The Thioglycollate Broth or Oxygen Determination Tests: Atlas page 7
|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
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 (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. Facultative anaerobes, grow throughout the media but heavier at the bottom and facultative aerobes grow thoughout but more at the top. The facultatives grow most densely where oxygen is present. The aerotolerants grow equally well thoroughout the tube... Usually this test should be read at about48 hrs. although some slower growing microbes may take more incubation time.
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.
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.
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