Biological Indicator Selection
By Nicole Robichaud
With the many types of biological indicators (BIs) available -sporestrips, self-contained biological indicators (SCBIs), glass ampoule liquid submersible BIs, stainless steel BIs, and Mesa UniQ Industrial Use BIs such as threads, microstrips, and paper discs - the process of selecting the appropriate BI to monitor a sterilization process may seem like a daunting task. However, with a quick evaluation of the load type and/or the sterilization parameters, the selection can be easily narrowed down. This Spore News will provide information on beginning the process of BI selection, enabling end-users to more accurately determine which type of BI is appropriate for monitoring their sterilization cycle.
Porous or Hard Goods
If the load consists of porous or hard goods, Mesa Labs recommends using MesaStrip, Spore Strips, EZTest® SCBI, or Smart-Read™ EZTest SCBI. Porous and hard goods are sterilized by direct contact with the steam generated by the autoclave.(1) The steam generated by the autoclave surrounds or penetrates the items and directly contacts the bioburden thus inactivating it. This is also true with the BIs used in autoclave loads containing porous or hard goods, steam surrounds and penetrates the BI and directly contacts the spores on the BI carrier material.
Strips and SCBIs function in essentially the same manner during a sterilization cycle; however, post sterilization, strips need to be aseptically cultured into growth media and if not handled properly can result in contamination of the spore strip or culture media which will cause a false positive result. Using an SCBI provides an alternative option for customers who do not have the capability to perform aseptic culturing as the system is ‘self-contained’ and this eliminates the chance of post-process contamination. Customers sterilizing items in dental offices, small clinics, or body art studios may choose to use a service such as Mesa Labs Mail-In Spore Testing where after sterilization, the BI is sent to Mesa for testing.
If the BI must be placed into a small area, such as tubing or a medical device, then a regular-sized spore strip or SCBI may be too large and instead, a Mesa UniQ™ BI for Industrial Use such as a microstrip, paper disc, thread or ProLine Tubing Process Challenge Device can be used.
Containers of Aqueous Liquids
If the load consists of containers of aqueous liquid, Mesa recommends one of our glass ampoule, liquid submersible BIs, ProSpore®, MagnaAmp®, or SterilAmp® II. Liquids are not sterilized by direct contact with the autoclave steam because the steam cannot reach the liquid inside the container.(2) In simple terms, the steam from the autoclave heats the container, which in turn heats the liquid contents and inactivates the bioburden. Likewise, the spores in the glass ampoule BI will never be contacted by the steam from the autoclave. The glass ampoule will be heated, which in turn heats the liquid media, inactivating the spores in the media. The volume of liquid inside the containers being sterilized will determine which liquid submersible BI is best to use.
For volumes of liquid greater than 150 mL, Mesa recommends the use of ProSpore or MagnaAmp. Volumes of liquid greater than 150 mL will experience a lag time to temperature in the geometric center of the liquid making this the most difficult to sterilize area and the ideal location to suspend the BI. ProSpore and MagnaAmp have a “neck” around which a fine gauge wire can be easily attached to suspend the ampoule in the liquid where it can monitor this most difficult to sterilize location.
For volumes of liquid less than or equal to 150 mL, Mesa recommends the use of SterilAmp II. Volumes of 150 mL or less will heat consistently throughout with minimal to no lag time for the geometric center of the liquid to heat to sterilization temperature. SterilAmp II is a small (26 x 6.5 mm or 18 x 6.5 mm) glass ampoule that will float on top of the liquid but since there is a minimal lag time for the geometric center of these small volumes to heat to temperature, SterilAmp II will adequately monitor the conditions that are experienced by the liquid.
In some instances, the liquid BI may not need to be placed into the liquid but rather can be placed at various locations in the autoclave next to the containers of liquid. Generally, ProSpore or MagnaAmp are used in this manner when sterilizing vials of product that are approximately the same volume as ProSpore (~ 4 mL) or MagnaAmp (~ 1 mL). In this way, the BI will function as a surrogate vial and will experience similar conditions as the product vials.
If the load consists of bags or containers of biohazardous waste, Mesa recommends the use of one of our glass ampoule, liquid submersible BIs MagnaAmp or ProSpore. Fluids inside of the bags or containers will coat and damage a spore strip or SCBI leaving the possibility of a false positive result, but a glass ampoule BI will not be affected by waste fluids. Near the bottom of the waste container will typically be the most difficult to sterilize area and so a fine gauge wire can be attached to the neck of the MagnaAmp or ProSpore for ease of placement into the waste and ease of retrieval of the BI after completion of the cycle.
The organism of choice for steam sterilization cycles at temperatures of 121 ᵒC or greater is Geobacillus stearothermophilus (ATCC 7953). For sterilization temperatures of less than 121ᵒC which are typically used for the sterilization of heat-sensitive aqueous liquid products, G. stearothermophilus may be too resistant and Bacillus subtilis 5230 (ATCC 35021) may need to be used.(2) For this purpose, Mesa recommends SterilAmp II 5230 which utilizes the same ampoule as SterilAmp II but contains Bacillus subtilis 5230.
Porous or Hard Goods
As with heat-sensitive liquids, G. stearothermophilus may be too resistant for cycles utilizing sterilization temperatures less than 121°C for heat-sensitive porous or hard goods and Bacillus subtilis 5230 (ATCC 35021) may need to be used.(3) For this application, Mesa recommends our Bacillus subtilis 5230 MesaStrip or the Mesa UniQ microstrip if a regular-sized strip is too large for the location.
Ethylene Oxide (EO) sterilization is generally used for items that would be damaged by heat or radiation.(4) BI selection for this process can be less complicated than Steam BI selection simply because EO is not used to sterilize liquids. For EO sterilization Mesa recommends the use of MesaStrip, Spore Strips, Releasat Biological Indicator Culturing Kit, or EZTest SCBI, each inoculated with Bacillus atrophaeus (9372).
Strips and SCBIs function in essentially the same manner during a sterilization cycle; however, post sterilization, strips need to be aseptically cultured into growth media and if not handled properly, this can result in contamination of the spore strip or culture media which will cause a false positive result. Using an SCBI provides an alternative option for customers who do not have the capability to perform aseptic culturing as the system is ‘self-contained’ and this eliminates the chance of post-process contamination.
If the BI must be placed into a small area of a device, then a regular-sized spore strip or an SCBI may be too large and instead, a Mesa UniQ BI for Industrial Use such as MicroStrips, Paper Discs, or Threads can be used.
When sterilizing large loads, it’s time-consuming and costly to place and retrieve BIs that are embedded in the load. However, after performing the appropriate validation activities with BIs embedded in the load, the user may be able to switch to one of Mesa’s Process Challenge Devices (PCD) for routine monitoring. A PCD is selected that provides an equal or greater challenge to the cycle as compared to the embedded BIs.(5) The PCDs are then placed on the outside of the load, making placement and removal an easy task.
Dry heat is used to sterilize items that will not be damaged by heat (such as glass or stainless steel) or items that cannot be sterilized by steam (such as non-aqueous liquids or powders).(6) The main consideration for dry heat BI selection is the temperature of the cycle.
For cycles operating at 180ᵒC or less, Mesa recommends Mesa Strips, Spore Strips, or Releasat Biological Indicator Culturing Kit, each inoculated with Bacillus atrophaeus (9372). The strips are packaged in a protective glassine envelope which can withstand the lower dry heat temperatures; however, at temperatures above 180ᵒC, the glue that holds the glassine envelope together can separate, leaving the strip susceptible to post-process contamination.
For cycles operating above 180 ᵒC, Mesa recommends DriAmp® which consists of silica material, inoculated with Bacillus atrophaeus (9372), and sealed in a glass ampoule. DriAmp can withstand temperatures in excess of 350ᵒC, and so it can even be used when it is necessary for a user to monitor sterilization with a BI in a dry heat depyrogenation cycle.
For dry heat sterilization of non-aqueous products such as oils, Mesa recommends the use of DriAmp. Just like aqueous liquids, the conditions of the product inside of the container must be monitored. Since DriAmp is a sealed glass ampoule, it’s ideal for placement in the product where a spore strip would be compromised.
Vaporized Hydrogen Peroxide (VH2O2)
ISO recommends the use of Geobacillus stearothermophilus on a non-cellulosic carrier to demonstrate the microbiocidal effectiveness of VH2O2 sterilization cycles used by process developers, sterilization equipment manufacturers, medical device manufacturers and end users of VH2O2 sterilizers and medical devices sterilized by VH2O2(7). For these processes, Mesa recommends one of our Geobacillus stearothermophilus, non-cellulosic carrier SCBIs, ExpoSure®, or EZTest. Exposure is ideal for US Healthcare settings and EZTest is ideal for all other settings.
When performing VH2O2 decontamination at ambient temperature and pressure, the USP recommends, Selection of the appropriate biological indicator (BI) and resistance should be based on experimentation within the user’s system.(8) With the numerous different types of VH2O2 systems and the infinite number of different cycles, Mesa’s line of Apex® VH2O2 BIs offers a wide variety of choices to suit any need. The Apex line consists of three types of BIs (stainless-steel discs packaged in Tyvek®, unpackaged stainless-steel ribbons, and the Apex EZTest SCBI), three different organisms (G. stearothermophilus 12980 or 7953, and B. atrophaeus) and a variety of populations.
Biological indicators may not be necessary for all radiation sterilization processes but may be required when sterilizing biological products such as tissues or cell preparations or other products that may provide spore protection.(9) When a BI is necessary, Mesa recommends the use of MesaStrip for radiation.
Alternative Sterilization Methods
Today there are a variety of alternative vapors and gases being used for decontamination and sterilization; however, BI resistance and population requirements for these processes are not specified in standards. Mesa offers MesaStrips for Chlorine Dioxide and Ozone but for other alternative modalities reach out to one of our experts for help in selecting a BI that will meet your needs.
During cycle development, the Contract Studies team at Mesa can assist in performing studies to determine if a BI from our extensive portfolio will be appropriate for your cycle validation and if not, they can assist with a custom solution that will be appropriate for your unique situation. For an outline of services offered by the Contract Studies Laboratory, see our white paper Spore News – Contract Studies.
While identifying the correct type of BI for a specific process is the initial step, there are other important variables to consider. What spore population do you require? For steam sterilization, United States Healthcare is required to use a BI with a spore population of at least 105 and generally speaking, outside of US Healthcare, a BI with a spore population of 106 is used. Do you need a BI with FDA 510(k) clearance? United States Healthcare must use a BI with FDA 510(k) clearance when reprocessing items for patient use. If you are unsure about requirements you must follow, it’s best to contact your regulatory agency for guidance.
- United States Pharmacopeia 2022, General Information, Chapter <1229.1> Steam Sterilization by Direct Contact
- United States Pharmacopeia 2022, General Information, Chapter <1229.2> Moist Heat Sterilization of Aqueous Liquids
- ANSI/AAMI/ISO 11138-3:2017 Sterilization of health care products-Biological indicators-Part 3: Biological indicators for moist heat sterilization processes
- United States Pharmacopeia 2022, General Information, Chapter <1229.7> Gaseous Sterilization
- ANSI/AMMI/ISO 11135:2014 Sterilization of health care products – Ethylene oxide – Requirements for development, validation and routine control of a sterilization process for medical devices
- United States Pharmacopeia 2022, General Information, Chapter <1229.8> Dry Heat Sterilization
- ISO 22441:2022 Sterilization of health care products – Low temperature vaporized hydrogen peroxide – Requirements for development, validation and routine control of a sterilization process for medical devices
- United States Pharmacopeia 2022, General Information, Chapter <1229.11> Vapor Phase Sterilization
- European Pharmacopoeia 10 – 5.1.2 Biological indicators and related microbial preparations