MicroVision 
 
Oxygen Requirements 
The presence or absence of molecular oxygen can be a critical factor in determining if a particular species of bacteria will grow in a given environment.  Organisms that require oxygen to live are called obligate aerobes .  Obligate anaerobes are unable to use molecular oxygen to obtain energy and usually find oxygen harmful.  Facultative anaerobes can use oxygen if it is available but can also grow when oxygen is absent.  In contrast, aerotolerant anaerobes cannot use oxygen for growth, but they do tolerate its presence well. Some bacteria require a lower oxygen concentration than what is in the air.  These organisms are classified as microaerophilic .  A variety of culture methods have been developed to satisfy these diverse oxygen needs. Thioglycolate broth, anaerobic jars, and candle jars create alternative environments to atmospheric oxygen.

Thioglycolate Broth 

Thioglycolate (thio) broth contains sodium thioglycolate, a reducing agent that creates anaerobic conditions when it reduces molecular oxygen to water. Dyes such as resazurin or methylene blue are usually added to the broth to provide a visual indication of the presence of oxygen. Resazurin is pink when oxidized and colorless when reduced.  Methylene blue is blue when oxidized and colorless when reduced.  Resazurin is present in the thioglycolate tubes shown below.  The pink band near the top of the broth results when oxygen diffuses in.  Strict aerobes will grow only in the pink band, microaerophiles will grow near the bottom of the band where the concentration of oxygen is lower.  The absence of pink in the rest of the tube indicates the absence of oxygen and a suitable environment for strict anaerobes.  Both facultative anaerobes and aerotolerant anaerobes will grow throughout the tube; however, facultative anaerobes will grow most densely where oxygen is present.

Anaerobic Jar 

The nutrient agar plates employed for routine culturing do not contain reducing agents. They can however be used to culture anaerobes if they are placed inside an anaerobe jar - a chamber from which the oxygen is removed.  Commercially available gas generator packets (e.g. GasPak) are placed in the jar along with plates or tubes containing conventional media destined to be incubated anaerobically.  The introduction of water, along with the palladium catalyst in the lid, induces the generation of hydrogen and carbon dioxide gases.  The hydrogen combines with the oxygen in the jar to produce water.  The lid, of course, is sealed shut.  In order to assure that oxygen actually was removed from the chamber, a strip of paper soaked in methylene blue dye is included in the jar.  It is blue when exposed to oxygen but will become colorless (white) when oxygen is absent. 

Candle Jar 

The candle jar can be used to decrease the concentration of oxygen in the culture environment.  Bacterial plates or tubes are placed in a jar along with a lighted candle.  The lid is sealed.  As the flame burns, oxgyen decreases and the flame goes out.  Oxygen is present, but at a lower percentage than in the atmosphere. The concentration of carbon dioxide increases as a result of the flame; this is also desirable for many microaerophiles. 

Click on the image to see an enlarged version. Growth in image 1 occurs between the blue lines as indicated by the arrow.  

1. thioglycollate 2. anaerobe jar 3. candle jar
 
© 1998