How Many Types of Biological Indicators Are There? Complete Classification and Applications

By -

Biological Indicators (BIs) are the gold standard tools used to monitor the effectiveness of sterilization processes. They contain specific microorganisms with known resistance to particular sterilization methods. The principle is simple — if the sterilization process can destroy these resistant microorganisms, it can effectively sterilize the load. But many professionals often ask, “How many types of Biological Indicators are there?” In this article, we’ll explain the complete classification, examples, and applications of each type in detail.

🧫 What Are Biological Indicators?

A Biological Indicator (BI) is a preparation of living microorganisms (usually bacterial spores) that provides a defined resistance to a specific sterilization process. BIs are used to validate and monitor sterilizers such as autoclaves, dry heat ovens, ethylene oxide chambers, hydrogen peroxide systems, and radiation sterilizers.

Each biological indicator contains a known population of resistant spores, such as Geobacillus stearothermophilus or Bacillus atrophaeus, which are exposed to sterilization conditions. The survival or inactivation of these spores confirms whether the sterilization process has achieved the required Sterility Assurance Level (SAL).

📘 Classification of Biological Indicators

The types of biological indicators can be classified based on different criteria — primarily by sterilization process, by physical form, or by the microbial species used.

1️⃣ Classification Based on Sterilization Process

According to the type of sterilization method, there are five major types of biological indicators:

  • 1. Steam Sterilization (Moist Heat) – Uses Geobacillus stearothermophilus spores (e.g., ATCC 7953) for autoclave validation at 121°C or 134°C.
  • 2. Dry Heat Sterilization – Uses Bacillus atrophaeus (formerly B. subtilis) spores resistant to dry heat at 160°C–180°C.
  • 3. Ethylene Oxide (EtO) Sterilization – Uses Bacillus atrophaeus spores for chemical sterilization validation.
  • 4. Hydrogen Peroxide (H2O2) Sterilization – Uses Geobacillus stearothermophilus spores resistant to vaporized H2O2.
  • 5. Radiation Sterilization – Uses Bacillus pumilus spores resistant to gamma or electron beam radiation.

👉 Each type of biological indicator is chosen based on the sterilization mechanism — thermal, chemical, or radiation — to match the specific resistance profile required for that process.

2️⃣ Classification Based on Physical Form

Biological indicators are also categorized by their presentation form — how the spores are contained and used during validation:

  • 1. Spore Strip Type – A filter paper or stainless steel strip impregnated with bacterial spores, sealed in a glassine or Tyvek pouch. Commonly used in laboratory validation.
  • 2. Self-Contained Biological Indicators (SCBI) – These consist of a spore carrier and growth medium in a single vial. After sterilization, the vial is incubated to detect growth. (e.g., Attest™ by 3M).
  • 3. Suspension Form – A liquid suspension of spores used for inoculating materials or for resistance performance testing.
  • 4. Ampoule Type – A glass ampoule containing spores in a liquid medium, used for steam and hydrogen peroxide systems.
  • 5. Strip + Sealed Tubes – Combination indicators designed for large-scale validation, containing multiple spore carriers sealed in test tubes for controlled testing.

3️⃣ Classification Based on Microbial Species Used

The resistance of biological indicators depends on the microorganism used. Commonly, the following bacterial spore-formers are employed:

Microorganism Used For Typical Conditions
Geobacillus stearothermophilus Steam and Hydrogen Peroxide Sterilization 121°C–134°C, 6 log spore population
Bacillus atrophaeus Dry Heat and Ethylene Oxide 160°C–180°C, chemical exposure
Bacillus pumilus Gamma and Electron Beam Radiation 25–50 kGy doses
Bacillus subtilis (old standard) Legacy dry heat and gas systems Used in older validation studies

🧠 How Many Types of Biological Indicators Are Commonly Used?

In general practice, there are five main types of Biological Indicators used in pharmaceutical and healthcare sterilization validation:

  1. Steam Sterilization BI (Geobacillus stearothermophilus)
  2. Dry Heat Sterilization BI (Bacillus atrophaeus)
  3. Ethylene Oxide Sterilization BI (Bacillus atrophaeus)
  4. Hydrogen Peroxide Sterilization BI (Geobacillus stearothermophilus)
  5. Radiation Sterilization BI (Bacillus pumilus)

These five types cover all critical sterilization methods used in pharmaceutical manufacturing, medical devices, and hospital sterilization units.

🧪 Applications of Biological Indicators

  • Validation of sterilization cycles in autoclaves and dry heat ovens.
  • Routine monitoring of sterilizers in pharmaceutical and hospital settings.
  • Quality control of sterilization equipment performance.
  • Resistance performance testing of spore preparations (as per USP <55> and ISO 11138).
  • Process development and requalification studies in sterile manufacturing.

📈 Key Parameters of Biological Indicators

  • Population: The number of spores per carrier (commonly 106).
  • D-value: The time required to reduce the microbial population by 90% at a specific temperature.
  • Z-value: The temperature change needed to change the D-value by one log cycle.
  • Survivor Curve: Used to determine the rate of microbial inactivation.

📚 References

  • USP <55> Biological Indicator Resistance Performance Tests
  • ISO 11138 Series — Biological Indicators for Sterilization Processes
  • FDA Guidance for Industry — Sterilization Process Validation
  • AAMI TIR12:2020 — Design and Use of Biological Indicators

✅ Summary

In summary, there are five major types of biological indicators, classified according to the sterilization process they monitor — Steam, Dry Heat, Ethylene Oxide, Hydrogen Peroxide, and Radiation. Each uses a specific spore-forming microorganism with defined resistance characteristics. Biological Indicators play a crucial role in ensuring sterilization reliability, patient safety, and regulatory compliance across the pharmaceutical and healthcare industries.

In short: Biological Indicators = Reliable Proof of Sterilization Efficiency ✔️

💬 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.

Popular posts from this blog

Non-Viable particle count (NVPC)

By -
Image
Particle mater in room air limits are in particles of 0.5 μm and larger per cubic meter. ISO = cubic meter (m 3 ) FD Standard 209 E = cubic feet (ft 3 )   ISO class equal to federal standard 209 E class .  1m 3  = 35.31 ft 3 1m = 100 cm Adapted from former Federal Standard No. 209 E,                ISO = international organization for standard 14644 part -1, may -1999 p ublished. Maximum concentration limits (particles M3 of air) for particles equal to and larger than the considered sizes shown below: (a) ISO Classification Number(N) 0.1µm 0.2µm 0.3µm 0.5µm 1.0µm 5.0µm ISO 1 b                    10 d                 2 d d d e ISO 2 10...
Read more

Alert and Action Limits

By -
In pharmaceuticals alert and action limits are very important, because these limits are effective control to the process. alert and action limits are in house limits, these limits must be less than guideline limits or define limits. Alert and action limits defined based on trend analysis. alert and action limits are like a barrier before the guideline limits. Alert Limit: To be alert the microbial counts reach to this alert level. It is nor necessary to take any action. Possible deviation from normal process condition This is a trigger, that something is going wrong means microbial count increasing that area. Action Limits: When microbial count reach this action limits then action is mandatory. To control the  microbial count in the area. Signal an apparent deviation from normal process condition And require immediate action. If not controlled, we might get area failure. These alert and action limits must be less than final limits. Example : A...
Read more

TNTC vs TFTC

By -
Image
Too Numerous To Count (TNTC) or Too Many To Counts (TMTC) In samples with very high bacterial concentration, that is not suitable for counting (unable to get accurate counts) and reports the results as "to numerous to count" (TNTC). While the best solution is to collect another sample and dilute to get a more accurate counts, this might not always to be possible. Ideally one plate with 200 to 250 colonies are used. Counts above 250 are considered "to numerous to count" (TNTC), because it is impossible to count the colonies. plates countable range 25 to 250. Too Few To Count (TFTC): If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count this count is called too few to count (TFTC).
Read more