Oxygen Requirements and Nitrate Test

Oxygen Tolerance and Bacterial Classification

• Understanding different oxygen requirements helps in identifying bacterial species.
• Classified based on their ability to tolerate or require oxygen:
• Obligate Aerobes:
  • Require oxygen for growth.
  • Participate in aerobic respiration.
  • Grow at the top of culture media where [O2] is high.
  • Have both catalase and superoxide dismutase to detoxify harmful forms of oxygen.
• Facultative Anaerobes:
  • Prefer aerobic conditions but can switch to anaerobic respiration or fermentation when oxygen is absent.
  • Grow throughout the medium but denser at the top due to higher ATP yield from aerobic respiration.
  • Also have catalase and superoxide dismutase.
• Obligate Anaerobes:
  • Cannot survive in oxygen; even small amounts are lethal.
  • Grow only at the bottom of culture media.
  • Lack catalase and superoxide dismutase.
• Microaerophiles:
  • Survive in lower than atmospheric oxygen levels (less than 21% O2).
  • Grow in the upper middle region of the medium.
• Aerotolerant Anaerobes:
  • Do not utilize oxygen but are not affected by its presence.
  • Grow uniformly throughout the medium.
  • Possess both catalase and superoxide dismutase.

Testing Oxygen Tolerance

• Fluid Thioglycollate Broth:
• Used to assess oxygen tolerance in bacterial species.
• Sodium thioglycolate and L-cystine reduce oxygen to water, assisting in the creation of an oxygen gradient.
• Resazurin serves as the oxygen indicator; red when oxidized, colorless when reduced.

Methods for Creating Anaerobic Environments

• Gas Pak Jar:
• Contains a packet with ascorbic acid, reducing O2 to create an anaerobic environment (0.1% O2).
• Candle Jar:
• A lit candle consumes some oxygen and creates a microaerophilic environment (approx. 10% O2).
• The combustion process generates CO2, beneficial for capnophilic microorganisms.

Reactive Oxygen Species (ROS) and Detoxification

• During aerobic respiration, toxic reactive oxygen species can be produced.
• Superoxide Radicals (O2-): Produced when oxygen is only partially reduced.
• Hydrogen Peroxide (H2O2): Can form when electrons are transferred directly to oxygen.
• Bacteria produce enzymes to detoxify ROS, such as:
• Superoxide Dismutase: Converts O2- to H2O2.
• Catalase: Converts H2O2 into harmless water and oxygen.

Catalase Test

• Determines the presence of catalase in bacteria.
• Reactions:
• Magically distinguishes catalase + organisms (e.g., Micrococcus) from catalase - (e.g., Streptococcus).
• Equation: 2H2O2 \xrightarrow{Catalase} 2H2O + O2

Nitrate Reduction Tests

• Participants can reduce nitrate (NO3-) during anaerobic respiration.
• Steps in the test:
  • Test for Nitrogen Gas (N2):
  • Check for gas in the Durham tube. Indicates presence of both enzymes (nitrate & nitrite reductase).
  • Test for Nitrite (NO2-):
  • If no gas, add Nitrate Reagents A & B. A red color indicates nitrate reductase; clear indicates need for further testing.
  • Test for Nitrate (NO3-):
  • If still clear after reagents, add zinc to the tube to reduce any unreacted nitrate to nitrite.
  • Color change confirms presence of nitrate. \n - Different bacteria can perform dissimilatory (reducing NO3- to N2) or assimilatory (reducing NO3- to NH4+) nitrate reduction.
• Final step of nitrate reduction confirms if ammonium (NH4+) is present after eliminating alternatives.