Resazurin Test

The Resazurin Test is a rapid, indirect method used to assess the microbial quality of milk by measuring the activity of bacteria present in the sample. It’s based on the reduction of a dye (resazurin) by bacterial metabolism, which causes a color change that correlates with microbial load. This test is particularly useful for evaluating raw milk freshness and hygiene at farms, collection centers, or processing plants. Here’s a detailed explanation:

Purpose of the Resazurin Test

• Microbial Quality: Estimates bacterial contamination, indicating milk freshness and storage conditions.

• Hygiene Check: Reflects milking and handling practices.

• Screening Tool: Quickly identifies milk that may spoil prematurely or fail stricter microbial standards.

Principle

• Resazurin is a redox-sensitive dye that starts as blue in its oxidized form. When added to milk, metabolically active bacteria reduce it through respiration or fermentation:

  • Blue (oxidized) → Pink (partially reduced, resorufin) → Colorless (fully reduced, dihydroresorufin).

• The rate and extent of color change depend on the number and activity of bacteria. Higher bacterial loads reduce the dye faster, signaling poorer milk quality.

Procedure

1. Sample Collection:

   • Take a representative sample of raw milk (e.g., 10 mL) from a well-mixed source (e.g., bulk tank).

2. Equipment:

   • Sterile test tubes or vials.

   • Resazurin solution (typically 0.005-0.01% w/v, prepared fresh or stabilized).

   • Water bath or incubator (37°C/98.6°F).

   • Pipettes and a comparator (optional, for color standards).

3. Steps:

   • Add a fixed volume of resazurin solution (e.g., 1 mL) to 10 mL of milk in a test tube.

   • Mix gently to ensure even distribution.

   • Incubate at 37°C (body temperature, optimal for bacterial activity).

   • Observe color change at set intervals (e.g., 10 minutes, 1 hour, or 3 hours, depending on protocol).

4. Observation:

   • Compare the color to a standard chart or scale (e.g., Lovibond comparator or visual guide).

Common Protocols

• 10-Minute Test:

  • Quick screening; assesses very high bacterial loads.

  • Blue: Excellent quality.

  • Pink: Moderate quality.

  • Colorless: Poor quality.

• 1-Hour or 3-Hour Test:

  • More sensitive; used for detailed grading.

  • Color assessed after longer incubation (e.g., 1 hour often correlates with TBC standards).

Interpretation (Example Scale)

• Blue (Unchanged): Very low bacterial activity (<50,000 CFU/mL), excellent quality.

• Blue-Purple: Low activity (50,000-200,000 CFU/mL), good quality.

• Pink: Moderate activity (200,000-1,000,000 CFU/mL), fair quality.

• Colorless: High activity (>1,000,000 CFU/mL), poor quality.

• Note: Exact CFU correlations vary by milk type, temperature, and test duration; calibration with plate counts is needed for precision.

Factors Affecting Results

1. Bacterial Load:

   • Higher counts (e.g., >10⁶ CFU/mL) reduce the dye quickly.

   • Psychrotrophs (e.g., Pseudomonas) may reduce slower if incubated at 37°C vs. their optimal cold growth.

2. Milk Condition:

   • Fresh milk (stored <4°C) shows slower reduction.

   • Spoiled milk (high acidity) accelerates reduction due to active bacteria.

3. Non-Bacterial Factors:

   • Somatic cells (e.g., from mastitis) can reduce the dye slightly.

   • Milk enzymes or leukocytes may interfere, though less significantly.

4. Temperature:

   • Incubation at 37°C speeds up bacterial metabolism; colder temps slow the test.

Applications

• Farm Level: Monitors hygiene and cooling efficiency.

• Collection Centers: Screens raw milk before transport, especially in regions without advanced labs.

• Processing Plants: Part of platform testing to reject heavily contaminated milk.

Advantages

• Rapid: 10-minute version gives quick results; 1-3 hours still faster than plate counts (48 hours).

• Simple: Minimal equipment (dye, tubes, incubator).

• Cost-Effective: Affordable for small-scale operations.

Limitations

• Indirect: Measures bacterial activity, not exact counts; doesn’t identify pathogens.

• Sensitivity: May miss low-level contamination or slow-growing bacteria.

• Interference: Non-microbial reduction (e.g., by somatic cells) can skew results.

• Subjectivity: Color interpretation varies without a comparator.

Correlation with Other Tests

• Total Bacterial Count (TBC): Rough correlation; e.g., pink after 1 hour often aligns with >200,000 CFU/mL.

• COB/Alcohol Tests: High bacterial activity (colorless resazurin) often precedes protein instability.

• pH/Acidity: Faster dye reduction aligns with lower pH (<6.6) from spoilage.

Practical Example

• Scenario: 10 mL milk + 1 mL resazurin, incubated 1 hour at 37°C.

  • Result: Stays blue—excellent quality, accepted.

  • Alternative: Turns pink—fair quality, flagged for further testing; colorless—rejected.