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What does each reagent do?

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In LifeCheckTM ATP testing, each reagent plays a critical role in ensuring accurate and reliable microbial quantification. This article explains the function, handling, and best practices for each reagent used in the workflow.

Why is Each Reagent Important?

Each reagent plays a vital role in delivering precise and reliable ATP test results:

Standard (Calibration)

The Standard is a stable ATP solution with a concentration of 1000 pg/mL . It serves as a calibration tool and ensures comparability of results over time.

Key Functions:

  • Provides a baseline for ATP quantification, allowing conversion of RLU (Relative Light Unit) results into ATP concentrations.
  • Compensates for variations in reagents, temperature, analyst technique, and luminometer sensitivity, ensuring results remain comparable over time.
  • Serves as a quick measure of Reagent Z activity and luminometer performance.

Usage Instructions:

  • Hold the bottle vertically.
  • Add two drops (100 µL) to a 12x55 mm test tube for calibration.
  • Mix gently before use.

Storage Best Practices:

  • Store unopened at room temperature for long-term stability.
  • Once opened, use within three months, then discard and replace.
  • Avoid leaving the bottle uncapped or exposing it to excessive light or heat.

Reagent X (Extraction)

Reagent X is a highly aggressive lysing agent designed to break open microbial cells and release ATP for measurement.

Key Functions:

  • Breaks open microbial cells, ensuring effective ATP extraction for accurate quantification.
  • Stabilizes ATP during the extraction process.
  • Reagent X is more aggressive than conventional ATP testing reagents, requiring dilution to avoid enzyme inhibition during the assay step.

Reagent Z (Detection)

Reagent Z, or Luminase, is the enzyme responsible for the bioluminescent reaction that quantifies ATP.

Key Functions:

  • Reacts with ATP to produce light, proportional to the ATP concentration, which is measured as RLU in the luminometer.
  • Provides sensitive and reliable quantification of ATP.

Usage and Handling:

  • Rehydrate the freeze-dried enzyme by adding the rehydration buffer.
  • Allow the mixture to dissolve for at least 5 minutes at room temperature.
  • Avoid contamination when removing the glass vial stopper.

Sensitivity Monitoring:

  • If the RLU reading for the Standard is less than 5,000 RLU, the activity of Reagent Z is too low, and a new vial should be rehydrated for optimal sensitivity.

Storage Best Practices:

  • Freeze-Dried: Store in the refrigerator or freezer for long-term stability.
  • Rehydrated: Store in the fridge for 2–4 weeks or in the freezer for 3months. Allow the enzyme to warm naturally to room temperature before use.
  • You can always test your enzyme and if RLU is >5000 it is good to use.

Reagent D (Dilution)

Reagent D is a dilution reagent critical for ensuring accurate assay performance.

Key Functions:

  • Dilutes the lysate from Reagent X to:
    • Neutralize dissolved materials that may interfere with the assay.
    • Prevent the high alkalinity of Reagent X from inhibiting Reagent Z during the light measurement step.

Rinse Reagent (Cleaning)

The Rinse Reagent is used to clean filters and surfaces during the testing process.

Key Functions:

  • Dissolves organic contaminants, such as hydrocarbons in oil and gas samples, on the filter surface without affecting ATP, which remains bound to the filter
  • Removes residues that may interfere with subsequent steps in the process

Soak Reagent (Stabilization)

The Soak Reagent stabilizes ATP during the testing process to ensure accurate quantification.

Key Functions:

  • Stabilizes free-floating ATP, ensuring it remains measurable.
  • Releases bound and complexed ATP, allowing for precise measurement while leaving intact cells unharmed.

Need Help or Want to Learn More?

Our team of experts is here to assist you with your microbiological monitoring needs. Whether you have questions, need technical support, or want to learn more about LifeCheckTM ATP testing, contact LuminUltra® Support to get started.

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