Carbohydrate Broths

• Components and Functionality:

  • Peptone broth with a sugar and a pH indicator.
  • pH Indicator: Phenol red is utilized as the pH indicator.
  • If pH < 7: solution or media appears yellow.
  • If pH > 7: solution or media appears red or magenta.

• Usage: Carbohydrate (CHO) broths are not primarily used to differentiate among all organisms, but they are especially important for distinguishing Gram-negative bacteria, specifically Gram-negative short rods utilizing lactose.

• Bacterial Metabolism:

  • Bacteria ferment sugars to produce either acid or gas.
  • If bacteria do not ferment the sugar:
  • They utilize the protein in the media.
  • This process produces ammonia, leading to an increase in the pH of the solution.

Observations on Carbohydrate Fermentation

• Gram-negative Bacteria:

  • Notable examples include E. coli, Enterobacter, Proteus, and Pseudomonas.

• Fermentation Results:

  • All bacteria except Pseudomonas ferment glucose.
  • E. coli and Enterobacter ferment lactose while producing gas.
  • Proteus and Pseudomonas do not ferment lactose.
  • The first three (except Pseudomonas) are part of the Enterobacteriaceae family (enteric group of bacteria).

Further Discussion on Carbohydrate Metabolism

• Micrococcus and Pseudomonas:

  • These organisms do not ferment any sugars due to their status as obligate aerobes.

• Gas Production during Fermentation:

  • Gas production indicates a strong fermentation. For instance, E. coli is identified as a strong fermenter of lactose.

• Color Change Observations:

  • When analyzing Enterobacter, a layer of red may form on top of the yellow broth due to the following:
  • Enterobacter is a weaker fermenter of lactose and does not significantly decrease the pH, thus lacking a buffering capacity in the phenol red broth.
  • After fermenting lactose, it starts using the protein as a substrate, producing ammonia, which increases the pH and reverts the broth slightly to red. This phenomenon is termed reversion of products.

Practical Steps for Laboratory Procedure

• Consolidate all nitrate broths together and transfer to a separate area of the test tube rack.
• Organize sugars in the following order: G (Glucose), L (Lactose), M (Mannitol), S (Sucrose) each attached to the appropriate bacterium label facing front.
• Capture an image of the arrangements and upload it to Canvas under the corresponding bacterium section.
• Repeat the procedure for other bacteria, including lab unknown cultures #1 (A & B).
• Ensure to upload lab unknown #1 images in the specified location.
• After utilizing the CHO broths for inoculating selective and differential media plus Nutrient Agar (NA) for catalase tests, retain the sugars for an additional lab period for further observation.