lab 15

Separation of Lactose Fermentation Profiles

  • Observational Contrast in Growth on Laboratory Plates

    • Significant differences in appearance noted between two sides of the laboratory bench.

    • Color variations indicate different growth characteristics.

    • Examples: A black plate indicates negative results under certain conditions.

  • Color Indicators and pH Levels

    • Presence of pink color on the plate suggests a low pH due to high acid concentration.

    • Low pH correlates with a lot of acid present.

Gram-Negative Bacteria Testing

  • Initial Point in Testing Flowchart

    • The catalyst test is the branching moment for samples derived from Enterobacter.

    • The first major decision point is the MAC test (MacConkey Agar test).

    • It aids in distinguishing five gram-negative lactose fermenters.

    • Possible profiles include MR (Methyl Red) profile, citrate profile, or other pathways.

    • Each student's flowchart may uniquely reflect their findings.

    • All four tests must eventually be documented in lab reports.

  • Indole Test

    • Focus: The breakdown of tryptophan by the enzyme tryptophanase.

    • Definition: The indole test checks for the presence of indole, indicating tryptophan metabolism.

    • Important for understanding bacterial profiles, specifically for E. Coli which tests positive.

    • When Kovac's reagent is added, a cherry red ring at the top signifies a positive reaction, confirming that indole is present.

    • Positive result: Proteus vulgaris gives a positive indole test while other species typically remain negative.

Methyl Red Test

  • Purpose: Determines if glucose fermentation occurs, distinguishing between mixed acid and 2,3-butanediol fermentation pathways.

    • Definition: Methyl red serves as a pH indicator that turns red in acidic conditions, confirming fermentation.

    • Method:

    • Add methyl red to the glucose medium.

    • Results:

      • Positive test results in a red color; indicates heavy acid production.

      • Negative test results in yellow or brown shades, indicating little to no fermentation.

Voges-Proskauer (VP) Test

  • Investigates fermentation pathways in bacteria.

    • If positive, it indicates 2,3-butanediol is present.

    • Procedure:

    • Two reagents A and B are added to differentiate from methyl red results.

Separation of Lactose Positive and Negative Fermenters

  • Lactose Positive Fermenters

    • Strong lactose fermenters are identified, including E. Coli and Citrobacter, which are MR positive and VP negative.

    • Lactose Negatives

    • Cannot ferment lactose but may still ferment glucose.

    • Positive results for the methyl red test in species like Proteus and Morganella.

Citrate Test

  • Tests if bacteria can utilize citrate as the sole nitrogen source.

    • Bromothymol blue is the pH indicator used in this test.

    • Turns blue upon positive reaction, indicating the breakdown of nitrogen and ammonia production.

    • Negative test results in a green coloration, showing no nitrogen source usage.

Differential Reagent Implications

  • Two types of pH indicators discussed:

    • Neutral red (low pH, indicating acid presence).

    • Phenol red (high pH, indicating basic conditions).

  • Example of Proteus mirabilis showing heavy growth and bright pink, linked to significant acid production which can cause bile precipitation in medium.

    • Indicative of one plausible identification method based on observed reactions.

General Test Observations

  • No memorization of species necessary; rather focus on:

    • Recognizing pH indicators and their results.

    • Identifying substrate use and resultant metabolic profiles.

    • Understanding which products are being formed and the gases produced.

    • Notes on bacteria's inability to ferment sugars but may use alternative pathways.