Biological Indicator Incubation and Reduced Incubation Time (RIT)
Introduction
Biological Indicators (BIs) are the gold standard of lethality indicators and to achieve final test results, a cultivation period is required. Cultivation is the act of introducing the spores to a growth medium and incubating at a defined time/temperature combination. Cultivation can be relatively simple with self-contained BIs (SCBIs) or more involved with standard spore strip BIs. SCBIs contain both the necessary growth medium and vial as on-board components allowing the act of cultivation to occur in any setting. Spore strip BIs require additional materials (tubes of media) and resources (aseptic environment) for culturing the spores.
Cultivating the test microorganisms is a standard microbiological practice; however, choosing an appropriate incubation period has not always been straightforward. This period must be sufficient in length to allow surviving injured spores time for outgrowth. In general, most labs recognize this period to be 14 days; however, the health care standards for BIs (containing widely recognized test microorganisms) have set the incubation time at 7 days. Shorter incubation periods are allowed provided the “Reduced Incubation Time” has been validated. There are two published validation methods available, one from the US Food and Drug Administration and the other from the International Standards Organization.
Standards & References
ISO 11138-1:2017 Sterilization of health care products — Biological indicators —Part 1: General requirements
7.3.2 A reference incubation time is commonly recognized to be seven days for established sterilization processes, such as moist heat and ethylene oxide, using well characterized test organisms, such as Geobacillus stearothermophilus and Bacillus atrophaeus, respectively.
ISO 11138-7:2019 Sterilization of health care products — Biological indicators —Part 7: Guidance for the selection, use and interpretation of results
8.2.3 The biological indicators should be removed from the sterilizer load as soon as possible after the process, without compromising the safety of personnel. They should be tested within a specified time interval that has been established for that product and process. The time intervals between preparation of the indicators and their use in the sterilization process, and between the end of the process and the culturing of the indicators should be justified in order to demonstrate that these intervals have no effect on the results of the biological testing of the sterilizer or sterilization process. These justified time intervals should not be exceeded. If the biological indicators are handled in a manner other than those stated by the manufacturer, the procedures should be validated to determine if they affect the performance of the biological indicator. Any established time intervals should be followed.
ISO 11138-7:2019 Sterilization of health care products — Biological indicators —Part 7: Guidance for the selection, use and interpretation of resultsIntroduction. A biological indicator incubation time is the minimum period of cultivation required before making a final determination that a biological indicator is negative (shows no growth).
Scope. This document is applicable to manufacturers of biological indicators (BIs) and to end users of BIs who intend to, if required by their quality system, establish, validate or confirm a RIT.
4.4. The RIT validation for a spore strip that includes a recovery medium as a kit or a self-contained BI does not have to be repeated by the end user as long as the end user uses the BI with same sterilizing agent and BI incubation temperature as that used in the validation and used in ethylene oxide. See ISO 11138-7:2019, 12.3.3.
Guidance for Industry and FDA Staff, Biological Indicator (BI) Premarket Notification [510(k)] Submissions
Attachment II. Examples of Validation of Biological Indicator Incubation Time
USP <55> BIOLOGICAL INDICATORS—RESISTANCE PERFORMANCE TESTS:
Recovery. After completion of the sterilizing procedure for BIs and within a noted time (NMT 4 h), aseptically remove and add each BI to a suitable medium (see media in Sterility Tests ⟨71⟩) to submerge the BI completely in a suitable tube. For self-contained BIs, the paper strip is immersed in the self-contained medium according to manufacturers’ instructions, within a noted time NMT 4 h.
Mesa Labs’ Recommendations
1.Culturing spore strip BIs
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culture positive control BIs last
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culture BIs in controlled environment
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use sterile forceps between BI units
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use protective equipment (lab coat and gloves)
2. In situations where BI results cannot be read at the labeled incubation time, cultivate BIs and incubate longer. The labeled incubation claim is a minimum time, and it is better to cultivate immediately (after treatment) than to delay cultivation in an attempt to meet the minimum incubation period. With SCBIs where prolonged incubation may result in growth medium evaporation, the vents in the caps should be sealed.
3. Read the BI results frequently throughout incubation and then subculture any growth-positive BIs immediately to avoid death of microorganisms.
4. It is costly and time consuming for BI users to verify labeled RIT claims. Consider avoiding this practice unless BIs are not used in accordance with label claims.
5.The ISO standard states that the BIs should be removed and tested from the sterilizer load as soon as possible and within a specified time interval. For BI resistance testing, Mesa has established an interval ≤ 4 hours, and recommend this time period for end users.
Additional Resources
- Spore News SN017 --Reduced Incubation Time, What’s the Point?
- Spore News SN067 —Sub-Culturing a Positive Biological Indicator for Identification
- Spore News SN071 --Review of the Newly Released ISO 11138-8 Standard on the Validation of a Reduced Incubation Time for Biological Indicators
SN078-V1