The presence or absence of molecular oxygen can be a critical factor in the ability of bacteria to grow in each environment. When bacteria use oxygen in cellular respiration and other chemical reactions, damaging superoxide and peroxides are produced. These highly reactive byproducts damage the cell unless they are quickly neutralized. Bacteria called aerobes produce ATP via aerobic energy production (cellular respiration) using oxygen as the final member of the electron transport chain. Aerobes produce enzymes such as catalase, peroxidase and/or superoxide dismutase that break down toxic forms of oxygen and their intermediate byproducts.

superoxide (superoxide dismutase enzyme) —–> hydrogen peroxide and oxygen

hydrogen peroxide (catalase enzyme) —–> hydrogen and oxygen

Bacteria called anaerobes produce ATP via anaerobic energy production using nitrate, sulfate, or a ferric ion as the final member of the electron transport chain. Anaerobes have no tolerance of oxygen since they cannot produce catalase, peroxidase and/or superoxide dismutase.

This extensive classification scheme has been developed to group bacteria according to their oxygen requirements:

Obligate Aerobes- Produce ATP via aerobic energy production. Require around 20% atmospheric oxygen.

Microaerophiles- Produce ATP via aerobic energy production or fermentation. Require between 5-15% atmospheric oxygen for growth.

Aerotolerant anaerobes-Produce ATP anaerobically, and can ferment. Oxygen can be present, but they do not utilize it for ATP production or fermentation.

Capnophiles–Produce ATP via aerobic energy production. Require between 5-15% atmospheric oxygen and increased (10%) carbon dioxide.

Facultative Anaerobes–Produce ATP via aerobic energy production, anaerobic energy production, or fermentation. These organisms grow equally well in aerobic or anaerobic environments.

Obligate Anaerobes –Produce ATP via anaerobic energy production or fermentation. organisms that die in the presence of atmospheric oxygen because they lack the enzymes needed to break down toxic forms of oxygen and their intermediate byproducts.

Thioglycollate is a medium designed to test the aerotolerance of bacteria. Along with nutrients, it contains a reducing agent, sodium thioglycollate, which combines with oxygen to produce water. Thioglycollate also contains a small amount of agar which helps retard oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth. Because the thioglycollate can eliminate the oxygen in the bottom of the tube, but not at the surface, varying concentrations of oxygen are found within the tube. On occasion, an Eh indicator is added to the media to indicate the presence or absence of oxygen. Eh refers to an electron indicator which shows the redox state of the medium, thus showing where the aerobic and anaerobic zones separate. For example, resazurin is pink in the presence of oxygen and colorless when reduced.

A candle jar can readily achieve microaerophilic and capnophilic conditions. After inoculated plates or tubes are placed in a jar, a lit candle is added and the lid on the candle jar is then tightly sealed. As the flame on the candle burns, it uses up much of the oxygen and produces carbon dioxide.

A Gas Pak jar is used to grow strict anaerobes that must be grown in an atmosphere without free oxygen present. Plates or tubes are placed in a jar along with Gas Pak envelopes that function as disposable hydrogen and carbon dioxide generators. The hydrogen combines with the oxygen in the jar to produce water. To indicate anaerobic conditions are in the Gas Pak jar, a strip of paper soaked in methylene blue dye is included in the jar. Methylene blue is colorless when oxygen is absent and blue when oxygen is present.

PRE-ASSESSMENT

Growth of microorganisms in various concentrations of oxygen

1.  Observe the TSA plates inoculated with E. coli, Clostridium and Pseudomonas in the various oxygen concentrations.

2.  Record growth (+) or no growth (-) on the worksheet.

Production of Catalase

1.  Obtain a glass slide and a small bottle of hydrogen peroxide.

2.  Using a sterilized inoculating loop, take a small amount of E. coli from a plate that was positive for growth and put the inoculum in the middle of a dry slide. Place the slide on a dark background for better observation.

3.  Place a drop of hydrogen peroxide on top of the inoculum.

4. The appearance of bubbles indicates the organism can produce catalase.

5.  Repeat this procedure with the Pseudomonas and Clostridium.

6.  Record the results on the worksheet.

Growth in thioglycollate tubed media

1.  Using the information gathered above, hypothesize where the bacteria would grow in the thioglycollate media. Record the reaction on the worksheet.

Growth on the media surface

Growth throughout the media

Growth only near the bottom half of the media

Classification based on the Oxygen requirements

1.  Using the information gathered above, classify the bacteria based on its ability to use oxygen. Record the results on the worksheet.

Obligate Aerobe–needs oxygen to grow

Facultative Anaerobe–able to grow in oxygen, but does not require it

Microaerophilic–grows in decreased amount of oxygen

Obligate Anaerobe–cannot grow in oxygen

Growth in thioglycollate Tubes

POST TEST

DISCOVERIES IN MICROBIOLOGY

GERTRUDE ELION

American biochemist and pharmacologist Gertrude Elion discovered the first successful antiviral drug, acyclovir, used to treat herpes simplex and varicella-zoster infections.  She also discovered the first immunosuppressive drug, azathioprine, used to prevent rejection of organ transplants.