MCB2010L Carb Catabolism pres

Carbohydrate Catabolism Overview

• Focuses on biochemical tests, acid-base reactions, and energy and metabolism.

• Refer to pages 268-269 for a foundational understanding.

• Exercises include tests for fermentation and metabolic differentiation:

  • 5-2: Phenol Red Fermentation Broth (pg. 279-286)

  • 5-3: Methyl Red & Voges-Proskauer Tests (pg. 287-294)

  • 5-7: Citrate Utilization Test (pg. 313-318)

  • 5-10: Starch Hydrolysis (Amylase) Test (pg. 331-336)

5-2 Phenol Red Fermentation Broth

• A differential fermentation medium.

• Distinguishes members of Enterobacteriaceae and identifies them from other Gram-negative rods.

• Helps identify Gram-positive fermenters (e.g., Streptococcus and Lactobacillus).

Composition

• Peptone: Protein source.

• Carbohydrate: Substrate (e.g., glucose, sucrose, lactose).

• Phenol Red: pH indicator.

  • pH < 6.8 = yellow (indicates acid production).

  • pH 6.8-7.4 = red (neutral pH).

  • pH > 7.4 = pink/magenta (indicates alkaline reaction).

• Durham Tube: Gas collector to indicate gas production.

Phenol Red Fermentation Rxn

• Indicates fermentation of glucose, lactose, and sucrose.

• Deamination of amino acids leads to ammonia production, alkalizing the medium.

Results Interpretation

Experimental Procedure

• Prepare 2 tubes of PR glucose and 2 tubes of PR sucrose.

• Inoculate with test organisms and incubate for 24 hours at 37°C.

• Document results based on Table 5-2 in the manual.

5-3 Methyl Red & Voges-Proskauer Tests

• MR-VP Medium: Combination medium for MR and VP tests.

• Differentiates members of Enterobacteriaceae.

Composition

• Contains peptone, glucose, and phosphate buffer (resists pH changes).

Methyl Red Test

• Detects mixed acid fermentation of glucose.

• Acidic products overcome phosphate buffer, permanently lowering the pH.

Results Interpretation

Voges-Proskauer Test

• Detects glucose fermentation producing acetoin and butanediol.

• Neutral pH characterizes the products.

Results Interpretation

pH Changes in MR–VP Broth

• MR-positive: stable acidic products permanently lower the pH.

• VP-positive: initial production of lactic acid activates 2,3-butanediol fermentation pathway.

Citrate Utilization Test

• Differentiates members of Enterobacteriaceae and other Gram-negative rods.

• Requires citrate permease enzyme for transport into the cell.

Composition

• Simmons citrate agar: defined medium.

• Citrate is the only carbon source; ammonium phosphate is the only nitrogen source.

Results Interpretation

• Medium contains bromothymol blue dye as a pH indicator.

  • Green at pH 6.9.

  • Blue at pH 7.6 indicates citrate utilization.

Procedure

• Inoculate Simmons citrate slants and incubate at 37°C for 48 hours.

Starch Hydrolysis (Amylase Test)

• Used to identify bacteria like Gardnerella vaginalis and differentiate certain genera:

  • Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and Enterococcus.

Enzymatic Activity

• Starch is a complex polysaccharide; it must be hydrolyzed extracellularly by exoenzymes:

  • α-amylase and β-amylase produce glucose and maltose.

Results Interpretation

• Iodine exposure to starch produces a dark brown to blue-black color.

  • Positive result: Clearing around growth indicates starch hydrolysis.

  • Negative result: No clearing indicates no amylase production.

Procedure

• Inoculate starch agar with E. coli and B. subtilis, incubate for 48 hours at 37°C, then assess results.