Laboratory Investigations in Microbiology

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Chapter 11: Effect of oxygen on bacterial growth

Perhaps one of the most serious environmental disasters occurred long ago on earth when a deadly poison gas was (deliberately?) released into the earth's atmosphere, killing many of its inhabitants. The poison gas? Oxygen! To you and me, oxygen is, of course, vital for respiration. At one point in time, though, the earth's atmosphere was likely completely anaerobic, containing such reducing compounds as hydrogen sulfide gas, ammonium, and hydrogen gas. Bacteria that lived then were presumably strict anaerobes. Then along came cyanobacteria - oxygen-producing photoautotrophs that gradually turned the atmosphere into what we are familiar with today. Although eventually algae and plants contributed to this process, one may say that it is ultimately still cyanobacteria (chloroplasts by endosymbiosis) that are at work.

Over time, organisms, of course, adapt or perish. Strategies that have evolved to deal with an oxygen-containing atmosphere include aerobic respiration, facultatively anaerobic metabolism, aerotolerance, and various enzymes to protect cells from the damaging oxygen molecules, such as catalase and superoxide dismutase. Organisms that could not adapt were banished into anaerobic environments (deep in soils, under water or in intestinal tracts!). This demonstration exercise will show you the four most common patterns of growth in relation to oxygen.

Obligate aerobes, of course, are bacteria that require oxygen to live. Oxygen is used in aerobic respiration as the sole electron acceptor, and its absence dooms the aerobe to death. Obligate aerobes are, not surprisingly, strictly respiratory (no fermentation) and carry the enzymes superoxide dismutase and catalase. Aerobes also have the enzyme cytochrome oxidase, which can be detected using the oxidase test. Examples of aerobes include Pseudomonas aeruginosa and Micrococcus luteus.

Obligate anaerobes, in contrast, are killed by the presence of oxygen. They have never developed a strategy for coping with this toxic compound or its byproducts, are catalase- and superoxide dismutase-negative, and can be found in nature only where little or no oxygen exists. Obligate anaerobes may be respiratory (using anaerobic respiration) or fermentative. Examples of obligate anaerobes include Clostridium botulinum and Veillonella parvula.

Facultative anaerobes have both a respiratory and a fermentative metabolism. For  respiration, they often have a branched electron transport chain that allows them to use multiple electron acceptors (oxygen, nitrate, sulfate). Because aerobic respiration is a much more efficient process than anaerobic respiration or fermentation, facultative anaerobes grow better with oxygen than without. Examples of facultative anaerobes include E. coli and Staphylococcus aureus.

Aerotolerant anaerobes are unaffected by oxygen. Like strict anaerobes, they do not have the ability to use oxygen. Unlike strict anaerobes, aerotolerant anaerobes are not killed by oxygen. Aerotolerant anaerobes are catalase-negative, but many do carry superoxide dismutase. These bacteria are always oxidase-negative and carry out a strictly fermentative type of metabolism. Examples in this category include Enterococcus faecalis and Streptococcus lactis.

Microaerophiles are a relatively recent discovery. These bacteria require oxygen in very small amounts and are killed by too much or too little of it. Examples include Helicobacter pylori and Campylobacter jejuni.

In order to test bacteria for their ability to grow without oxygen, an anaerobic culture chamber is required. Oxygen is removed from the chamber when it reacts with hydrogen gas to form water. By inoculating agar plates with bacteria that are incubated inside and outside of the chamber, one can determine an organism's preference for oxygen by comparing the amount of growth of these bacteria with or without oxygen.

Materials & Methods

Materials used in the demonstration exercise
Procedure (demonstration)
  1. Inoculate 2 TSA plates with a single streak of each of the four bacteria listed
  2. Place one plate into the anaerobic jar. Add 10 ml of water to the Gas-Pack to start hydrogen production. Place a litmus indicator strip into the chamber as well.
  3. Seal the jar tightly. Incubate the jar and the second agar plate for 2 - 7 days.
  4. Inoculate each anaerobic agar deep with one of the 4 cultures using an inoculating needle. Incubate 2 - 7 days.
  5. After incubation: examine the TSA plates inside and outside the anaerobic jar for growth. Examine the anaerobic deeps for growth.

 

Data Sheet & Review Questions

 

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