Microbiology Chapter 17: Red Phenol Broth

Chapter 17: Red Phenol Broth

• Purpose: Differentiates gram-negative bacteria based on their ability to ferment carbohydrates. The durham tube shows gas production.
• Fermentation: Bacteria fermenting carbs produce weak acid, lowering pH and changing color from red to yellow. This is a positive result.
• Deamination: Some bacteria which are negative for fermentation break down peptones into amino acids, excreting ammonia which turns the broth pink. This is a negative result for fermentation.
• Cellular Respiration: If carbs are utilized, but no color change occurs, the broth remains neutral, indicating a negative fermentation result.
• Phenol Red Broth components: Substrates (lactose, sucrose, glucose, mannitol) and a pH indicator (phenol red) are included. The indicator turns yellow or pink (positive) when fermented, red (negative) otherwise.
• Durham Tube Purpose: Detects gas production during fermentation.
• Results Interpretation: Determine if organisms utilize fermentation, not just for fermentation, but also consider the production of gas, cellular respiration and deamination as well.
• False Positives: Incubation > 24 hours at 37°C can lead to a false positive, due to depletion of the fermentable sugar and subsequent ammonia production from peptone metabolism.

Chapter 18: Nitrate Broth

• Purpose: Differentiates bacteria based on their ability to reduce nitrate or nitrite during anaerobic respiration. Determines if nitrate is used as a final electron acceptor.
• Aerobic Respiration vs. Anaerobic Nitrate Reduction: Aerobic respiration uses oxygen as the final electron acceptor, while anaerobic respiration using nitrate results in the reduction of nitrate to nitrite.
• Nitrate Broth Substrate: Potassium nitrate (acts as a nitrogen source for possible reduction).
• Reagents in Nitrate Reduction Test: Reagent A (sulfanilic acid) tests for nitrite; Reagent B (α-naphthylamine) tests for nitrite. Reagent C (zinc powder) is used as a catalyst if the initial tests are inconclusive.
• Positive Result: Pink/red precipitate after reagents A and B. Indicates partial/complete nitrate/nitrite reduction which is positive for anaerobic respiration.
• Negative Result: No precipitate after reagents A and B. If a positive response was not observed after 30 seconds, then reagent C is added to reduce nitrate to nitrite and observe for pink/red precipitate. A negative result indicates that the bacteria is unable or unwilling to use nitrate during anaerobic respiration.
• Enzymes: Nitrate reductase reduces nitrate to nitrite.
• Interpreting Results: Results indicate organisms' ability to utilize partial or complete nitrate reduction.

Chapter 19: Motility Medium

• Purpose: Identifies bacterial motility and differentiates based on respiration type (aerobic or anaerobic).
• Results Interpretation: Observe for motility (growth away from inoculation streak) and note if TTC is reduced by metabolizing bacteria (shows oxidation/reduction with red/pink) to identify if there is respiration.
• Redox Indicator: Tetrazolium salt (TTC). Oxidized form is colorless, reduced form (formazan) is red or pink due to bacterial metabolism.

Chapter 21: Mannitol Salt Agar (MSA)

• Purpose: Selective and differential medium for isolating and differentiating staphylococci.
• Selective Agent: 7.5% sodium chloride (NaCl); selects for salt-tolerant staphylococci.
• Substrate: Mannitol (sugar alcohol) for fermentation.
• Fermentation: If mannitol is fermented, acid is produced, decreasing the pH, and changing the phenol red indicator from red to yellow.
• Differentiation: Differentiates pathogenic from non-pathogenic Staphylococcus by observing for mannitol fermentation and colour change.

Chapter 22: Coagulase Test

• Purpose: Differentiates Staphylococcus aureus (coagulase-positive) from other staphylococci (coagulase-negative).
• Substrate: Rabbit plasma.
• Enzyme: Coagulase (enzyme detected).
• Result: Positive result = clot formation. Negative result = no clot formation.

Chapter 23: Blood Agar

• Purpose: Differential and enriched medium to determine hemolysis patterns (how bacteria affect red blood cells).
• Hemolysis Types:
  • Beta: Complete hemolysis (clearing around the colonies).
  • Alpha: Partial hemolysis (greening around the colonies).
  • Gamma: No hemolysis (no change around the colonies).
• Virulence: Hemolysis production is linked to virulence. Bacteria that produce hemolysins are more virulent and harmful to the host.

This quiz focuses on Chapter 17, detailing the Red Phenol Broth test used to distinguish gram-negative bacteria based on their fermentation abilities. Participants will explore concepts such as fermentation, pH changes, and gas production using Durham tubes. Interpret results of cellular respiration and deamination to understand bacterial behavior.