Nitrate Reductase Test Overview

Oct 17, 2024

Microbiology Lab: Nitrate Broth and Nitrate Reductase Test

Introduction

  • Presenter: Kevin Tokoff
  • Purpose: Discuss nitrate broth/nitrate reductase test
  • Key Elements:
    • Importance of learning the flowchart as it's not provided in exams/quizzes
    • Explanation of the test results and underlying mechanisms

Test Reactions

  • Two Simultaneous Reactions:
    1. Nitrate Reductase: Converts nitrate (NO3-) to nitrite (NO2-)
      • Mnemonic: Nitrate (A-T-E) comes before nitrite (I-T-E) alphabetically
    2. Nitrite Reductase: Converts nitrite (NO2-) to ammonia (NH3) and nitrogen gas (N2)

Enzyme Presence Scenarios

  • Possible enzyme combinations in bacteria:
    1. Only nitrate reductase
    2. Both nitrate and nitrite reductase
    3. Neither enzyme present

Procedure

  1. Initial Reagents:
    • Add 16 drops of sulfonylic acid (solution A) and alpha-naphthalamine (solution B) to nitrate broth
  2. Possible Immediate Outcomes:
    • Turns Red: Positive for only nitrate reductase
    • No Color Change: Requires further testing

Further Testing with Zinc Dust

  • If no color change occurs after initial reagents, add zinc dust:
    • Still No Color Change After Zinc: Indicates both enzymes are present
    • Turns Red After Zinc: Indicates neither enzyme is present

Interpretation of Results

  • Immediate Red Color Change:
    • Only nitrate reductase is present
  • No Initial Change, Red After Zinc:
    • Neither enzyme is present
  • No Change Even After Zinc:
    • Both enzymes are present

Theoretical Explanation

  • Nitrate and Nitrite Importance:
    • Immediate Red Color: Due to nitrite presence reacting with reagents to form a red dye
  • Mechanism for No Initial Color Change:
    • Both enzymes present: Nitrate converted to nitrite, then to nitrogen and ammonia (no nitrite left)
    • Neither enzyme: Broth remains loaded with nitrate, zinc converts nitrate to nitrite turning it red

Key Takeaways

  • Learn and understand the flowchart for predicting results
  • Know how reagents and zinc dust are used to determine enzyme presence
  • Mechanism Knowledge: Optional for exams but helps in understanding results

Conclusion

  • Understanding the flow chart and result prediction is vital
  • Remember the sequence: initial reagents, possible color changes, and addition of zinc dust

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