STERILIZATION INDICATORS

There are two types of sterilisation indicators they are:

 Biological indicators
 Sterilisation indicators

BIOLOGICAL INDICATORS:

A biological indicator may be defined as a characterised preparation of a specific

microorganism that provides a defined and stable resistance to a specific sterilisation
process.Microorganisms widely recognised as suitable for biological indicators are spore
forming bacteria because , with the exception of ionizing radiation processes, these
microorganisms are significantly more resistant than normal microflora. A biological
indicator can be used to assist in the performance qualification of the sterilisation equipment
and in the development and establishment of a validated sterilisation process for a particular
article.

Benefits of Biological Indicators in Sterilization Process

Biological indicators, depending on the specific type, can be used for various sterilization
processes using steam, hydrogen peroxide gas, ethylene oxide and more.

Several factors such as operator experience, load preparation and sterilizer condition can
impact the sterilization cycle. Biological indicators provide a direct measure of the lethality
of the process and so the use of biological indicators to routinely monitor sterilizers provides
assurance in the efficacy of the sterilization process.

BIs are typically used within process challenge devices (PCD) that are designed to represent
the most challenging products routinely processed. A passing result for the Biological
indicators within this defined challenge demonstrates that the sterilizer is effective in killing
a large number of highly resistant bacterial spores, providing users with a level of assurance
in their sterilization process.

Biological Indicator Testing Procedure

A biological indicator is made up of a carrier material, on which bacterial spores with a
defined resistance to the sterilization process have been applied. The carrier material is
enclosed within a glassine envelope or a vial. The Biological indicators is exposed to the
 sterilization process and then incubated under defined growth conditions to determine
whether any spores survived the process. If no spores survive, none grow and the test is a
pass. If growth is detected, the test is a fail.

As per AAMI, Process challenging devices (PCD) containing biological indicators should be
used for routine monitoring of steam sterilization cycles at least weekly, preferably daily, and
in every load containing implants. Sterile Processing Department staff should follow the
manufacturer's directions for the appropriate placement of the PCD containing the BI in the
sterilizer.

Besides routine sterilizer monitoring, PCDs containing BIs are also used for other
applications as listed below:

 Routine load release of nonimplant loads

 Routine load release of implant loads. AAMI recommends using a PCD containing a
Biological indicators and a Class 5 integrating indicator. Sterile Processing staff can
also use a PCD containing a Biological indicators and a Class 6 challenge pack. In
either case, loads containing implants should be quarantined until the BI results are
known, unless it is an emergency situation.

 Qualification of sterilizers after installation, relocation, major repair, malfunction, and

sterilization process failures

 Periodic product quality assurance testing

Regardless of the type of process or application, Sterile Processing staff should always follow
the manufacturer's instructions for use (IFUs) when using a biological indicator to monitor
sterilization.

Biological Indicators for Steam Sterilizer (Autoclave)

Biological indicators (within a PCD) are often used for routine monitoring, qualification and
load monitoring of a steam sterilizer. Biological indicators are designed to demonstrate
whether the conditions during a steam (autoclave) cycle were adequate to achieve a defined
level of microbial inactivation. For steam sterilization, BIs are typically used within PCDs
and AAMI recommends weekly, preferably daily, routine monitoring of sterilizers and in
every load containing implants.

The PCD containing the BI and/or a chemical indicator should be placed in the most
challenging location in the chamber. In a steam sterilizer, this is typically on the bottom shelf
 near the drain. Upon completion of the cycle, the PCD is taken out and the BI is incubated.
After incubation the BI result is read. Incubators that are also readers will provide a final pass
or fail result within a specified amount of time.

Types of Biological Indicators for Sterilization Processes

There are various types of biological indicators depending on the need and sterilization
process:

 Self-contained biological indicators (SCBI) – These indicators combine the carrier

material with spore and growth medium into a single vial, eliminating the need for aseptic
transfer. Following sterilization, the vial is activated allowing the spores to mix with the
growth medium and incubated to allow spore growth. Some SCBI designs use an enzyme
based detection system that can detect spore growth sooner that most on the market, with
shorter incubation times.
Irrespective of the design, biological indicators are used during a steam sterilization process,
hydrogen peroxide sterilization process or ethylene oxide process, to ensure the efficacy and
lethality of the process.

Biological Indicators – Carriers

A carrier is a vessel (plastic, glass, filter paper, etc) which contains spores and is sealed so as
to preserve the viability of the microorganisms. The carriers are well made and designed not
to be degraded by the sterilisation process the Biological Indicator is meant to monitor, as it
would compromise Biological Indicator performance. The packaging itself also undergoes
aseptic manufacturing procedures to ensure it does not contain any contaminants which
would affect the stability or the performance of the Biological Indicator.

Biological Indicators – Spore Suspension

Biological Indicators can also come in a spore suspension, which is then directly applied to
the product, packaging, or surface that will undergo the sterilisation procedure. Simulations
of inoculated products are sometimes used in sterility monitoring, but it is usually a
requirement to show the final product, which will receive the inoculation, does not alter the
effectiveness of the Biological Indicator, thereby invalidation results.
Biological Indicators – Self-Contained Indicators
Self-contained indicators have packaging which contains the recovery growth medium for the
microorganisms’ incubation period following sterilisation exposure. Self-contained
Biological Indicators containing both the bacteria spores and the growth medium are an
enclosed system, which provides sterilisation resistance.

Sterilisation resistance of the self-contained indicator’s system depends on the length of time
it takes for the sterilant to penetrate the package. Ease of penetration is controlled through
composition and design at the manufacturing level. These types of indicators often contain a
dye to indicate negative or positive growth in bacteria spores following incubation, and the
sample is ready for incubation directly following the sterilisation process.

Biological Indicators Versus Enzyme Indicators

Though Biological Indicators have long held the market in sterilisation
monitoring, Enzyme Indicators are set to replace them as a faster, cheaper alternative. A
variety of different industries depend on Biological Indicators to provide sterilisation
monitoring, and stopping research or production while waiting on Biological Indicator results
to come back and be frustrating, not to mention costly. Enzyme Indicators are able to provide
instant, quantifiable results, which means not to stop cleanroom or asepticenvironment
operations waiting for spores to incubate.
Enzyme Indicators use the thermostable enzyme Adenylate Kinase (isolated
from thermophilic bacteria) as it has predictable sterilisation resistance and a quantifiable
inactivation profile. Enzyme Indicators emit light that can then be measured with a
lumeometer, giving measurable data as compared to the positive or negative results received
with Biological Indicators. And unlike Biological Indicators, the results with Enzyme
Indicators are immediate. Protak Scientific is currently testing the commercial viability of
Enzyme Indicators, and is striving towards bringing this fast, reliable, and accurate
technology to the scientific community.
 Some of the Biological indicators

CHEMICAL INDICATORS:

Chemical indicator, any substance that gives a visible sign, usually by a colour change, of the
presence or absence of a threshold concentration of a chemical species, such as an acid or an
alkali in a solution. An example is the substance called methyl yellow, which imparts a
yellow colour to an alkaline solution. If acid is slowly added, the solution remains yellow
until all the alkali has been neutralized, whereupon the colour suddenly changes to red.
Like most indicators, methyl yellow is visible even if its concentration is as low as a few
parts per million parts of solution. Used at such low concentrations, indicators do not have
any influence on the conditions for which they are recommended. The common application of
indicators is the detection of end points of titrations.The colour of an indicator alters when the
acidity or the oxidizing strength of the solution, or the concentration of a certain chemical
species, reaches a critical range of values. Indicators are therefore classified as acid-base,
oxidation-reduction, or specific-substance indicators, every indicator in each class having a
characteristic transition range. Methyl yellow, an acid-base indicator, is yellow if
the hydrogen ion (acid) concentration of the solution is less than 0.0001 mole per litre and is
red if the concentration exceeds 0.0001. Ferrous 1,10-phenanthroline, an oxidation-reduction
indicator, changes from red to pale blue when the oxidation potential of the solution is
increased from 1.04 to 1.08 volts; and diphenylcarbazone, an indicator for mercuric ion,
changes from yellow to violet when the mercuric ion concentration is increased from
0.000001 to 0.00001 mole per litre. Each of these indicators thus has a relatively narrow
transition range, and each is capable of giving a sensitive, sharp indication of the completion
of a reaction, that is, the end point.Although the visible change of the indicator is usually a
colour change, in some cases it is a formation or disappearance of a turbidity. If, for example,
a soluble silver salt is added to a solution of cyanide that contains a trace of iodide, the
solution remains clear until all the cyanide has reacted to form the soluble silver cyanide
complex ion. Upon the addition of more silver, the solution becomes turbid because insoluble
silver iodide forms. Iodide is therefore an indicator for excess silver ion in this reaction.

CHEMICAL INDICATOR:

A chemical indicator is a substance that undergoes a distinct observable change when

conditions in its solution change. This could be a color change, precipitate formation, bubble
formation, temperature change, or other measurable quality.

Another type of indicator that may be encountered in chemistry and other sciences is a
pointer or light on a device or instrument, which may show pressure, volume, temperature,
etc. (OR)
the condition of a piece of equipment (e.g., power on/off, available memory space).
The term "indicator" comes from the Medieval Latin words indicare (to indicate) with the
suffix –tor.

Examples-of-indicators:
A pH indicator changes color over a narrow range of pH values in solution.Thereare many
different pH indicators, which display different colors and act between certain pH limits. A
classic example is litmus paper. Blue litmus paper turns red when it's exposed to acidic
conditions, while red litmus paper turns blue under basic conditions.

 Fluorescein is a type of adsorption indicator. The dye is used to detect the completed
reaction of the silver ion with chloride. Once sufficient silver is added to precipitate
chloride as silver chloride, excess silver is adsorbed onto the surface. Fluorescein
combines with adsorbed silver to produce a color change from greenish-yellow to red.
  Other types of fluorescent indicators are designed to bond to selected molecules. The
fluorescence signals the presence of the target species. A similar technique is used to
label molecules with radioisotopes.
 An indicator may be used to identify the endpoint of a titration. This may involve the
appearance or disappearance of a color.
 Indicators may indicate the presence or absence of a molecule of interest. For
example, lead tests, pregnancy tests, and nitrate tests all employ indicators.

DESIRABLE-QUALITIES-OF-CHEMICAL-INDICATOR:
To be useful, chemical indicators must be both sensitive and easily detectable.It need not,
however, show a visible change. The type of indicator depends on how it's being used. For
example, a sample analyzed with spectroscopy may employ an indicator that wouldn't be
visible to the naked eye, while a test for calcium in an aquarium would need to produce an
obvious color change.

Another important quality is that the indicator doesn't change the conditions of the sample.
For example, methyl yellow adds a yellow color to an alkaline solution, but if acid is added to
the solution, the color remains yellow until the pH is neutral. At this point, the color changes
from yellow to red. At low levels, methyl yellow does not, itself, change the acidity of a
sample.

Typically, methyl yellow is used at extremely low concentrations, in the parts per million
range. This small amount is sufficient to see a visible change in color, but not enough to
change the sample itself. But what If an enormous amount of methyl yellow were added to a
specimen? Not only might any color change be invisible, but the addition of so much methyl
yellow would change the chemical composition of the sample itself.

In some cases, small samples are separated from larger volumes so that they can be tested
using indicators that produce significant chemical changes.

Chemical indicator application for use:

Since the first steam sterilizer was created by Charles Chamberland in 1880, researchers have
worked on methods to monitor sterilization processes. It was not until the late 1940s and
early 1950s that biological and chemical indicators were used routinely to monitor the
process. At that time, it was stated that no single quality-assurance monitoring method
assures that any item is sterile: rather, it assures that the conditions for sterilization to occur
were met. That very statement remains valid in today’s healthcare environment.
As sterilization processes have evolved and become more complex, so have the monitoring
systems that we use. Sterility assurance programs may consist of the following tools that
healthcare management can use:

 Physical monitoring
 Chemical indicators/integrators
 Biological indicators
 Sterilizer preventative maintenance
 Load record keeping This article discusses chemical indicators/integrators (CIs).
Chemical indicators are defined by the Association for the Advancement of Medical
Instrumentation (AAMI) as “…sterilization process monitoring devices designed to respond
with a chemical or physical change to one or more of the physical conditions within the
sterilizing chamber. CIs are often used to detect sterilizer malfunction/failures resulting from
improper loading of the sterilizer, incorrect packaging, deficiencies of the sterilizing agent, or
malfunction of the sterilizer itself.”

Remember, the “pass” reading of a CI does not mean that the item or items in the sterilizer
load are sterile; it means that the parameter or parameters for sterilization that the CI was
designed to measure have been met. The use of CIs is only one portion of an effective quality
assurance program. They should be used in conjunction with a biological indicator (spore
test), physical monitors, a sterilizer preventative maintenance program, and accurate record
keeping for each sterilization load.

Chemical Indicator Classifications:

AAMI ST-60 defines five classes of CIs and the specific performance requirements for each.
The most basic of chemical indicators, they are also known as throughput indicators. These
CIs are intended for use with individual items to be sterilized. They are designed to
demonstrate that the item has been exposed to a sterilization process and to distinguish
between processed and non-processed items. Examples of Class 1 indicators are tape and the
indicators found on paper or plastic peel pouches.
 Process Indicators (Class 1): The most basic of chemical indicators, they are also
known as throughput indicators. These CIs are intended for use with individual items
to be sterilized. They are designed to demonstrate that the item has been exposed to a
sterilization process and to distinguish between processed and non-processed items.
Examples of Class 1 indicators are tape and the indicators found on paper or plastic
peel pouches.

 Indicators for Use in specific Tests (Class 2): Also known as specialty indicators,
Class 2 indicators are designed for use in specific test procedures as defi ned by
relevant sterilization standards. Examples of Class 2 indicators are the Bowie Dick
and Dart products used in steam sterilizers. These indicators test for the presence of
air in the steam sterilizing chamber. A positive test would mean that air, which
inhibits the conditions necessary for sterilization, has either not been removed during
the cycle or has entered the chamber through leaks in the system. Maintenance must
be performed on the steam sterilizer to prevent the presence of air in the chamber
during a sterilization cycle.

 Single-parameter Indicators (Class 3): These indicators react to one of the critical
process parameters of sterilization and indicate exposure to a sterilization cycle at
stated values of the chosen parameter. Critical parameters typically chosen for steam
sterilization processes are time or temperature.

 Multi-parameter Indicators (Class 4): These indicators are more accurate by design
than Class 3 indicators. They react to two or more critical parameters of the
sterilization process and indicate exposure to the sterilization cycle at stated values of
the chosen parameters. Time and temperature are examples of steam sterilization
parameters, and time and concentration of ethylene oxide are chosen for EO
sterilization.

 Integrating Indicators (Class 5): These indicators, known as “integrators,” are

designed to react to all critical parameters over a specified range of sterilization
cycles. Their performance has been correlated to the performance of a biological
indicator (BI) under its labeled conditions for use. This class of indicator can be used
 in place of the BI in many applications, thereby reducing the overall cost of sterile
processing.

Use of Chemical Indicators:

When considering which class of indicator to use, you must consider your external and
internal chemical monitoring needs for your various sterilization systems.
External indicators are simply used to distinguish between processed and unprocessed items.
A Class 1 indicator in the form of tape, an indicating label, or the indicator legend on a paper
or plastic peel pouch is appropriate and should be placed on each package that is intended for
sterilization.

The external CI usually exhibits a simple visual color change that shows the package has
been exposed to physical conditions present during a sterilization process.
Internal indicators need to be used within each package that will be sterilized. Depending on
the complexity of the pack and devices within them, a Class 3, Class 4, or Class 5 CI can be
used. All internal CIs should be placed in an area of the package that is determined to be the
least accessible to sterilant contact. This may not always be in the center of the package.
AAMI states that a Class 5 indicator may serve as the basis for the release of processed items,
excluding implants.
Integrator challenge packs utilize a Class 5 chemical integrator within an appropriate
challenge pack to monitor sterilization loads. The challenge packs are placed within the
sterilization load, outside the packs, in the sterilization chamber, and have been correlated to
the kill achieved in the AAMI 16 towel BI test pack. The result of the chemical integrator
may be used to release the load, excluding implantable devices.

Results of external indicators and chemical integrator challenge packs

can be read at the end of the sterilization cycle when the package is retrieved from the
sterilizer. However, internal indicators must be interpreted at the time of use. Therefore all
healthcare personnel should be trained on the proper interpretation of a positive and negative
result and what to do if a negative result occurs.
References:

1. Applied microbiology- Vinita kale ,kishore bhusari

2. http://chemicalindicators.britanicca.com

3. microbiology-pelczar

4. http://biologicalindicators.mesalabs.com