What is Biological Indicator?
A Biological Indicator (BI) is a standardized preparation of specific bacterial spores with a known, high resistance to a specific sterilization process. They are used to prove that a sterilization cycle successfully delivered the required lethality by placing them in the most difficult-to-sterilise locations (cold spots) within a load.
1. Types of Biological Indicators
Spore Strips
- Filter paper inoculated with spores.
- Usually packaged in glassine envelopes.
Spore Suspension
- Liquid suspensions used to directly inoculate product surfaces, packaging, or liquids.
- Useful where strips cannot fit.
Self-Contained Biological Indicators (SCBIs)
- Combines spore carrier and crushable growth media in one vial.
- Minimizes false positives by eliminating aseptic transfer steps.
Ampoules
- Sealed glass vials containing spores and culture media.
- Ideal for monitoring liquid loads.
Spore Discs
- Made of borosilicate paper or stainless steel.
2. Microorganisms Used in Biological Indicators
2.1 Geobacillus stearothermophilus
- Reason for Selection:
- Exceptionally high resistance to moist heat.
- Consistent inactivation kinetics.
- Thermophilic (heat-loving), non-pathogenic, and highly resistant to moist heat.
- Applications:
- Steam sterilization (autoclaves, SIP).
- Vaporized hydrogen peroxide (VH₂O₂).
- Advantages:
- Industry standard, widely available in strips, ampoules, and suspensions.
- Offers predictable resistance levels (D-values 1.5–3.0 minutes at 121°C).
2.2 Bacillus subtilis
- Resistance Profile:
- Not suitable for standard steam sterilization (low resistance).
- D-value at 121°C ≈ 0.5 minutes (compared to 1.5–3.0 minutes for G. stearothermophilus).
- Applications:
- Used in steam sterilization for heat-sensitive liquid loads.
- Rationale:
- Lower resistance allows gentler sterilization cycles.
- Prevents product degradation while ensuring microbial kill.
3. Biological Indicators vs Sterilization Methods
| Sterilization Method | Biological Indicator Microorganism | Rationale for Use | Guideline Reference |
| Steam (Moist Heat, Autoclave, SIP) | Geobacillus stearothermophilus | Thermophilic spores highly resistant to moist heat | ISO 11138-3; USP <55>; FDA Aseptic Processing Guidance |
| Ethylene Oxide (EtO) | Bacillus atrophaeus(formerly B. subtilis var. niger) | Spores resistant to gaseous sterilization | ISO 11138-2; USP <55>; EMA GMP Annex 1 |
| Dry Heat (Depyrogenation, Hot Air Ovens) | Bacillus atrophaeus | Resistant to dry heat conditions | ISO 11138-4; USP <55>; WHO GMP |
| Vaporized Hydrogen Peroxide (VH₂O₂) | Geobacillus stearothermophilus | Resistant to oxidative sterilization | ISO 11138-1; FDA Guidance |
| Radiation (Gamma, E-beam) | Bacillus pumilus | Spores resistant to ionizing radiation | ISO 11138-5; USP <55> |
4. Regulatory Guideline References
ISO 11138 Series
- Sterilization of health care products — Biological indicators.
USP (United States Pharmacopeia)
- USP <55>: Biological Indicators—Resistance Performance Tests.
- USP <1035>: Biological Indicators for Sterilization.
Ph. Eur. (European Pharmacopoeia)
- Chapter 5.1.2: Biological indicators and related microbial preparations used in sterile product manufacture.
EN 866 Series
- Biological systems for testing sterilizers and sterilization processes.
ISO 14161 / EN ISO 14161
- Guidance for selection, use, and interpretation of BI results.
ISO 18472
- Sterilization of health care products — Biological and Chemical Indicators — Test Equipment.
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