Guidance for Industry and FDA Staff 
 
Biological Indicator (BI) Premarket 
Notification [510(k)] Submissions
 
 
 
Document issued on: October 4, 2007 
 
 
The draft of this document was issued May 21, 2001   
 
This document supersedes FDA Guide for Validation of Biological Indicator 
Incubation Time, January 1, 1986 
 
For questions regarding this document contact Dr. Sheila Murphey at 240-276-3700 or by email at 
sheila.murphey@fda.hhs.gov 
 
        U.S. Department Of Health and Human Services 
Food and Drug Administration 
Center for Devices and Radiological Health 
 
Infection Control Devices Branch 
Division of Anesthesiology, General Hospital, Infection Control, and Dental Devices 
Office of Device Evaluation  Contains Nonbinding Recommendations 
 
Preface 
 
Public Comment 
 
Written comments and suggestions may be submitted at any time for Agency consideration to the 
Division of Dockets Management, Food and Drug Administration, 5630 Fishers Lane, Room 
1061, (HFA-305), Rockville, MD, 20852.  Alternatively, electronic comments may be submitted 
to http://www.fda.gov/dockets/ecomments.  When submitting comments, please refer to Docket 
No. 2001D-0193.  Comments may not be acted upon by the Agency until the document is next 
revised or updated. 
 
Additional Copies 
 
Additional copies are available from the Internet at: 
http://www.fda.gov/cdrh/ode/guidance/1320.pdf.  You may also send an e-mail request to 
dsmica@fda.hhs.gov to receive an electronic copy of the guidance or send a fax request to 240-
276-3151 to receive a hard copy.  Please use the document number (1320) to identify the 
guidance you are requesting.  Contains Nonbinding Recommendations 
Table of Contents 
 
 
1.  INTRODUCTION  1 
  THE LEAST BURDENSOME APPROACH  2 
2.  SCOPE  2 
3.  DEFINITIONS  3 
4.  DEVICE COMPARISON  5 
5.  DESCRIPTION AND SPECIFICATIONS  6 
6.  FDA-RECOGNIZED STANDARDS  7 
7.  PERFORMANCE CHARACTERISTICS  7 
A.  Viable Spore Population Assay  8 
B.  Resistance Characteristics Study  8 
C.  Carrier and Primary Packaging Materials Evaluation  9 
D.  Holding Time Assessment  10 
E.  Recovery Protocols  10 
F.  Mail-In Protocols  10 
8.  SHELF LIFE  10 
9.  INCUBATORS  11 
10.  TEST PACKS  11 
11.  LABELING  11 
A.  Intended Use  11 
B.  Description  12 
C.  Instructions for Use  12 
D.  Precautions  12 
12.  REFERENCES  13 
ATTACHMENT I  BI 510(K) CHECKLIST  14 
ATTACHMENT II - RECOMMENDED VALIDATION OF BIOLOGICAL INDICATOR 
INCUBATION TIME  15 
  Contains Nonbinding Recommendations 
Guidance for Industry and FDA Staff 
 
Biological Indicator (BI) Premarket 
Notification [510(k)] Submissions 
 
This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic.  
It does not create or confer any rights for or on any person and does not operate to bind FDA or the 
public.  You can use an alternative approach if the approach satisfies the requirements of the 
applicable statutes and regulations.  If you want to discuss an alternative approach, contact the FDA 
staff responsible for implementing this guidance.  If you cannot identify the appropriate FDA staff, call 
the appropriate number listed on the title page of this guidance.  
1.  Introduction 
FDA regulates biological indicators (BI) intended to monitor sterilizers used in health care 
facilities as class II medical devices requiring premarket notification (510(k)).  21 CFR 
880.2800(a).  This guidance document provides information that will help manufacturers prepare 
510(k)s for BIs used with conventional sterilization methods.  FDA believes that providing this 
information will promote a consistent and efficient regulatory process.   
 
The effective performance of sterilizers used in health care facilities is important to help prevent 
nosocomial infections.  BIs can provide sterilizer users with information on the effectiveness of 
sterilizer processes.  The use of comprehensive, scientifically sound criteria to evaluate BIs helps 
ensure the performance of these devices.  This guidance document draws upon long-standing 
scientific principles used to evaluate BIs.  It was produced through interaction with industry, 
government, academia, and health care professionals. 
 
This document supplements other FDA documents regarding the specific content requirements 
of a 510(k) submission.  You should also refer to: 21 CFR 807.87; the guidance, Format for 
Traditional and Abbreviated 510(k)s;1 and the section of CDRHs Device Advice, Premarket 
Notification 510(k).2
 
In addition, The New 510(k) Paradigm - Alternate Approaches to Demonstrating 
Substantial Equivalence in Premarket Notifications,3 describes options for submitting either 
                                                 
1   http://www.fda.gov/cdrh/ode/guidance/1567.html.  
2   http://www.fda.gov/cdrh/devadvice/314.html. 
3   http://www.fda.gov/cdrh/ode/parad510.html.  
page 1  Contains Nonbinding Recommendations 
an Abbreviated 510(k) or a Special 510(k), in lieu of a Traditional 510(k).  FDA believes an 
Abbreviated 510(k) provides the least burdensome means of demonstrating substantial 
equivalence for a new device, particularly once FDA has issued a guidance document addressing 
that device. Manufacturers considering certain modifications to their own cleared devices may 
lessen the regulatory burden by submitting a Special 510(k).
 
The Least Burdensome Approach 
The issues identified in this guidance document represent those that we believe need to be 
addressed before your device can be marketed.  In developing the guidance, we carefully 
considered the relevant statutory criteria for Agency decision-making.  We also considered the 
burden that may be incurred in your attempt to follow the guidance and address the issues we 
have identified.  We believe that we have considered the least burdensome approach to 
resolving the issues presented in the guidance document.  If, however, you believe that there is a 
less burdensome way to address the issues, you should follow the procedures outlined in the A 
Suggested Approach to Resolving Least Burdensome Issues, 
http://www.fda.gov/cdrh/modact/leastburdensome.html. 
 
FDA's guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agency's current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  
The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required. 
2.  Scope 
This document provides guidance concerning the content and format of 510(k) submissions for 
BIs intended to monitor sterilization processes used in health care facilities (product code, FRC).  
These are Class II devices identified under 21 CFR 880.2800(a): 
 
A biological sterilization process indicator is a device intended for use by a health care 
provider to accompany products being sterilized through a sterilization procedure and to 
monitor adequacy of sterilization.  The device consists of a known number of 
microorganisms, of a known resistance to the mode of sterilization, in or on a carrier and 
enclosed in a protective package.  Subsequent growth or failure of the microorganisms to 
grow under suitable conditions indicates the adequacy of sterilization.   
FDA encourages you to contact the Division of Small Manufacturers, International, and 
Consumer Assistance (DSMICA) or the Division of Anesthesiology, General Hospital, Infection 
Control, and Dental Devices (DAGID) representatives with any questions you may have before 
submitting a 510(k) for a BI. 
page 2  Contains Nonbinding Recommendations 
Exclusions 
This document does not address the following sterilization process indicators or related 
products: 
  physical/chemical sterilization process indicators (JOJ, LRT, MTC) identified under  
21 CFR 880.2800(b)  
  BIs intended for use in a manufacturing setting 
  BIs intended for use in liquid chemical sterilization (product code MRB). 
 
3.  Definitions 
The following are definitions of terms used throughout the document.  Many of the definitions 
have been standardized by organizations such as the International Organization for 
Standardization (ISO), the American National Standards Institute (ANSI), and the Association 
for the Advancement of Medical Instrumentation (AAMI).  Complete citations for these 
standards are in section 12, References. 
 
Biological Indicator (BI):  Microbiological test system providing a defined resistance to a 
specified sterilization process (AAMI, 2006a). 
 
Carrier:  Supporting material onto which indicator organisms are deposited (AAMI 2006b). 
 
Chemical Indicator:  System that reveals change in one or more predefined process variables 
based on a chemical or physical change resulting from exposure to a process.  (ISO, 2006) 
 
D-value: (decimal reduction value):  Time or radiation dose required to achieve inactivation of 
90% of a population of the test microorganism under stated exposure conditions (AAMI 2006a).4
 
Death Rate Curve (or Survivor Curve):  The graphic representation of inactivation against 
increasing exposure to stated conditions (AAMI, 2006a). 
 
Inactivation:  Loss of the ability of indicator organisms to germinate, outgrow, and/or multiply 
under specified culture conditions (AAMI, 2006a).  
 
Inoculated Carrier:  Specified material onto which a defined number of test organisms have 
been deposited (AAMI, 2006a). 
 
                                                 
4   This is the definition for D-value in the standard cited.  However, radiation sterilization is not discussed in this 
guidance document because it is not a sterilization method generally used in health care facilities. 
page 3  Contains Nonbinding Recommendations 
Medical Device:  As defined in Federal Food, Drug, and Cosmetic Act (the Act) (21 USC 
321(h)): 
 
[a]n instrument, apparatus, implement, machine, contrivance, implant, 
in vitro reagent, or other similar or related article, including any 
component, part, or accessory, which is- 
 
1.  recognized in the official National Formulary, or the United 
States Pharmacopoeia, or any supplement to them,  
 
2.  intended for use in the diagnosis of disease or other 
conditions, or in the cure, mitigation, treatment, or prevention 
of disease, in man or other animals, or  
 
3.  intended to affect the structure or any function of the body 
of man or other animals, and  
 
which does not achieve its primary intended purposes through 
chemical action within or on the body of man or other animals and 
which is not dependent upon being metabolized for the achievement of 
its primary intended purposes.   
 
Pack, Primary:  System that protects the inoculated carrier from damage and contamination 
without preventing penetration of the sterilizing agent(s) (AAMI, 2006b). 
 
Pack, Secondary:  Container system in which BIs are packed for transport and storage (AAMI, 
2006b). 
 
Resistometer:  A test instrument designed to rapidly produce and precisely control critical 
parameters associated with a given sterilization process.5, 6
 
Sterile:  Free from viable microorganisms (AAMI, 2006a). 
 
Sterility Assurance Level (SAL): Probability of a single viable microorganism occurring on 
product after sterilization (AAMI, 2006a). 
 
Sterilization:  Validated process used to render a product free of all forms of viable 
microorganisms (AAMI, 2006a). 
                                                 
5   In addition to routine quality system testing of indicator performance consistency, a resistometer is used to 
characterize cause and effect relationships associated with a given sterilization process and devices used to evaluate 
the efficacy of the sterilization process. 
6   Resistometers were formerly referred to as Biological Indicator Evaluator Resistometer (BIER) or Chemical 
Indicator Evaluator Resistometer test systems (AAMI 2002). 
page 4  Contains Nonbinding Recommendations 
 
Survivor Curve:  The graphic representation of inactivation against increasing exposure to 
stated conditions (AAMI, 2006c). 
 
 
Survival/Kill Window:  Extent of exposure to a sterilization process under defined conditions 
when there is a transition from all BIs showing growth (survival exposure) to all BIs showing no 
growth (kill exposure) (AAMI, 2006c).  
 
Test Pack (Process Challenge Device):  Item designed to simulate products to be sterilized and 
to constitute a defined challenge to the sterilization process and used to assess the effective 
performance of the process (AAMI, 2006a) 
 
Z-value:  For a thermal sterilization process, the change in exposure temperature that 
corresponds to a 10-fold change in D-value (AAMI, 2006b). 
4.  Device Comparison 
FDA recommends that you include a section or table comparing the new device to the legally 
marketed predicate device.  The following table is an example of the type of information that you 
should provide.   (See Section VI below for additional information you should provide in your 
510(k)). 
 
Table 1  Device Comparison 
ELEMENT  NEW DEVICE  PREDICATE 
Intended use 
  method of sterilization     
  process parameters 
Organism 
  spore 
   
  species 
  strain 
Viable spore population     
Resistance characteristics: 
  D-value 
   
  Z-value (thermal only) 
  Survival/Kill Window 
Shelf-life     
page 5  Contains Nonbinding Recommendations 
5.  Description and Specifications 
You should provide a detailed description of the BI.  The description should include the general 
characteristics of the device design as well as the manufacturing specifications.  The bulleted list 
below is an example of the types of information that you should provide for your device.   
  indicator organism, genus, species, and strain 
  type of BI, e.g., self contained or paper strip 
  intended sterilization process, cycle, and parameters 
  resistance characteristics: D-values, survival/kill times, Z-values (thermal only) 
  specification for spore population 
  shelf-life 
  carrier material and primary packaging material 
  description of the culture medium (for self contained BIs) 
  identification of the media, growth, and culture conditions (for non self contained 
BIs). 
 
Generally, the following spores are used for the sterilization methods listed below in Table 2. 
 
Table 2  Recommended Spores for Sterilization Process 
STERILIZATION PROCESS  SPORE (INDICATOR ORGANISM) 
Geobacillus stearothermophilus 
Steam 
(formerly Bacillus stearothermophilus) 
Bacillus atrophaeus 
Dry Heat 
(formerly Bacillus subtilis var. niger) 
Bacillus atrophaeus 
Ethylene Oxide 
(formerly Bacillus subtilis var. niger) 
Geobacillus stearothermophilus 
Hydrogen Peroxide 
(formerly Bacillus stearothermophilus) 
 
For BIs used in sterilization methods other than those listed, you should demonstrate that the BI 
used for validation is the most resistant to the process.  Alternatively, you should reference 
published scientific studies indicating that the subject BI is the most resistant to the process.  If 
you use an organism for a sterilization process that is not specified in Table 2, you should 
page 6  Contains Nonbinding Recommendations 
provide test data to demonstrate that the subject microorganism is at least as or more resistant 
than that listed in Table 2.    
6.  FDA-Recognized Standards 
If any part of the device design or testing relies on a recognized standard, we recommend that 
you include either:  
 
  a statement that testing will be conducted and meet specified acceptance criteria before 
the device is marketed or 
 
  a declaration of conformity to the standard.7  
 
Because a declaration of conformity is based on results from testing, we believe you cannot 
properly submit a declaration of conformity until you have completed the testing the standard 
describes.  For more information, please refer to section 514(c)(1)(B) of the Act and the FDA 
guidance, Use of Standards in Substantial Equivalence Determinations; Final Guidance for 
Industry and FDA.8
 
Recognized standards that apply to BIs are discussed in the following sections. 
7.  Performance Characteristics 
FDA recommends that you provide the following information in your 510(k). 
  clear statement of the study objective(s) 
  description of the equipment and media used to evaluate the BI, including the results 
of the growth promotion studies 
  enumeration of viable spore populations 
  resistance characteristics of the final finished product, as appropriate to the 
sterilization method (e.g., D-value, Z-value, survival/kill window)  
  sterilization methods, cycle parameters, and exposure times 
  determination of growth inhibition of carrier and primary packaging 
  assessment of sterilant neutralization effectiveness, if neutralization is required 
  positive/negative controls 
  summary of study results 
  conclusions reached based upon study results. 
                                                 
7   See Required Elements for a Declaration of Conformity to a Recognized Standard (Screening Checklist for All 
Premarket Notification [510(k)] Submissions), http://www.fda.gov/cdrh/ode/reqrecstand.html. 
8   http://www.fda.gov/cdrh/ode/guidance/1131.html. 
page 7  Contains Nonbinding Recommendations 
 
FDA recommends that performance studies include statistically valid sample sizes and are 
conducted on at least 3 different spore lots, prepared from different spore crops.  You should 
conduct the performance studies discussed below. 
A.  Viable Spore Population Assay 
You should provide assay results to demonstrate that the product meets its specification for 
the spore population.  For examples of assay methods, refer to the applicable United States 
Pharmacopeia (USP) standards and FDA recognized standards (see 12.  References).  See 
also Table 3 Recommended Minimum Populations and Resistance Characteristics. 
B.  Resistance Characteristics Study 
You should provide the resistance characteristics of the BI in the specified sterilization 
process and cycle, using a resistometer.  We recommend that you perform all testing on only 
final finished products.  We also recommend that the resistometer used for the 
characterization of the BI conform with the current ANSI/AAMI standard for resistometers.  
If no resistometer exists for the sterilization process being evaluated, we recommend using 
actual production sterilizers with the critical cycle parameters controlled to the extent 
possible during testing.  You should document that testing parameters were adequately 
controlled and explain how this was achieved.  If you conduct resistance characteristics 
studies using sterilizers that do not routinely monitor all critical parameters, we recommend 
that you add external monitors to document evidence of adequate control. 
D-value 
For test methods for determining BI resistance characteristics, please refer to 
ANSI/AAMI ST 59 or equivalent method. 
 
For non self-contained BIs, you should plot the D-values for a representative range (e.g., 
115C, 120C, 125C, 130C) to determine the D-value for different thermal exposure 
temperatures.  If the curve demonstrates linearity, the BI is suitable for the full range of 
cycle temperatures.   
 
For self-contained BIs, you should calculate the D-value at each proposed temperature.    
Z-value 
You should provide Z-values for thermal processes.  FDA generally recommends that BIs 
have a minimum Z-value of 10C. 
 
To determine the Z-value, you should construct a thermal resistance curve.  The thermal 
resistance curve is a semi-logarithmic graph of D-value vs. temperature.   The Z-value 
can be obtained from the slope of the curve by calculating the number of degrees of 
temperature for a 1-logarithm change in D-value.  The greater the number of 
page 8  Contains Nonbinding Recommendations 
temperatures studied, the more accurate the resulting Z-value.  FDA recommends that 
studies use at least three different temperatures (Pflug, 1990). 
Survival/Kill Window 
You should conduct a study to verify the calculated survival/kill window.  USP provides 
an acceptable test method.  The minimum expected survival time and maximum expected 
kill time can be calculated from the following equations (USP): 
 
Minimum Expected Survival Time 
( )
Survival Time = Not Less Than  D-value   log  viable spore population - 2  
10
 
Maximum Expected Kill Time 
( )
Kill Time = Not More Than D-value   log  viable spore population + 4    
  10
Table 3  below lists examples of the resistance characteristics of 510(k) cleared BIs with 
minimum populations, D-values, and the survival times.  
 
Table 3 - Recommended Minimum Populations and Resistance Characteristics 
STERILZATION  VIABLE SPORE  D-VALUE  Z-VALUE  SURVIVAL 
CYCLE  POPULATION  TIME 
Steam 121C  105 1.5 min  10C  5 min 
Steam 132C  105 10 s  10C  1 min 
Steam 134C or 135C  105 8 s  10C  40 s 
Ethylene Oxide  106 3 min  A  15 min 
600 mg/L, 60% RH, 
54C 
Dry heat 160C  106 3 min  A  12 min 
A Not applicable. 
 
C.  Carrier and Primary Packaging Materials Evaluation 
You should evaluate the effects of carrier and packaging materials on the resistance 
characteristics of the BI.  Please refer to ANSI/AAMI/ISO 11138-1 Annex B for examples of 
methods that may be used. 
page 9  Contains Nonbinding Recommendations 
D.  Holding Time Assessment 
Holding time is the length of time that the exposed BI is held before incubation.  You should 
evaluate the effect of the labeled holding time on the resistance characteristics and spore 
recovery.   
E.  Recovery Protocols 
You should identify the recovery media and evaluate all recovery protocols to validate the 
incubation time.  Generally, FDA recommends 7 days as the conventional incubation time 
for BIs used to monitor the traditional sterilization processes listed in Table 2.  To validate 
incubation times of less than 7 days, see Examples of Validation of Biological Indicator 
Incubation Time in Attachment II. 
 
For self contained BIs, you should also evaluate the effect of the sterilization process on the 
recovery medium. 
F.  Mail-In Protocols 
Some manufacturers market BIs as part of a monitoring service; the user mails the exposed 
BI to the manufacturer (or laboratory) to incubate and read.  The handling required by the 
monitoring service may impact adversely upon the BI resistance.  Therefore, labeling for BIs 
marketed as part of a monitoring service should detail the handling protocol.  The handling 
protocol should be validated. 
8.  Shelf Life  
You should demonstrate that the specifications (e.g., D-value, spore population) are maintained 
throughout the labeled shelf life of the device.  For all studies, FDA recommends that you use at 
least 3 lots from 3 different spore crops of BI and a statistically significant sample size.  You 
should use storage conditions described in the device labeling.   
 
You should provide the following information about shelf life: 
 
  labeled shelf life 
 
  dates and length of time for each testing interval, e.g., 0, 6 months, 1 year, 2 years 
 
  resistometer vessel results for each testing interval demonstrating that the established 
endpoints are maintained. 
 
BIs may be unstable at elevated temperatures; therefore, FDA believes accelerated aging studies 
may be inappropriate.   
 
In lieu of complete real time shelf life data, the FDA will consider preliminary real time shelf life 
data (e.g., 6 months) along with a detailed protocol and sampling plan for an ongoing real time 
page 10  Contains Nonbinding Recommendations 
(e.g., 2 years) study that you intend to continue after your device is cleared.  These study 
protocols should include the shelf life information listed above.  You should document the 
results in your design history file as a part of the Quality System Regulations (21 CFR 820.30).   
9.  Incubators 
BI labeling may recommend a suitable incubator.  Alternatively, labeling may recommend 
incubation conditions and allow users to select an appropriate incubator.  FDA considers 
incubators without specific claims to be general purpose laboratory equipment regulated under 
21 CFR 862.2050 as Class I exempt.  A 510(k) may be required for incubators that are dedicated 
for a specific BI or make specific performance claims that go beyond incubation.  
 
However, incubator performance is an important aspect in BI validation.  Some (typically self-
contained) BIs are marketed as part of a system that includes an incubator.  A 510(k) for this 
kind of BI should include a certification that any incubator sold as part of a system maintains the 
operating temperature specifications under all potential loading conditions over the 
recommended incubation time.   
10.  Test packs 
BIs may be used in test packs to simulate products being sterilized.9 Test packs are intended to 
simulate products and constitute a defined challenge to the sterilization process that is equal to or 
greater than the most difficult item routinely processed.  For BIs indicated for use in specific test 
packs, you should demonstrate that the performance of the BI in that test pack is equivalent to 
the performance of the AAMI reference BI in the same test pack in their respective sterilization 
processes.  You should also demonstrate that the BI test pack provides a greater challenge to the 
process than the BI itself.  
 
11.  Labeling 
The premarket notification must include labeling in sufficient detail to satisfy the requirements 
of 21 CFR 807.87(e).  The following suggestions are aimed at assisting you in preparing labeling 
that satisfies the requirements of 21 CFR Part 801.10
A.  Intended Use 
We recommend that the intended use describe:  
  method of sterilization  
                                                 
9  These test packs are also called process challenge devices (PCDs).  
10   Although final labeling is not required for 510(k) clearance, final labeling must comply with the requirements of 
21 CFR Part 801 before a medical device is introduced into interstate commerce.  Labeling recommendations in this 
guidance are consistent with the requirements of part 801. 
page 11  Contains Nonbinding Recommendations 
  sterilization cycle 
  exposure time 
  temperature 
  sterilant concentration  
  any other information specific to the intended use of your device.    
B.  Description 
We recommend that the labeling provide a description of the device.  The description should 
include: 
  genus, species, and strain 
  lot/batch number 
  type of BI (paper strip, self-contained, size of paper carrier)  
  D- value and method used to determine it (see also D.  Precautions below) 
  Z-value (thermal processes only) 
  survival/kill time under the conditions specified 
  total viable spore count, including whether the count had been determined after 
preliminary heat treatment 
  expiration information. 
C.  Instructions for Use  
We recommend that your instructions for use include: 
  sterilization method, cycle, and exposure time 
  instructions for interpreting results 
  instructions for disposal, e.g., sterilize by steam 121C for not less than 30 minutes 
  instructions for spore recovery, e.g., incubation time, culture conditions, type of 
media. 
 
For self contained BIs, we also recommend that the labeling state whether the supplied 
bacteriological medium will meet requirements for growth promoting ability. 
D.  Precautions 
The labeling should advise users that the manufacturers D-value cannot be duplicated in the 
healthcare facility, for example: 
 
page 12  Contains Nonbinding Recommendations 
The D-value is reproducible only under the exact test laboratory resistometer conditions 
under which it was determined.  The user would not necessarily obtain the same result. 
 
12.  References
 
AAMI 2006a. Association for the Advancement of Medical Instrumentation (AAMI).  
Sterilization of health care products-Vocabulary.  ANSI/AAMI/ISO TIR11139:2006. 
AAMI. 2006b. Association for the Advancement of Medical Instrumentation (AAMI). 
Sterilization of health care products-Biological indicators-Part 1: General requirement.  
ANSI/AAMI/ISO ST11138-1:2006.  
 
AAMI.2006c. Association for the Advancement of Medical Instrumentation (AAMI). 
Sterilization of health care products- Biological indicators- Part 4: Biological indicators for dry 
heat sterilization processes. ANSI/AAMI/ISO ST11138-4:2006.  
 
ISO. 2006. International Organization for Standardization (ISO). Sterilization of health care 
products Biological and Chemical Indicators Test Equipment. ISO 18472:2006.  
 
Pflug, I. J. 1990. Microbiology and engineering of sterilization processes.  Environmental 
Sterilization Laboratory, 100 Union Street, Minneapolis, MN 55455. 
 
United States Pharmacopeia (USP) & National Formulary.  USP 30 (2007) or current edition of 
the following: 
 
USP Biological Indicator for Dry-Heat Sterilization, Paper Carrier 
USP Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier 
USP Biological Indicator for Steam Sterilization, Paper Carrier 
USP Biological Indicator for Steam Sterilization, Self Contained 
 
page 13  Contains Nonbinding Recommendations 
Attachment I.  Summary of FDA Recommendations  
Below is a summary of the recommendations provided in this guidance document.  We 
recommend that you also refer to Format for Traditional and Abbreviated 510(k)s.11
 
Device Comparison  
Description And Specifications 
  Genus, species, and strain 
  Design, e.g., paper strip, self contained 
  Spore population and concentration 
  Carrier material 
  Packaging, primary and secondary 
  Culture medium 
  Resistance characteristics 
Voluntary Consensus Standards BI meets or will meet  
Labeling  
  Description of design, e.g., paper strip, self contained 
  Identification of the genus, species, and strain 
  Conditions for use, including sterilization process parameters (sterilizing agent, concentration, 
temperature) and (for steam) gravity versus dynamic air removal (pre-vacuum), etc. 
  Heat shock spore population and concentration 
  D-value and method used for D-value determination.  Z-value, if appropriate.  Survival/kill times or 
any other applicable resistance characteristics  
  Shelf-life and manufacture or expiration date  
  Storage conditions with temperature and humidity ranges  
  Instructions for interpreting results and spore recovery 
  Instructions for disposal, method, any neutralizers needed, or hazardous wastes associated with 
product use 
  The reproducibility of the D-value 
  Whether bacteriological medium is adequate to promote growth (self contained designs only) 
Performance Characteristics  
  Viable spore population assays 
  D-value 
  Z-value 
  Survival/Kill Time 
  Carrier and primary packaging materials evaluation 
  Holding time assessment 
  Recovery Protocol, medium and incubation time 
  Effect of sterilization process on recovery medium (self contained BIs only) 
  Validation of accessories (e.g., incubator, test pack) if any 
  Validation of mail-in protocols, if any 
                                                 
11  http://www.fda.gov/cdrh/ode/guidance/1567.html. 
Page 14  Contains Nonbinding Recommendations 
Attachment II.  Examples of Validation of Biological 
Indicator Incubation Time
 
These recommendations are appropriate for all biological indicators, self contained or on a strip, 
which are intended to accompany products being sterilized through a sterilization procedure and 
to monitor adequacy of sterilization. 
 
The incubation period for BIs may be reduced from the standard seven or more days, provided 
that the validation studies demonstrate that the revised numbers of days of incubation are 
sufficient according to appropriate methodology. 
 
Methodology 
 
FDA recommends that manufacturers use the following methodology to reduce the incubation 
time for BIs from the standard seven or more days. 
  Identify the partial cycle in which 30 -80% of the BIs survive. A partial cycle is 
one in which all sterilization parameters, except the time parameter, are met. 
  Obtain a minimum of 100 BIs from each of 3 different lots (for a minimal sample 
size of 300 BIs). 
  Expose the BIs to 3 partial sterilization cycles using 100 indicators from each lot 
per each partial cycle. Only the BIs from one lot should be used in each partial 
cycle.  Lots should not be mixed. 
  It is preferable to run BIs in a device load.  However, because of the inherent 
difficulties of achieving a partial cycle kill under such circumstances, partial 
cycles can be run without device loads. 
NOTE: During all sterilization validation studies, the effects of the sterilant in 
combination with the device material on the indicator organism should be 
determined.  If the materials are judged to have a significant effect on organism 
destruction, the indicators should be exposed to the sterilant in conjunction with 
the devices during the partial cycle studies. 
  Place the BIs in the growth media no more than 8 hours after removal from the 
sterilization chamber or removal from the sterilized load of devices.  Incubate all 
of the BIs for a minimum of 7 days.   
  Prepare a growth chart to record the number of positive BIs on either a daily basis 
or for the particular time interval of interest. 
  Using the number of BIs that test positive on day 7 as the base of 100% grow out 
(denominator data), determine from the growth chart the number of BIs that have 
more than 97% of the base number of BIs (numerator) that test positive in each 
partial cycle for the proposed incubation time to be acceptable. 
  The greatest number of days of incubation time to obtain more than 97% positive 
BIs (based on the 7 day incubation time) in any one of the partial cycles is the 
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minimum incubation time for the BI.  For this method, averaging the three (or 
more) partial cycle incubation times is not recommended (see examples below). 
  If there are fewer than 30% survivors or more than 80% survivors in any one run, 
this particular run should not be used and should be repeated to achieve the 
desired number of survivors (see examples below). 
 
Example #1:  The following Table provides examples of the number of positive BIs needed to 
achieve 97% growth. 
Table A  Examples of 97% Grow Out of the Biological Indicator 
Numerator Data 1 30  31  32  33  33  34  35  36  37  38   
Denominator Data2 30  31  32  33  34  35  36  37  38  39   
Percent Growth  100%  100%  100%  100% 97% 97% 97% 97%  97%  97%   
Numerator Data  39  40  41  42  43  44  45  46  47  48   
Denominator Data  40  41  42  43  44  45  46  47  48  49   
Percent Growth  97%  97%  97%  97%  97% 97% 97% 97%  97%  97%   
Numerator Data   49  50  51  52  53  54  55  56  57  58   
Denominator Data  50  51  52  53  54  55  56  57  58  59   
Percent Growth  97%  97%  97%  97%  97% 97% 97% 97%  97%  97%   
Numerator Data   59  60  61  62  63  64  65  65  66  67   
Denominator Data  60  61  62  63  64  65  66  67  68  69   
Percent Growth  97%  97%  97%  97%  97% 97% 97% 97%  97%  97%   
Numerator Data  68  69  70  71  72  73  74  75  76  77  78 
Denominator Data   70  71  72  73  74  75  76  77  78  79  80 
Percent Growth  97%  97%  97%  97%  97% 97% 97% 97%  97%  97%  97% 
1  The numerator is the number of positive BIs that is greater than 97% of the denominator.  If 
the numerator is equal to or greater than the one listed for the corresponding denominator (based 
on the total number of positive BIs on day 7), the length of the incubation time when this occurs 
is acceptable. 
 
2  The denominator is the total number of positive BIs on day 7 of incubation. 
 
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Example #2 - A manufacturer would like to reduce its biological indicator incubation time to 3 
days.  Using the methodology described above, the following data was generated. 
 
Table B  This Manufacturers Test Data  
 
  Day 1  Day 2  Day 3  Day 4  Day 5  Day 6  Day 7 
Partial cycle #1               
Numerator      0    56   57    57    58    59    59 
Denominator    59    59    59    59    59      59    59 
Percent Growth    -        94.9%  96.6% 96.6%  98.3%  100%  100% 
  Day 1  Day 2  Day 3  Day 4  Day 5  Day 6   Day 7 
Partial cycle #2               
Numerator    1   34   35   35  35   35   35 
Denominator   35   35   35    35  35    35    35 
Percent Growth  2.9%  97.1%  100%  100%  100%  100%  100% 
  Day 1  Day 2  Day 3  Day 4  Day 5  Day 6  Day 7 
Partial Cycle #3               
Numerator     0    79    81  Test invalid because percent 
Denominator        positive biological indicators is 
Percent Growth        outside the 30-80% growth. 
  Day 1  Day 2  Day 3  Day 4   Day 5  Day 6  Day 7 
Partial Cycle #4               
Numerator    0    47   48   49    49   49   49 
Denominator    49   49   49   49   49    49    49 
Percent Growth     0  95.9%  98.0% 100%  100%  100%  100% 
 
The 3-day reduced incubation time is not validated because, of the three valid partial cycles, not 
all achieved 97% growth in 3 or fewer days.  However, based upon the criteria listed in bullet 7 
of the test methodology, the data validate a 5-day reduced incubation time. The 5-day incubation 
time in this example is the greatest number of days, from all the valid partial cycles, needed to 
grow out more than 97% of the denominator BIs. 
 
 
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