Biological Indicators – Confirmation of Viable Spore Count and Resistance Performance Test

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Biological Indicators (BIs) are essential tools in sterilization validation and routine monitoring. They provide direct evidence of microbial inactivation and confirm the efficacy of sterilization cycles. The two key parameters that define the performance of a biological indicator are the viable spore count and the resistance performance test. These parameters ensure that each BI lot is consistent, reliable, and capable of challenging the sterilization process effectively.

1. Introduction

Biological Indicators contain a known population of highly resistant bacterial spores, usually of species such as Geobacillus stearothermophilus (for steam sterilization) or Bacillus atrophaeus (for ethylene oxide and dry heat sterilization). The purpose of testing viable spore count and resistance performance is to verify the quality and consistency of the BI before use in sterilization validation or monitoring.

2. Objective

The main objectives of this study are:

  • To determine the actual number of viable spores per carrier (viable spore count).
  • To evaluate the resistance of the spores to the sterilization process (D-value and survival-kill time).
  • To ensure batch-to-batch consistency of biological indicators used in sterilization processes.

3. Materials and Equipment

  • Biological Indicators (spore strips or spore suspensions)
  • Sterile forceps and scissors
  • Tryptic Soy Agar (TSA) or suitable recovery medium
  • Incubator (55–60°C for G. stearothermophilus)
  • Steam autoclave or sterilizer
  • Test tubes, pipettes, sterile water for dilution
  • Spectrophotometer (optional for optical density check)

4. Test Method for Viable Spore Count

  1. Take 10 Biological Indicator strips from the lot to be tested.
  2. Cut each strip aseptically into small pieces and transfer to a sterile tube containing 10 mL sterile water or buffer.
  3. Vortex or sonicate gently to dislodge spores from the carrier.
  4. Prepare serial dilutions (10⁻¹ to 10⁻⁶) in sterile water.
  5. From each dilution, plate 1 mL on TSA plates in triplicate.
  6. Incubate at 55–60°C for 48 hours (for G. stearothermophilus) or at 30–35°C (for B. atrophaeus).
  7. Count the colony-forming units (CFU) and calculate the mean viable spore count per carrier.

Calculation:

Viable Spore Count (CFU/carrier) = Average CFU × Dilution Factor / Volume Plated

Acceptance Criteria:

  • The mean viable spore count shall be within ±0.5 log₁₀ of the labeled claim (e.g., 10⁶ spores per carrier).
  • Coefficient of variation (CV) should not exceed 20%.

5. Resistance Performance Test

The resistance performance of BIs is measured using the D-value and survival–kill time method.

5.1 D-Value Determination

D-value is the time (in minutes) required to reduce the spore population by 90% (one log reduction) under specified sterilization conditions.

  1. Expose sets of BI carriers to a series of increasing sterilization times (e.g., 0, 1, 2, 3, 4, 5 minutes at 121°C for steam sterilization).
  2. After each exposure, transfer carriers aseptically into recovery media tubes.
  3. Incubate all tubes at 55–60°C for 7 days and observe for growth (turbidity or color change).
  4. Plot survivor curve (log number of survivors vs. exposure time).
  5. Calculate the D-value from the negative reciprocal of the slope of the survivor curve.
D-value (min) = (t2 - t1) / (log N1 - log N2)

5.2 Survival–Kill Time Test

This test defines the time intervals where all units survive (survival time) and all are killed (kill time).

  • Survival time: The longest exposure time showing 100% survival (all positive).
  • Kill time: The shortest exposure time showing 100% kill (all negative).

Acceptance Criteria:

  • Survival–kill time difference should typically be between 1.5 and 3 minutes for 121°C steam BIs.
  • D-value should be within the manufacturer’s specified range (e.g., 1.5–2.5 minutes for 121°C steam).

6. Interpretation of Results

The BI batch is considered satisfactory if:

  • Mean viable spore count is within the acceptable range of the label claim.
  • D-value and survival–kill time are consistent with the manufacturer’s validated specifications.
  • No abnormal results (e.g., early kill or survival anomalies) are observed.

7. Documentation and Traceability

  • Record batch number, date of test, equipment used, sterilization conditions, and incubation details.
  • Maintain calibration records of autoclave, thermocouple, and incubator.
  • Attach graphs, calculations, and test observations in the BI qualification report.

8. Conclusion

Confirmation of viable spore count and resistance performance is crucial to ensure the reliability of biological indicators used in sterilization validation. A properly qualified BI lot assures that sterilization cycles consistently achieve microbial inactivation and that process control remains validated over time. Routine verification strengthens sterility assurance and compliance with GMP and ISO 11138 standards.

9. References

  • ISO 11138 – Biological Indicators for Sterilization Processes
  • USP <55> – Biological Indicators – Resistance Performance Tests
  • FDA Guidance for Industry – Sterilization Process Validation

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.

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