Protein-Catabolism-Student

Fermentation of Carbohydrates

  • Fermentation Process: Involves breakdown of carbohydrates.

Phenol Red Indicator

  • Yellow Color: Indicates carbohydrate metabolism, meaning acids were produced from sugar breakdown.

    • Phenol red is yellow at acidic pH.

  • Fuchsia/Pink Color: Indicates peptone utilization, showing ammonia (a base) production.

    • Phenol red appears pink at basic pH.

Fermentation Test Results Interpretation

  • Yellow Color Change:

    • Indicates (+) fermentation.

  • Red/Pink Color Change:

    • Also indicates (-) fermentation and (+) peptone utilization.

  • Gas Production:

    • Significant sized bubbles in the Durham tube indicate (+) gas production.

    • Tiny bubbles or no bubbles indicate (-) gas production.

True Fermenters vs. Non-Fermenters

  • Observation: Determine which organisms are true fermenters based on test results observed.

  • Procedure: Share fermentation tubes and assess results for un-inoculated organisms.

  • Disposal: Remove tape from test tubes and dispose of them correctly in the designated area.

Protein Catabolism Part 1

  • Context and readings covered on pages 125-130.

Gelatinase/Gelatin Hydrolysis Test

  • Ingredients: Beef extract, Peptone, Gelatin.

  • Purpose: To differentiate organisms based on their capability to produce the exoenzyme gelatinase.

  • Mechanism: Gelatin hydrolysis by gelatinase changes the medium from solid to liquid.

Hydrolysis Process

  • Products: Gelatin → Amino Acids + Ammonia.

  • Enzyme Type: Gelatinase acts as an exoenzyme.

Gelatinase Test Incubation

  • Inoculation Method: Using a needle, not a loop.

  • Incubation Conditions: 4-7 days at room temperature (25°C).

  • Justification:

    • Growth is slow at lower temperatures, requiring extended incubation.

    • Gelatin melts between 25-40°C and should remain solid at 37°C to ensure test integrity.

Gelatinase Test Results Interpretation

  • Liquid Medium: Indicates both gelatinase production (+) and gelatin hydrolysis (+).

  • Solid Medium: Indicates negative results (-) for both gelatinase production and gelatin hydrolysis.

Agar vs. Gelatin

  • Agar:

    • Melts at 100°C.

    • Not broken down by bacteria and cannot serve as a nutrient.

  • Gelatin:

    • Melts at 25-40°C and can be degraded by some bacteria, providing nutrition.

Urease Test

  • Ingredients: Urea, Yeast extract, Phenol red.

  • Purpose: Differential test for urease production.

  • Mechanism: Urease hydrolyzes urea into ammonia and carbon dioxide; ammonia changes the pH indicator to pink/fuchsia.

Urease Test Results Interpretation

  • Hot Pink Color Change: Indicates positive results for urease production and urea hydrolysis.

  • Orange/Yellow or No Color Change: Negative for both urease and urea hydrolysis.

Protein Catabolism Part 2

  • Context and readings covered on pages 131-136.

Deamination vs. Decarboxylation

  1. Deamination: Removal of the amino group from amino acids resulting in ammonia production.

  2. Decarboxylation: Removal of carbon dioxide from amino acids leading to amine formation.

Motility, Indole, Ornithine (MIO) Test

  • Ingredients: Peptone, Glucose, pH indicator (bromocresol purple), Tryptophan, Ornithine.

  • Purpose: To evaluate motility, decarboxylation ability of ornithine, and indole production.

MIO Results Processing

  • Order of Reading:

    1. Assess motility.

    2. Check ornithine decarboxylation via media color change.

    3. Test for indole production using Kovac's reagent for color change.

Indole Production

  • Indole Test: Indicates red ring formation when Kovac’s reagent is added if indole is present.

  • Indole Reaction: Tryptophan → Indole + Pyruvic Acid + Ammonia.

MIO Results Interpretation

  • Growth Moving from Inoculation Point: Indicates (+) motility.

  • Growth Only Along Inoculation Point: Indicates (-) motility.

  • Red Layer After Kovac's Reagent: Indicates (+) for indole and tryptophanase production.

  • No Color Change: Indicates (-) for indole and tryptophanase, yellow indicates acidic reaction, not anything regarding the test.

Phenylalanine Slant

  • Differential Test Purpose: Tests the production of the endoenzyme deaminase.

  • Mechanism: Deaminase removes amine group from phenylalanine, producing phenylpyruvic acid.

Phenylalanine Slant Results Interpretation

  • Dark Green Color Change: Indicates (+) presence of phenylpyruvic acid and deaminase activity.

  • No Color Change: Indicates (-) for both phenylpyruvic acid and deaminase activity.

Sulfur in Amino Acids

  • Context: Some amino acids possess sulfur; specific bacteria can release hydrogen sulfide (H2S).

  • Detection Method: Heavy-metal salt with ferrous ion is required in the medium to detect H2S production.

Peptone Iron Deep Test

  • Purpose: Differential test for hydrogen sulfide (H2S) production where cystine and iron ion interact if H2S is produced.

Peptone Iron Deep Results Interpretation

  • Black Color Change: Indicates (+) for H2S production.

  • No Black Color Change: Indicates (-) for H2S production.

Today’s Lab Activities

  • Required Media: 2 of each - Urea Broth, MIO, Gelatinase Test, Peptone Iron Deeps, Phenylalanine Slants.

  • Inoculate: Using proper organisms for tests.

  • Incubation:

    • Urea and Phenylalanine Slants, Peptone Iron, MIO: Incubator.

    • Gelatinase: Room temperature.

  • Label: Include Name, Date, Lab Section, Media Name, and Organism Name for each tube.