OBJECTIVES: Students should be able to 1. distinguish between the categories of bacteria based
on responses to oxygen 2. explain the biological basis for each category 3. culture aerobes, facultative anaerobes,
microaerophiles, and aerotolerant anaerobes 4. describe how the anaerobic glove box works
Bacteria are often categorized according to their growth responses to atmospheric oxygen. This may vary from species that can grow only in the presence of oxygen to those that can grow only in the absence of oxygen. The strict or obligate aerobes must have oxygen to metabolize. The strict or obligate anaerobes must reside in oxygen-free environments in order to survive. Their metabolism does not use molecular oxygen and they cannot protect themselves from the toxic byproducts that oxygen forms within the cell. Thus in the presence of even trace amounts of oxygen these cells will die. Facultative anaerobes can grow in either the presence or absence of oxygen. They have two forms of metabolism, one is aerobic and the other is anaerobic. They use oxygen if it is available and switch to the anaerobic pathways if it is not. Aerotolerant anaerobes are indifferent to the presence of oxygen. They only have anaerobic metabolism, but can remove the toxic byproducts of oxygen. Thus these anaerobes can survive in oxygen even though they don't use it for energy production.Microaerophiles can grow without oxygen, but prefer to grow in low oxygen concentrations. High levels of oxygen inhibit enzymes critical for growth and are toxic to microaerophiles.
Fluid thioglycollate broth is a reducing medium, that is, it contains compounds that react with molecular oxygen keeping the free levels low. It also contains the indicator dye reazurin which turns pink in the presence of oxygen. Since oxygen is present at the surface of the medium, the upper layer is usually pink whereas the dye is colorless in the remainder of the tube. Agar is also included in this medium to give it a semisolid consistency. This prevents the movement of inoculated organisms. Fluid thioglycollate broth has something for every microbe. Strict aerobes will grow only at the top of the tube, strict anaerobes only at the bottom, facultative and aerotolerant anaerobes throughout the tube, and microaerophiles somewhat below the surface.
Anaerobic jars such as the GasPak are vessels in which an anaerobic environment is generated after inoculated media are sealed into the chamber. Anaerobiasis is achieved by adding water to commercially available gas generator envelopes that are placed in the jar just prior to sealing. Chemicals in the envelope produce hydrogen gas and carbon dioxide. The hydrogen combines with free oxygen in the chamber to produce water. The carbon dioxide is required for the growth of certain organisms. A methylene blue indicator strip is usually placed in the jar. It turns colorless when the oxygen has been removed.
The candle jar is used to create microaerophilic conditions. It is a large screw-capped container into which the medium is placed along with a candle. The candle is lit and the jar is sealed. The candle will burn and reduce the oxygen concentration.
MATERIALS
Cultures (in broth)Clostridium sporogenes (strict anaerobe)
E. coli (facultative anaerobe)
B. megaterium (strict aerobe)
N. sicca (microaerophile)
PROCEDURES
FLUID THIOGLYCOLLATE
1. Check to make certain that no more than 20% of the upper portion of the medium is pink.2. Inoculate the tube to the bottom and gently rotate between the palms of your hands to disperse the organism. Do not shake or oxygen will be added to the medium.
3. Incubate the tubes for 48 hours.
4. Record the pattern of growth.
ANAEROBIC JAR
1.Streak the organism on the appropriate agar medium.
2. Place the plate in the anaerobic jar. When all plates are in the container, the indicator strip (it should be blue) and the gas generator envelope are added. Water is added with a pipette. The jar is sealed and placed in an incubator for 48 hours. The indicator should be checked to make certain that all oxygen has been removed.
3. A second plate should be placed in the incubator as an aerobic control.
4. Observe for growth.
CANDLE JAR
1. Streak the organism on the appropriate agar medium
2. Place the plate in the glass candle jar. When all plates are in the jar, the instructor will place a candle in the jar, light the candle and close the lid. When the candle has stopped burning, place the jar into an incubator for 48 hrs.3. Observe for growth.
Assessing your performance
Fluid thioglycollate 1. Is less than 20% of the upper portion of the agar pink
? 2. Is growth of C. sporogenes visible only at the bottom
of the tube? 3. Is growth of E. coli visible throughout the tube? 4. Is growth of B. megaterium visible only at the surface
of the tube? 5 Is growth of N. sicca visible just below the surface of
the tube? Anaerobic jar/candle jar 1. Is there growth of C. sporogenes and E. coli from the
anaerobic jar? 2. Is there growth of E. coli and B. megaterium from the
control aerobic plate? 3. Is there growth of E. coli and N. sicca from the
candle jar? 4. Are all plates labeled correctly?
Questions
1. Complete the following chart
Organism
Metabolizes oxygen
Growth in presence of oxygen
Growth in absence of oxygen
Detoxifies byproducts of oxygen
Aerobe
Facultative anaerobe
Strict anaerobe
Aerotolerant
Microaerophile
2. Compare the results with growth on the aerobic control plate and describe the oxygen requirements of your organism.
3. If an organism is a strict anaerobe, it cannot survive in the presence of oxygen even for a short time. How might you culture these organisms?
4. An aerobe, facultative anaerobe, strict anaerobe, and an aerotolerant anaerobe are inoculated on a medium in a sealed container with oxygen present. Explain what will happen.