Biological Indicator Growout Times: The Impact on Establishing a Reduced Incubation Time Protocol - Pharmaceutical Technology

Latest Issue

Latest Issue
PharmTech Europe

Biological Indicator Growout Times: The Impact on Establishing a Reduced Incubation Time Protocol
The authors provide a review of test methodology and standards, including current industry and regulatory proposals, for biological indicator growout times.

Pharmaceutical Technology
Volume 36, Issue 5, pp. s42-s45

Status of ISO/AAMI Working Group 4 activity

In 2009, in conjunction with the AAMI BI Working Group ad hoc committee, a study was initiated to examine growout times of Geobacillus stearothermophilus BIs exposed at 121 C in moist-heat sterilization cycles. The data were generated using a technology that continuously monitors incubated BIs and automatically records growth-positive results (20). The system facilitated accumulation of approximately 4000 data points. Per the ISO BI Working Group directive, the study was published in 2010 and is the first in a series of planned publications designed to provide a data-based scientific rationale for a proposed RIT test methodology. Observations and conclusions from this first study include (21):

  • Delayed growout of a BI is believed to be due to either delayed spore germination or an increase in the generation time of the vegetative cell. The study concluded that for sterilant stressed populations of spores, longer germination times would result in delayed outgrowth as opposed to longer generation times.
  • Most Probable Number (MPN) estimates of surviving organisms and Poisson Distribution described by Halverson and Ziegler can be used to estimate the number of surviving organisms on each positive BI (22).
  • BI growout time is related to the number of surviving spores per unit. The time between the first positive and the last positive in each group of BIs increased as the predicted population decreased.
  • Growout times appear to follow a normal distribution for BIs with several hundred surviving organisms. However, BIs with a few surviving organisms have more highly variable and longer grow-out times.
  • Estimates of the surviving number of organisms can be used to determine the number of generations necessary to produce 1,000,000 cells/mL and show visible turbidity in broth culture.

An additional study examined growout times for Bacillus atrophaeus BIs exposed to ethylene oxide (EO) and chlorine dioxide gas-sterilization processes, and Geobacillus stearothermophilus BIs exposed to hydrogen-peroxide vapor sterilization process and moist-heat processes at 132–135 C. The resulting data were consistent with the observations and conclusions from the initial study. A final proposed test method based on the aggregated data is planned to be submitted by the AAMI WG4 ad hoc committee to the ISO and AAMI BI working groups for review, discussion, and possible modification for inclusion into the ISO TS 16342 draft document. The target date for submission is fourth quarter 2012.

Philip M. Schneider is a senior consultant with LexaMed, 705 Front Street, Toledo, OH 43604,
and is Convener of ISO/TC 198 WG4 Biological Indicators. John R. Gillis is owner of J.R. Gillis & Associates, 2303 Nelson Road, Bozeman, MT 59715,
and is an Expert Member of ISO/TC198 WG4 Biological Indicators.


1. ISO TS 11139:2006 (ISO, Geneva, 2006).

2. ISO 11138 Series (ISO, Geneva, 2006).

3. I SO 14161:2009 (ISO, Geneva, 2009).

4. ISO 11138-1:2006 (ISO, Geneva, 2006)

5. P.J. McCormick, Memorandum presented to AAMI BI Working Group, 2001.

6. R.C. Kralovic, Pharm. Tech. 6, (11) 100–103 (1982).

7. H.W. Winckels and P.G.F. Boumans, proceedings of the EUCOMED Conference: Sterilization Validation of Medical Devices and Surgical Products (Copenhagen, 1984).

8. P.J. McCormick, Memorandum to Lois Jones, AAMI Biological Indicator Incubation Time Validation Task Group, June 28, 1999.

9. FDA CDRH, Guide for Validation of Biological Indicator Incubation Time (June 5, 1985).

10. ANSI/AAMI ST19:1986 (AAMI, Arlington, VA, 1986).

11. ANSI/AAMI ST21:1986 (AAMI, Arlington, VA, 1986).

12. USP 22–NF 17 General Chapter <1211>, "Sterilization and Sterility Assurance of Compendial Articles," 1990.

13. USP 34–NF 29 General Chapter <1211>, "Sterilization and Sterility Assurance of Compendial Articles," 2011.

14. ISO 11138-1: 1994 (ISO, Geneva, 1994).

15. CEN, EN 866-1: 1997 (CEN, Brussels, 1997).

16. ANSI/AAMI ST59: 1999 (AAMI, Arlington, VA, 1999).

17. ANSI/AAMI ST34: 1991 (AAMI, Arlington, VA, 1991).

18. H.F. Bushar, Memorandum presented to AAMI BI Working Group, 1997.

19. ISO TS CD 16342: 2000 (ISO, Geneva, 2000).

20. Smart-Read EZTest Biological Indicator Monitoring System, Mesa Labs, Bozeman, Montana.

21. J.R. Gillis et al., Pharm. Tech., 34 (1), online (2010).

22. H.O. Halverson and N.R. Ziegler, J. Bacteriology 25, 101–121 (1933).


blog comments powered by Disqus
LCGC E-mail Newsletters

Subscribe: Click to learn more about the newsletter
| Weekly
| Monthly
| Weekly

Which of the following business challenge poses the greatest threat to your company?
Building a sustainable pipeline of products
Attracting a skilled workforce
Obtaining/maintaining adequate financing
Regulatory compliance
Building a sustainable pipeline of products
Attracting a skilled workforce
Obtaining/maintaining adequate financing
Regulatory compliance
View Results
Eric Langer Outsourcing Outlook Eric LangerBiopharma Outsourcing Activities Update
Cynthia Challener, PhD Ingredients Insider Cynthia Challener, PhDAppropriate Process Design Critical for Commercial Manufacture of Highly Potent APIs
Jill Wechsler Regulatory Watch Jill Wechsler FDA and Manufacturers Seek a More Secure Drug Supply Chain
Sean Milmo European Regulatory WatcchSean MilmoQuality by Design?Bridging the Gap between Concept and Implementation
Medicare Payment Data Raises Questions About Drug Costs
FDA Wants You!
A New Strategy to Tackle Antibiotic Resistance
Drug-Diagnostic Development Stymied by Payer Concerns
Obama Administration Halts Attack on Medicare Drug Plans
Source: Pharmaceutical Technology,
Click here