EDUCATION

Self-Study Guide

Indicators,
Disinfection and
Sterilization

One Integrated Approach to Healthcare.

Indicators, Disinfection and Sterilization

Accreditation/Contact Hour
This Study Guide is provided by STERIS Corporation, an approved Administrator Education Unit (AEU) provider by the Board
of Ambulatory Surgery Certification (BASC) provider # 1417 and an approved provider of continuing nursing education by the
California Board of Registered Nursing (CBRN) – provider number CEP # 11681.

This Study Guide is approved for:

• 1 contact hour(s) of continuing education credit by:
o ACI (Association for Advancement of Medical Instrumentation (AAMI) Credentials Institute);
o CBRN (California Board of Registered Nursing) provider # CEP 11681;
o CBSPD (Certified Board for Sterile Processing and Distribution); and
o IAHCSMM (International Association of Healthcare Center Service Materiel Management).

Authors of this educational material are clinical education employees of STERIS Corporation and do not receive additional compensation for this activity. STERIS
Corporation is providing this educational content. Specific products pictured are used to support this material and do not constitute commercial support.

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Description of the Study Guide Topic
No matter where a medical procedure is performed, whether in
a physician’s office, clinic or hospital, devices are used. Many
of these devices are reusable, and it is the responsibility of
healthcare workers to ensure that they are cleaned, disinfected
and/or sterilized between each patient use.
In order to ascertain that devices are disinfected and/or sterilized,
it is not sufficient to only use a particular antimicrobial product
or process. Staff should be trained, equipment maintained, and
a quality process in place to ensure consistency. An important
part of this is the use of quality control monitors and indicators.
These include physical monitors, chemical indicators (CIs),
biological indicators (BIs), spore test strips and solution test
strips. These monitors and indicators are considered in more
detail within this study guide.
Key Terms (Blue Bold Font)
To provide focus for the reader on vocabulary introduced and used
extensively throughout this study guide to help assure that the
learner understands the basic language associated with this lesson.
Objective:
Upon completion of this study guide program, the participant
should be able to:
1. Describe the difference between disinfection and
sterilization.
2. Identify different quality control monitors available
for high-level disinfection and sterilization.
Indicators, Disinfection and Sterilization
Chemical solutions used for high level disinfection are classified
and assigned designated levels depending on their ability to
inactivate certain types of microorganisms (e.g., low or high-
level). Sterilization requires the complete inactivation of all
microorganisms in a defined process. The choice of disinfection
or sterilization is based on whether a device contacts intact skin,
mucous membranes, or was introduced into a sterile cavity of the
body. Non-critical items are described as items that into contact
intact skin, but not mucous membranes. They require low level
disinfection. Semi-critical items contact mucous membranes
or non-intact skin. They require high level disinfection or if
feasible, sterilization. Critical items are described as any device
that enters sterile tissue or vasculature and require sterilization.
In this classification, disinfectants are used to reduce the level of
microorganisms on surfaces and medical devices. The different
levels of disinfection refers to their ability to inactivate various
types of microorganisms. In the HICPAC (Healthcare Infection
Control Practices Advisory Committee), 2008 “Guidelines
for Disinfection & Sterilization in Healthcare Facilities” high
level disinfectants are described as being effective against all
pathogenic microorganisms and small concentration of bacterial
spores; such products are therefore expected to be effective
against bacteria, viruses, fungi, mycobacteria and bacterial spores
(although they require longer exposure times to be sporicidal).
Sterilization is the process by which all forms of microbial life
including bacteria, viruses, spores, and fungi are completely
destroyed in a defined process.

Intended Audience:
This study guide is a self-study program intended for use
by, Peri-operative Services, Sterile Processing, Reprocessing
Department, Infection Prevention, and other healthcare
professionals interested in this topic.

Disinfection and Sterilization

Disinfection is defined as the antimicrobial reduction of
microorganisms, and at its highest level should be the removal
of all or most pathogenic microorganisms. This definition is A variety of disinfection and sterilization products are used to
broad to cover a variety of applications including disinfection of render surfaces and devices safe for use. Products are required
medical devices, as well as, environmental surface disinfectants. to be verified and validated for safety and efficacy for use by the
Dr. Earle H. Spaulding of Temple University, in an article on the manufacturer. This includes the need to register the product/
disinfection of surgical instruments with a chemical solution, process for use in a particular country or region.
proposed “a strategy for sterilization or disinfection of inanimate
In the United States, for example, chemical disinfectants
objects and surfaces based on the degree of risk involved in their
used on environmental surfaces must be registered with the
use.” He then classified medical devices based on how the devices
Environmental Protection Agency (EPA) and products/processes
were used and how they should be consequently disinfected and/
for the reprocessing of medical devices are registered with
or sterilized.

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Indicators, Disinfection and Sterilization
the Food and Drug and sensors, as well as, chemical indicators, chemical test strips,
Administration biological indicators and spore test strips.
(FDA). This is the
minimum requirement Physical Monitors
but does not ensure that Physical monitors verify that the parameters of the disinfection
the products/processes and/or sterilization cycle/process have been met. They can
are used correctly for the include time, temperature and pressure recorders; displays, chart
reprocessing of devices in recordings, digital printouts and gauges depending on the process.
healthcare facilities. These are commonly used on washer-disinfectors and thermal
So what means are (steam or dry heat) sterilizers, but are also used in low temperature
available to the healthcare disinfection and sterilization processes. With chemical usage
worker to help ensure there can be sensors to detect the level of chemicals both inside
that disinfection or the equipment, as well as in the work environment. Physical
sterilization has been monitors can provide real-time assessment of the disinfection/
achieved correctly? In sterilization process and provide a permanent record in the case
part, professionals use of chart recordings, digital printouts and computer generated
quality control monitors reports. They are used to detect processor malfunctions as soon
and indicators. One example includes the use of temperature as they may occur so that appropriate corrective actions can be
probes for monitoring thermal-based disinfection and taken. In some cases, they are used in pairs, in a process called
sterilization processes, to ensure that the correct temperature is independent monitoring. One set of probes controls the process
achieved during the exposure time. Another example is the use (e.g., exposure to temperature over time) and the other set checks
of chemical monitors or indicators that change in physical form that the controlling probes are functioning correctly (e.g., the
or color. temperature is within a certain range). In all cases, these types of
sensors and monitors require periodic calibration to ensure they
Monitors (such as solution test strips) used in the process of are functioning and recording correctly.
disinfection can tell us that the disinfectant is present at an
effective concentration or an effective temperature over time Chemical Indicators:
needed to achieve disinfection. In the 1930’s, chemical indicators were first introduced to
monitor steam sterilization processes. The first chemical
indicators used simple chemical reactions that occurred when
the indicator was exposed to specific sterilization conditions.
Unexposed
These simple indicators often provided very little information
about the steam sterilization process itself, but were able to
detect certain procedural failures. However, as with many
Pass other medical practices and devices, advancements in the
understanding of these processes have led to advancements
Solution Test Strip in chemical indicators for all types of sterilization processes.
Sterilization monitors or sterility assurance products (commonly Today’s indicators use complex chemical reactions and provide
referred to as biological & chemical indicators, BIs or CIs significantly more information about physical and chemical
respectively) can indicate that a sterilization processes. The term ‘chemical indicator’ is often
sterilizer appears to function correctly used as a term to describe any type of chemical based indicator
or that sterilization has been acheived system, but is predominantly used and defined as applying to
on a device or instrument set. sterilization processes. They are commonly referred to as CIs.
Disinfection and sterility assurance The classification systems of sterilization process CIs have
products attempt to capture errors varied internationally. For example, in the United States there
associated with equipment, people were no classifications for chemical indicators until December
and utilities. Healthcare professionals 1988, when the Association for the Advancement of
use a combination of products to Medical Instrumentation (AAMI) published a technical
ensure that the critical parameters of report entitled “The Selection and Use of Chemical Indicators
a disinfection or sterilization process for Steam Sterilization Monitoring in Healthcare Facilities.”
were met and effective. These include Chemical indicators (CIs) are defined by AAMI as a “Device
physical monitors, such as, gauges used to monitor the presence or attainment of one or more of
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the parameters required for a satisfactory sterilization process, or
used in specific tests of sterilization equipment”.
In addition the internationally recognized standard, ANSI/
AAMI/ ISO 11140:2015 defines a CI as a test system that
reveals change in one or more predefined process variables
based on a chemical or physical change resulting from exposure
to a process. It has been adopted by many countries including
the United States and describes types of CIs and specifies
performance requirements for each of them. The standards
and performance parameters apply to all sterilization processes
including steam, ethylene oxide and hydrogen peroxide gas
sterilization processes. They are:
1. Process Indicators (Type 1): These chemical indicators
are used to indicate that an item has been exposed to the
sterilization process and to distinguish between
processed and unprocessed items. They are often referred
to as external CIs. Examples of Type 1 indicators are
indicator tape and the external indicators found on
peel pouches.
2. Indicators for Use in Specific Tests (Type 2): These
indicators are designed for use in specific test procedures
as defined by sterilization standards, such as ANSI/
AAMI ST79 or ANSI/AAMI ST58. An example of
Type 2 indicators is the Bowie-Dick (air removal) test
used for steam sterilization. This indicator tests for the
presence of air in the sterilizer chamber and a positive
test would mean that air, which inhibits steam from
contacting all surfaces of an item, has either not been
removed during the cycle or has entered the chamber
through leaks in the system.
3. Single Critical Process Variable Indicators (Type 3): These
indicators are designed to react to one of the critical
process variables of the sterilization process and to indicate
exposure to a sterilization cycle at stated values of the chosen
variables. As an example, an ethylene oxide sterilization
process requires humidity, a specific temperature, ethylene
oxide at a specific concentration for a specified time. A type
3 indicator would show that the ethylene oxide concertation
was achieved or that the correct temperature was reached.
4. Multi-Critical Variable Indicators (Type 4): These indicators
are designed to react to two or more of the critical process
variables of sterilization and to indicate exposure to indicate
exposure to the cycle at stated values of the chosen varible.
As previously mentioned, ethylene oxide requires several
parameters to be achieved for successful sterilization, a type
3 would demonstrate that two of these parameters, such as
time and ethylene oxide concentration, were achieved.
5. Integrating Indicators (Type 5): These indicators also
known as “integrators” react to all of the critical process
variables over a specified range of sterilization cycles
for a sterilization process and their performance has
Indicators, Disinfection and Sterilization
been correlated to the performance of a biological indicator
(BI) under the labeled conditions for use. Using steam
sterilization as an example, a type 5 integrating indicator
would require all the critical parameters of temperature, time
and saturated steam needed to kill a biological indicator in a
steam sterilization cycle. Additionally, the indicator can be
used in several sterilization cycles. This may include several
exposure times for a given temperature. A type 5 may
be used on 3 minute 270°F gravity immediate use steam
sterilization (IUSS), 4 minute 270°F prevacuum terminal
steam sterilization and 10 minute 270°F IUSS cycles, for
example.
6. Emulating Indicators (Type 6): These indicators are also
known as cycle-specific indicators. Type 6 indicators must
demonstrate that it requires all the critical process variables
of sterilization for a specific validated sterilization cycle.
Whereas the type 5 integrating, could be used in three
different sterilization cycles, a different type 6 indicator (or
ink spot) is used for each of the three cycles. The difference
between them being the required exposure time. This is
what makes these cycle-specific indicators different from
the type 5 integrating indicators.
Type 5: Integrating Indicator
Type 6: Emulating Indicator
Healthcare personnel should select CIs that are suitable for
use in a specific sterilization cycle and all CIs should be used
according to the manufacturer’s instructions. The Type of CI
chosen should depend upon how many critical process variables
are to be monitored and how much information is desired about
the sterilization process.
Type 4: Multi-Critical Process Variable CIs, Type 5: Integrating
CIs, and Type 6: Emulating CIs provide more information about
the process than Type 3: Single-Variable CIs and can provide
additional quality assurance for the individual monitoring
of such items as complex devices, surgical trays, and rigid
sterilization container systems.
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Indicators, Disinfection and Sterilization
CIs are recommended to be placed in every package, tray and In the United States, the FDA states that “BIs can provide
rigid container. A “pass” response of a CI does not prove that sterilizer users with information on the effectiveness of the
the item monitored by the indicator is sterile. It indicates that sterilization processes.” A BI is an indicator intended for use by
that sterilization parameters measured by the CI were achieved healthcare personnel to accompany products being sterilized
within the item. The CI should be placed in an area of the through a sterilization cycle and to monitor the adequacy of
package, tray or rigid container considered the least accessible sterilization.
to steam penetration. The CI is retrieved at the time of use
and is interpreted by the user. The user should be trained and
knowledgeable about the performance characteristics of the
monitoring system. If the interpretation of the CI suggests
inadequate processing, the contents of the package, tray or rigid
container should not be used.
The use of CIs is part of an effective quality assurance program.
CIs should be used in conjunction with physical monitors and BIs
when appropriate, to demonstrate the efficacy of the sterilization
process. Guidelines can vary in their recommendations of when
the various types of indicators should be used depending upon
the geographical location in which it will be used.
Any microorganism can be used for biological monitoring for
the effectiveness of a disinfection/sterilization process, but BIs
Solution Test Strips
are primarily used with bacterial endospores since they are
Solution test strips or chemical monitoring devices are similar considered some of the hardiest microorganisms to inactivate
to chemical indicators and are specifically labeled in the United and are therefore a challenge to the process. The type of bacterial
States to be used with specific high level disinfectant or liquid spores can vary depending on its resistence to the sterilization
chemical sterilants. They are used to determine whether the process.
concentration of the active ingredient used in these solutions
The most commonly used BI in healthcare today contains
is at or greater than the product’s minimum recommended
endospores of Geobacillus stearothermophilus, formerly
concentration (MRC) or minimum effective concentration
called Bacillus stearothermophilus. G. stearothermophilus
(MEC). Many of these types of disinfectants/sterilants
is a nonpathogenic spore-forming bacterium that is able to
are designed for multiple uses both for manual immersion
withstand the high temperatures of steam sterilization when
and within automated reprocessing machines (e.g., washer/
in its spore form. This same organism is also used to monitor
disinfectors and automated endoscope reprocessors). There
hydrogen peroxide gas and ozone sterilization processes because
is a risk that the concentration of the active ingredient(s) will
it is highly resistant to these sterilization methods as well.
decrease over time with dilution by water, the presence of organic
or other extraneous materials, evaporation of the solution Another organism used in healthcare facilities is Bacillus
and exposure of the solution to light or other factirs. The test atrophaeus, formerly called Bacillus subtillis var. niger. This
strip is generally designed to be dipped into the solution and organism is resistant to ethylene oxide and dry heat sterilization
indicate a manufacturer specified color change at the MRC. It is methods.
recommended that high level disinfectants and liquid chemical A biological indicator
sterilants should be tested prior to before each use. consists of a carrier
If the test strip indicates that the concentration of the active material on which high
ingredient is below the MRC or MRC (does not indicate the populations of bacterial
required color change) the solution should not be used. Individual spores (endospores), with
test strips and disinfectant/sterilant products can vary and close a known resistance to the
attention should be given to manufacturer’s instructions. sterilization process have
been applied. The carrier
Biological Indicators: is exposed to the
sterilization process and
ANSI/AAMI/ISO 11138-1:2017 describes a biological indicator
then incubated under
(commonly referred to as a BI) as “A test system containing viable
defined growth
microorganisms providing a defined resistance to a specified
conditions to test if any spores have survived. If no spores survive,
sterilization process”.
the test is considered a pass, but if growth is observed the test is
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a fail.

A biological indicator may be designed to monitor one specific
form of sterilization using just one type of bacterium, or it may
be populated with multiple types of organisms and be used for
several sterilization applications. The typical biological indicator
design includes a carrier, containing endospores which is placed
in a protective glassine envelope.
Following sterilization, the carrier is carefully removed from the
protective glassine envelope and transferred to a vial of sterile
medium provided, which is places in an incubator to allow any
surviving spores to grow over time. This must be accomplished
aseptically to prevent the introduction of environmental
bacteria into the medium potentially resulting in growth of
contaminating bacteria and providing a false positive result.
Self-contained biological indicators (SCBI) combine the paper
strip and the growth medium into a single vial eliminating the
need for aseptic transfer. This design can be exposed to the
sterilization process and then activated to allow the growth
media to mix with the spore-containing paper strip, minimizing
any cross-contamination risk.
After sterilization processes, the BIs must be incubated within
the growth medium for a specified period of time to detect if any
spores have survived the sterilization process. Growing organisms
are detected through pH indicators in the growth medium that
change colors due to the organisms metabolic processes. These
metabolic processes of growing organisms change the pH from
neutral to acidic conditions which in turn results in the color
change.
Incubation times can vary depending on the design of the
BI (e.g., 24 hours to 7 days). Some self-contained biological
indicators have been designed to combine specialized media with
the spores to allow the detection of growth sooner. Typically,
self-contained biological indicators have a 1 to 2-day incubation
periods for detection of growth.
Several early detection systems have been developed to allow even
faster detection of spore growth. One system utilizes a photo eye
within the incubator that can detect the change in color of the
media before the human eye can see it. The incubation time
required to detect the replication of a low number of injured
spores using this system is about 10 hours.
There are also systems utilizing enzyme-based read-out
technology that monitors the activity of an enzyme associated
Indicators, Disinfection and Sterilization
with the spore. The enzyme’s activity can be detected before
the bacterium grows and divides. This gives a result within 20
minutes to four hours, depending upon the indicator organism
being used and the type of sterilization process being monitored.
The growth media of these self-contained biological indicators
contains a modified sugar. After the sterilization process, the
media is released to the bacterial spores within the vial. A
natural occurring enzyme within the bacteria works on the
modified sugar to release a fluorescent component. A specialized
reader is used to detect the fluorescent material. Presence of
the fluorescent material shows a positive result. Though the
fluorescent response is the final read for the BI, some enzyme-
based rapid-readout BIs also include an optional pH indicator in
the growth media.
Biological indicators are intended to demonstrate whether the
conditions were adequate to achieve a defined level of microbial
inactivation during a sterilization process (generally using
bacterial spores as the most resistant to that process). A negative
BI does not prove that all items in a sterilization load are sterile
or that the sterilization conditions were met. The specific
recommendations on the use of BIs can vary from country to
country. All recommend routine microbial testing of the process.
However, the frequency between them can be very different.
These guidelines also recommend that BI testing is conducted
for sterilizer qualification after installation, relocation, major
repairs and after sterilization process failures. Another
recommendation is that every load containing an implant (or
device that remains within the patient following surgery) should
also contain a BI. The implant should be quarantined until the
full result of the incubated BI is known.
Healthcare professionals should use biological indicators that
have been recommended by the manufacturer of the sterilization
system and in this case cleared by the US-FDA for the specific
sterilization process.
Spore Test Strips
A recently introduced term in the United States for a type of
indicator is a spore test strip. A spore test strip is defined as a
carrier or strip with a known number of spores, at least 500,000
per strip, of known resistance to a particular liquid chemical
sterilant in a liquid chemical sterilant processing system.
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Indicators, Disinfection and Sterilization

This is a type of spore-containing indicator for testing liquid

chemical sterilization processes. In other respects, it is similar to
a biological indicator, in that it contains a known concentration
of a resistant microorganisms on a carrier. It is used to expose
the carrier to the liquid chemical sterilization process and
then incubated afterwards to detect the presence or absence of
growth. A “no growth” result from the spore test strip after the
specified predetermined incubation period indicates that the
liquid chemical sterilization process achieved the conditions
necessary to kill the specified minimum number of viable spores
on the test strip (e.g., 500,000spores/strip). Spore test strips do
not confirm the expected full performance of the liquid chemical
sterilant processing cycle.

Summary:
Healthcare professionals are responsible to ensure that every
reusable item used with patients is safe for use. This can include
that the item is cleaned, disinfected and/or sterilized after each
use. A combination of products and process controls can be used
to ensure that all of the critical parameters of the disinfection
and/or sterilization process are effective. These include quality
control monitors and also include physical monitors, chemical
indicators, biological indicators, spore test strips and solution
test strips. Healthcare professionals using these monitors need
to be trained in the correct usage and interpretation of the
results. As technology progresses new quality control monitors
will become available for use. Healthcare facilities should ensure
that staff responsible for these products has the knowledge and
expertise to determine which monitors are selected and how they
are effectively used.

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 Indicators, Disinfection and Sterilization
Glossary:
American National Standards Institute (ANSI):
ANSI, the American National Standards Institute, is a
nonprofit organization founded in 1918. ANSI facilitates
the development of U.S. National standards. It represents
over 270,000 companies and organization and 30 million
professionals worldwide.

Association for the Advancement of Medical

Instrumentation (AAMI): AAMI, the Association
for the Advancement of Medical Instrumentation, is a
nonprofit organization founded in 1967. It is a unique
alliance of more than 6,000 members from around the world
united by one mission — to increase the understanding
and beneficial use of medical instrumentation through
effective standards, educational programs, and publications.

Food and Drug Administration (FDA): The

FDA, Food and Drug Administration is an agency within
the department of Human Services in the USA. It is
responsible for protecting and promoting public health
through the regulation and supervision of food safety,
tobacco products, prescription drugs and medical devices
just to name a few. It is also responsible for premarket
approval of medical devices as well as overseeing the
manufacturing, performance and safety of these devices.

International Organization of Standardization

(ISO): ISO, International Organization of Standardization,
is a non-profit organization founded in 1947. ISO is
the world’s largest developer of voluntary International
Standards. Consisting of 164 countries and 3,335
technical committees, ISO strives to break down barriers
to international trade through development of state of the
art specifications for products, services and good practice,
helping to make industry more efficient and effective.

Minimum Effective Concentration (MEC): MEC is

the lowest concentration of active ingredient within a disinfectant
or sterilant solutions at which the product is still effective.

Minimum Required Concentration (MRC): MRC is

the lowest concentration of active ingredient within a disinfectant
or sterilant solutions as recommended by the manufacturer.

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Indicators, Disinfection and Sterilization

References:
• Association for the Advancement of Medical Instrumentation. (2017). ANSI/AAMI ST79:2017 Comprehensive guide to
steam Sterilization and sterility assurance in health care facilities. Arlington, VA: Author
• Association for the Advancement of Medical Instrumentation. (2010). ANSI/AAMI ST58:2010, Chemical sterilization and
high-level disinfection in health care facilities. Arlington, VA: Author
• Food and Drug Administration. (2007). Guidance for industry and FDA staff - Biological Indicator (BI) Premarket Noti-
fication [510(k)] Submissions. Retrieved from https://www.fda.gov/regulatory-information/search-fda-guidance-documents/
biological-indicator-bi-premarket-notification-510k-submissions
• Healthcare Infection Control Practices Advisory Committee.(2019). Guidelines for disinfection & sterilization in healthcare
facilities. Retrieved from https://www.cdc.gov/infectioncontrol/pdf/guidelines/disinfection-guidelines-H.pdf (Original pub-
lished 2008)
• Association for the Advancement of Medical Instrumentation. (2017). ANSI/AAMI/ISO 11138-1:2017. Sterilization of health-
care products-Biological indicators-Part 1: General requirements. Arlington, VA: Author
• Association for the Advancement of Medical Instrumentation. (2014). ANSI/AAMI/ISO 11140-1:2014 Sterilization of health-
care products - chemical indicators -part 1: general requirements. Arlington, VA: Author
• Association for the Advancement of Medical Instrumentation. (2014). ANSI/AAMI/ISO 14161:2014 Sterilization of health-
care products - Biological indicators - Guidance for selection, use and interpretation of results. Arlington, VA: Author
• Association for the Advancement of Medical Instrumentation. (2013). ANSI/AAMI/ISO 15882: 2008/(r) 2013. Sterilization of
healthcare products - Chemical indicators - Guidance for selection, use and interpretation of results. Arlington, VA: Author
• Spaulding EH. Chemical disinfection and antisepsis in the hospital. J Hosp. Res. 1957:9:5-31.

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Last updated: 09/2019
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