3M™ Attest™ Rapid Readout
Biological Indicators
Living
Sterility
Assurance
A historical perspective on Biological Indicators.
The concept of sterilization was born in the mid 18th century
when Lazzaro Spallanzani demonstrated that sealed flasks
boiled for a few minutes had living “animalcules,” but those
boiled for an hour did not.4
About a century later, Robert Koch became the first to use
bacterial spores as biological indicators (BI), as they are known
today. Koch conducted a series of experiments in which a roll
of flannel contaminated with spores was exposed to dry heat.
When exposed at 140-150°C for four hours, the spores survived
and germinated. When exposed to moist heat at 120°C for 30
minutes, they did not.5
1768
1870’s
The role of spores
and the basic science of BIs
as a tried and trusted measure
of lethality continues today.
1875
Evolution of BIs.
Early BIs, primarily spore strips with a known population of
spores, had a readout time of 7 days, which made it difficult
to act on the results. What’s more, use of spore strips required
the operator to be well trained in aseptic technique. Accidental
contamination during the transfer of spore strips into media
could cause a false positive result.
1880’s
1878
The problem was alleviated in the early 1970s, when 3M first
introduced self-contained biological indicators (SCBIs) which
had the spore strip and nutrient medium within a single container,
requiring minimal handling by the end user. In addition, the
readout time was reduced to 24-48 hours, which made the
results more actionable.
Increased demand for even faster instrument turnaround time
in the latter half of the 20th century led to the introduction of
rapid readout BIs which provided results in 1, 3 or 4 hours.
Today, this technology continues to provide the end user with
a direct measurement of cycle lethality.
The monitoring value provided by BIs continues to be
unparalleled. Several studies involving a side-by-side
comparison of different types of monitors for sterilization
effectiveness, have demonstrated the superiority of spores
in being able to detect failures such as superheated steam,
inability to reach temperature, and inadequate air removal
that are often missed by other monitors.
The bottom line
BIs possess a unique ability to
identify sterilization process failures
which are not picked up by other
monitoring technologies.
Spallanzani demonstrates that sealed flasks boiled
for a few minutes have living animalcules, but
those boiled for an hour do not.
Tyndall demonstrates that
bacteria have a heat stable
and heat labile form based on
his observations of the time it
took for effective sterilization
by boiling.
Cohn describes spores of Bacillus subtilis and
demonstrates their heat resistant properties.
Koch employs bacterial spores as a biological
indicator in a roll of flannel. Readout time is
7 days.
Sternberg provides early
evidence that studying the
destruction of spores
provides a safety margin
to the sterilization process.
1970’s
3M introduces self-contained biological
indicators (SCBIs) with a reduced readout
time of 24-48 hours.
19902006
3M introduces rapid readout
self-contained BIs for sterilization
monitoring, providing the end user
with actionable results within
1-4 hours (1990: 1 hr BI for
Steam – Flash, 1995: 3 hr BI
for Steam, 2006: 4 hr BI for EO).
2005
Studies by Bryce et al., Rutala et al.,
Gurevich et al. and Schneider et al.
demonstrate the superiority of spores
in being able to pick up failures that are
often missed by other monitors.3,6,7,8
Science supported by recommended practice.
As a direct measure of lethality, the BI has become the “Gold
Standard” for use in identifying sterilization process failures. Even
marginal failures caused by the inability to reach temperature,
inadequate air removal, or superheated steam can be detected
by BIs. For these reasons, and more, many facilities are choosing
to follow standards closely and in some cases taking standard
practice to the next level when it comes to monitoring with BIs.
The Cost basis for increasing the frequency
of use of BIs.
s3TATISTICSFROMTHE#ENTERSFOR$ISEASE#ONTROLAND0REVENTION
(CDC) show that an estimated 1.7 million HealthcareAssociated Infections (HAIs) occurred in 2002 and were
associated with approximately 99,000 deaths.9 To put this into
perspective the loss of life is roughly equivalent to that of a fully
loaded Boeing 747-400 series going down every other day for
one year. According to the CDC, HAIs are now the sixth leading
cause of death in the U.S., killing almost twice as many people
as breast cancer and HIV/ AIDS combined.10
s4HECOSTSASSOCIATEDWITH(!)SCANBEACRUSHINGBURDEN
on hospital budgets. Effective October 2008, the Centers
for Medicare and Medicaid Services no longer reimburses
healthcare facilities for costs related to certain HAIs that could
reasonably have been prevented through the use of evidencebased guidelines.
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infection after admission. The CDC estimates the annual cost of
dealing with these infections to be $6.7 billion, further
contributing to the mounting costs of healthcare within the
United States.11
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Taking best practices
To the next level
The concept of every load monitoring.
Healthcare facilities realize the benefits of monitoring with BIs as
a best-in-class solution. Many have taken their monitoring efforts
to the next level by moving from standards practice to a BI in
every load. Rationale for this change includes improving patient
safety with one high standard of care and monitoring, as well
as reducing the cost and impact of recalls should a sterilization
failure occur. (To learn more about this, ask your 3M
representative for information on Every Load Monitoring.)
CDC (Centers for Disease Control)
Guideline for Disinfection and Sterilization in
Healthcare Facilities, 200812
“If a sterilizer is used frequently (e.g., several loads per day),
daily use of biological indicators (BI) allows earlier discovery
of equipment malfunctions or procedural errors and thus
minimizes the extent of patient surveillance and product recall
needed in the event of a positive biological indicator. Each
load should be monitored if it contains implantable objects.
If feasible, implantable items should not be used until the
results of spore tests are known to be negative.”
“Biological indicators are recognized by most authorities as
being closest to the ideal monitors of the sterilization process
because they measure the sterilization process directly by using
the most resistant microorganisms (i.e., Bacillus spores), and
not by merely testing the physical and chemical conditions
necessary for sterilization.”
AAMI (Association for the Advancement of
Medical Instrumentation) ST7913
“Biological indicators are intended to demonstrate whether
the conditions were adequate to achieve sterilization.” “BIs are
the only sterilization process monitoring device that provides a
direct measure of the lethality of the process.” (10.5.3.1)
AAMI recommends weekly, but preferably daily, routine
sterilizer efficacy monitoring and sterilizer qualification testing
with a biological indicator process challenge device (BI PCD).
BIs within PCDs should be used to monitor every load
containing implants. BIs should also be used for periodic
quality assurance testing. (10.5.3.2)
For loads containing an implantable device, AAMI states that,
“Patient safety could be adversely affected by the implantation
of a nonsterile device. The sterilization of implantables should
be closely monitored and each load containing implants should
be quarantined until it is verified that BI testing has yielded
negative results.” (10.6.3)
AORN (Association of periOperative
Registered Nurses) Perioperative Standards
and Recommended Practices, 200914
“XVI.h.2. Steam sterilizers: Geobacillus stearothermophilus
BIs should be used for routine load release, routine sterilizer
efficacy monitoring, sterilizer qualification testing and periodic
product quality assurance testing. Routine sterilizer efficacy
monitoring should be done weekly, preferably daily, as follows:
sEACHLOADCONTAININGANIMPLANTABLEDEVICESHOULDBE
monitored with a BI and quarantined until the results of
the BI testing are available, and
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cycles for sterilizer qualification testing.
If a steam sterilizer is intended to be used for multiple types of
cycles (eg, gravity-displacement, dynamic air-removal, flash),
each sterilization mode should be tested.”
Biological Indicators:
Living Organisms That Provide
A Reliable Test Of Lethality.
Biological indicators (BIs) provide direct evidence that sterilization process conditions are sufficient to kill spores.
The theory being, if your process is effective enough to kill a large population of highly resistant spores, it will also
kill a lower number of less resistant organisms on the medical devices.
Plastic sleeve —
Macroporous
material
Cap filter
Glass ampoule —
Plastic cap
Flexible polypropylene sleeve
Spore strip
Label —
Containing the lot number and
a throughput chemical indicator
Growth medium contained in
a crushable glass ampoule
3M™ Attest™ 1292 Rapid Readout Biological Indicator
How a self-contained BI works.
A self-contained biological indicator (BI) consists
of a known population of bacterial spores of a
high resistance to the mode of sterilization
being monitored. For example, Geobacillus
stearothermophilus is the most resistant spore
for steam, hydrogen peroxide gas plasma, and
ozone sterilization. Bacillus atrophaeus is the
most resistant spore for ethylene oxide (EO)
and dry heat sterilization.
The self-contained 3M™ Attest™ BIs consist
of a spore strip (spores that are coated on a
paper strip) enclosed in a plastic vial along with
growth medium contained in a crushable glass
ampoule. The cap is designed to allow sterilant
to penetrate into the plastic vial, killing the spores
and demonstrating that sterilization conditions
were met.
4 Hour
EO
1294 Green Cap
3 Hour
Steam
1292 Brown Cap
1 Hour
Steam
1291 Blue Cap
How an Attest Rapid Readout
BI works.
3M™ Attest™ Rapid Readout Biological
Indicators are similar to conventional self-contained
BIs. 3M Attest rapid readout BIs for steam detect the presence of
G. stearothermophilus by detecting the activity of alpha-glucosidase,
an enzyme present within G. stearothermophilus. The presence of
the enzyme is detected by reading fluorescence produced by the
enzymatic breakdown of a non-fluorescent substrate. This
fluorescence is detected by the 3M™ Attest™ Auto-reader/
incubator. A fluorescence change indicates a steam sterilization
process failure. Non-fluorescence indicates inactivation of the enzyme
and an effective sterilization process. 3M Attest rapid readout BIs also
indicate the presence of viable G. stearothermophilus organisms
by a visible color change reaction formed by acidic by-products.
Defining Sterility Assurance Level
(SAL).
Sterility is defined as being free from all living
organisms.1 Since it is not practical to test every
device for the absence of microorganisms, the
concept of sterility is expressed as a statistical
probability. The sterility assurance level, or SAL,
defines the probability of a non-sterile unit for
a validated sterilization process. Often this is
expressed as a probability of 10-6, or one chance
in one million that a device is not sterile.
Biological indicators are used to develop and
validate sterilization processes, and thus estimate
SAL. A negative BI result indicates that sufficient
lethality has been achieved to kill a large population of highly resistant spores whose resistance
is far greater than the routine microbial bioburden
on reprocessed medical devices.
It’s not just about time.
Both Medical Device Manufacturers and
Sterilizer Manufacturers use this concept of
SAL to do validation of the sterilization cycle.
The typical procedure is to find out the time required to kill a
million spores and then double it. Given this, at the beginning of
the process all spores are expected to be alive or survive. By
the middle of the process all spores should be killed. Based on
this rationale, it is perfectly normal for a BI to be inactivated at
half the stipulated full cycle time. A negative BI result indicates
that enough lethality has been achieved to inactivate a large
population of highly resistant spores. BIs can also be depended
upon to detect if there was a gross or even marginal failure
due to, for example, inadequate air removal, failure to reach
temperature or superheated steam.2,3
Biological Indicator Load Control for The Steam Process.
Process Monitoring
Frequency of Use*
With What
3M Product Options
Load Control
Biological Indicator (BI) usage:
Nonimplant loads – May use a
BI within PCD
3MTM AttestTM Rapid Readout Biological
Indicators and Test Packs (results in 1 to
3 hours)
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The sterilization assurance
that Biological Indicators
can deliver
Nonimplant loads – Within a PCD, may be used to
monitor nonimplant loads.
Implant loads – Within a PCD, should be used in
every load containing implants. The PCD should also
contain a Class 5 Integrating Indicator.
Routine sterilizer efficacy testing – Within a PCD,
should be used weekly, preferably daily (each day the
sterilizer is used) for routine sterilizer efficacy testing.
(The PCD may also contain a CI.) Should be run in
each sterilization type used, and for flash sterilization
each type of tray configuration in routine use should
be tested separately.
Implant loads – A PCD
containing a BI and Class 5
integrating indicator in every load
3M recommends – A rapid
readout biological indicator process
challenge device in every load
3MTM AttestTM Biological Indicators and
Test Packs (results in 24 to 48 hours)
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3MTM ComplyTM (SteriGageTM)
Chemical Integrator Test Pack
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monitored with BI Test Pack or PCD)
Sterilizer qualification testing – Within a PCD,
should be used for sterilizer qualification testing
(after sterilizer installation, relocation, malfunction,
major repair, sterilization process failures). (The
PCD may also contain a CI.) One BI PCD should be
run three times consecutively in an empty chamber,
except for table-top sterilizers, where the test should
be run three times consecutively in a full load.
Each type of cycle used should be tested. In flash
sterilization choose one tray type to test.
* Association for the Advancement of Medical Instrumentation, Types and applications for use of sterilization monitoring devices, Table 8, ANSI/AAMI ST79:2006/A1:2008
References:
1. Association for the Advancement of Medical Instrumentation, Sterilization of health care products—Chemical indicators – Part 1: General requirements,
ANSI/AAMI/ISO 11140-1:2005.
2. Sailaja Chandrapati and Martha Young. To Kill or Not to Kill – A biological indicator story. Managing Infection Control, November, 2008.
3. Schneider, P., Reich, R., Kirckof, S., Foltz, W. Performance of various steam sterilization indicators under optimum and sub-optimum exposure conditions.
American Journal of Infection Control, Volume 33, Issue 5:S55-S67, 2005.
4. Guardino, R. F. Early History of Microbiology and Microbiological Methods. Encyclopedia of Rapid Microbiological Methods Editor. Miller, M.J. Vol 1, Chap 1: 2005.
5. Perkins, J. L. 2nd Edition (1978). Principles and methods of sterilization in Health Sciences. Charles C. Thomas Publisher, Springfield, IL.
6. Bryce, E.A., Roberts, F.J., Clements, B., MacLean, S. When the biological indicator is positive: Investigating autoclave failures. Infec Control Hosp Epidemiol.18: (9) 654-658:1997.
7. Rutala, W.A., Gerge, M.F., Weber, D.J. Evaluation of a rapid readout biological indicator for flash sterilization with three biological indicators and three chemical indicators.
Infec Control Hosp Epidemiol, 14:390-394, 1993.
8. Gurevich, I., Jacobsen, E., Cunha, B.A. Unreliability of chemical integrators compared to spore tests for sterilization monitoring. Am. J Infec Control. 24: 405-406:1996.
9. Centers for Disease Control and Prevention (CDC). Estimates of Healthcare-Associated Infections. www.cdc.gov/ncidod/dhqp/hai.html
10. Binder, Leah (The Leapfrog Group). Infections Caused by Health Care. Transcript of congressional testimony to the House Oversight and Government Reform
Committee (April 16, 2008).
11. Graves N. Economics and Preventing Hospital-Acquired Infection. Emerg Infect Dis [serial online], April 2004. Available from:
http://www.cdc.gov/ncidod/EID/vol10no4/02-0754.htm
12. Centers for Disease Control Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008 http://www.cdc.gov/ncidod/dhqp/pdf/guidelines/Disinfection_Nov_2008.pdf
13. Association for the Advancement of Medical Instrumentation. ANSI/AAMI ST79:2006/A1:2008 - Comprehensive Guide to Steam Sterilization and Sterility Assurance in Health Care
Facilities. Arlington, VA.
14. AORN (Association of periOperative Registered Nurses) Perioperative Standards and Recommended Practices, 2009 Edition, Sterilization.
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expert insight
on Biological
Indicators.
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3M Health Care
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