribosomal RNA sequence is increasingly used to identify known species, however that method is most reliable for identifying well characterized species of clinical importance. It is less useful for characterizing isolates from environmental samples and even less useful for the study of newly discovered species.

A second valuable "application" is the opportunity to put the concept of an experimental control into practice.


To determine relationship to oxygen we streak duplicate agar plates with one or more isolates, incubating one plate aerobically and one plate in an anaerobic jar under otherwise identical conditions. After two or three days' incubation we open the jars an immediately compare the aerobically incubated plates with plates incubated in the jars. Good aerobic growth combined with no anaerobic growth suggests an obligate aerobe. A "lawn" on the agar surface in an anaerobically incubated plate suggests a facultative anaerobe. Lack of aerobic growth indicates that the culture was not viable or that there was an error in technique.

             Description: anaerobic_plates
Fig. 1. (left) GasPak jars for anaerobic systems. (right) Bacterial growth patterns on three sets of duplicate plates incubated for 72h, one set grown aerobically (top row) and corresponding anaerobically incubated plates (bottom row). Note that in all cases anaerobic growth is sparse compared with aerobic growth.

Setting up the incubations

  1. Mark off sections and label two sets of plates (test plates, to be incubated anaerobically and control, to be incubated aerobically), prepared with an agar medium that we know supports growth of the isolates to be tested. Two or more isolates can be streaked onto the same plate. Dilution streaking is not necessary.

Conduct the following procedures quickly and efficiently. Your cultures will begin to grow as long as they are in aerobic conditions, regardless of relationship to oxygen. Leaving the plates exposed to oxygen for too long may result in false positives for anaerobic growth.

  1. Systematically prepare duplicate streak plates, taking care to ensure that each plate is streaked identically and labeled identically to ensure comparison of aerobically and anaerobically incubated cultures. To save time you may go directly from the first plate to the second without picking up additional inoculum. There will be plenty on the loop, however to avoid falsely identifying a facultative anaerobe as an obligate aerobe because the inoculum was lighter, you should streak the plate to be incubated anaerobically first, followed by the plate to be streaked aerobically.

  2. Prepare the anaerobic jars

    1. Place a drop of water on the blue part of an indicator strip and tape the strip on the inside of the jar where it is visible (blue part shows).
    2. Place the test plates into the rack in an anaerobic jar, inverted. Try to fill up each jar to conserve on generator packets, which are expensive. Join one or more other teams if necessary. Small jars take up to 14 plates and require one generator packet. Large jars take up to 42 plates and require three generator packets.
    3. Open a generator envelope and insert the packet into a rack.  Quickly place the rack into the jar, attach lid, tighten, and label the jar.
    4. For fastest growth place test & control plates in the 30 degree incubator for at least 48-72 hours. Room temperature is okay if they grow vigorously at that temperature

Reading results

Assemble representative from all teams with plates in a jar before you open it. Aerobic growth will commence after you expose the plates to oxygen. You must make your determinations within an hour.

  1. Open the jar, line up your test and control plates for quick comparison. Reject any test for which there was no aerobic growth. You will have to re-test that isolate if it is still viable.

  2. Assess the presence or absence of anaerobic growth

    1. Anaerobic growth is indicated by an obvious "lawn" covering the inoculated section of agar. Growth is always more sparse than the corresponding aerobic growth and pigmentation is usually absent, even for species that produce pigmented colonies aerobically.
    2. If all or most of the agar is bare, you probably have an obligate aerobe. Report "anaerobic growth not observed." You may see some material at the start of the streak, where a large amount of colony material was placed on the agar. Lack of growth over the "tracks" made by the loop over the rest of the section supports the conclusion that you have an aerobe.
    3. Occasionally you may see individual colonies pop up on the agar, but not a continuous lawn. This observation suggests that your isolate is an obligated aerobe, contaminated by a facultative anaerobe.

  3. After making and recording your assessments discard the generator packets in the regular trash, rinse out the jars and place them back on the table to be used again.

Identifying a microaerophil

We rarely encounter microaerophils in our samples because by incubating our spread plates aerobically we would suppress growth of any microaerophils that may be present, if they are there at all. If you do have one, its growth pattern in a broth culture will give it away.

In a tube of liquid media we have aerobic conditions in the top half centimeter or so. If there is anything in the tube that consumes oxygen, then conditions become more and more anaerobic as we descend to the bottom. Starting about a half centimeter below the tube surface and ending perhaps a centimeter further down, conditions become ideal for microaerophilic growth. If you happen upon a broth tube that is clear except for a turbid region a bit below the surface, you may indeed have a microaerophil.

Created by David R. Caprette, Teaching Professor, Rice University ( 18 Feb 2017 for the course BIOC 318, Laboratory Studies in Microbiology. Please feel free to copy and/or modify these materials for use in your own academic or other nonprofit program. If you don't mind letting me know of such intentions I'll be happy to hear from you.
Last updated 26 Feb 2017