LECTURE 6 Sterilization

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STERILIZERS AND

STERILIZATION

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Introduction

USP described five sterilization processes:


• Steam (moist) Sterilization
• Dry Heat Sterilization
• Filtration Sterilization
• Gas Sterilization
• Ionization Sterilization

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Methods of Sterilization

Moist Heat Dry Heat Chemical (cold) Radiation Filtration

Ethylene oxide
Saturated heat autoclaves Batch sterilizers
Vaporized hydrogen peroxide
Superheated water autoclaves Continuous tunnel sterilizers Electromagnetic particulate Membranes
Hydrogen peroxide/steam
Air oven steam autoclaves
Other gases

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Introduction … cont.

The sterilization processes are commonly used for parenteral products,


except gas and ionizing radiation, which are widely used for devices
and surgical materials.

To assist in the selection of the appropriate sterilization method, certain


basic information must be considered:
1- The nature and amount of product
2- Whether the product and container-closure system will have a
predominately moist or dry environment during sterilization.

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Introduction … cont.

For sterilization purposes, microorganisms can be categorized into three


general categories:
1- Easy to kill with either dry or moist heat
2- Susceptible to moist heat, but resistant to dry heat (Bacillus subtilis)
3- Resistant to moist heat, but susceptible to dry heat
(Colstridium sporogenes)
Sterilization tests are performed to verify that an adequate sterilization
process has been carried out. Validation of sterilization cycle also gives
assurance of process (including the performance of the equipment and
personnel)

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Hardest to Kill Prions (Creutzfeldt-Jakob Disease (CJD), mad
cow disease)
Spores (C. difficile)
Mycobacteria (Tb)
Non-enveloped viruses (norovirus)
Fungi (Candida)
Vegetative bacteria (MRSA, VRE)
Enveloped viruses (HIV, HBV)
Easiest to Kill
General Facts

The procedure to be used for sterilizing a drug, a pharmaceutical


product, or a medical device is determined to a large extent by the
nature of the product. It is important to remember that the same
sterilization technique cannot be applied universally because the
unique properties of some materials may result in their destruction
or modification.

Methods of inactivating microorganisms may be classified as


either physical or chemical. Physical methods include: moist heat,
dry heat and irradiation. Sterile filtration is another process, but it
only removes, not inactivates, microorganisms, chemical methods
include the use of either gaseous or liquid sterilants

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General Facts… (cont.)

Heat stable reusable medical devices that enter the blood stream or enter
normally sterile tissue should always be reprocessed using heat-based methods
of sterilization (e.g., steam autoclave or dry heat oven).

Laparoscopic or arthroscopic telescopes (optic portions of the endoscopic set)


should be subjected to a sterilization procedure before each use; if this is not
feasible, they should receive high-level disinfection. Heat stable accessories to
the endoscopic set (e.g., operative instruments) should be sterilized by
heat-based methods (e.g., steam autoclave or dry heat oven).

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General Facts … (cont.)

Reusable devices or items that touch mucous membranes should, at a minimum,


receive high-level disinfection between patients. These devices include reusable
flexible endoscopes, anesthesia breathing circuits, and respiratory therapy
equipment.

Medical devices that require sterilization or disinfection must be thoroughly


cleaned to reduce organic material or bioburden before being exposed to the
germicide, and the germicide and the device manufacturer's instructions should be
closely followed.

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STEAM STERILIZATION
Terminology

• Bioburden- The degree of contamination with microorganisms and organic


parameters
• Bioresistance- Factors such as heat and/or moisture sensitivities and product
stability
• Biostate- The nutritional, physical, and reproductive phase of
microorganisms
• Bioshielding- Characteristics of the packaging materials
• Bowie Dick test (DART)- A test run daily that validates the vacuum function
of the sterilizer. The test should be run in an empty load at the same time
every day
• Contaminate- To render unfit for use through introduction of a substance
which is harmful or injurious

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Terminology … cont.

• Contamination- A state of being soiled or infected by contact with infectious


materials
• Culture- A growth of microorganisms on a medium
• Culture Medium- Any substance or preparation used for the growth and
cultivation of microorganisms
• Incubate- To maintain under optimum environmental conditions that are
favorable for growth
• Incubation period- The period between the time infection occurs and the
appearance of the first symptoms
• Incubator- an Apparatus for maintaining a constant and suitable
temperatures for the growth and cultivation of microorganisms
• Nonpathogenic- Not capable of producing disease
• Pathogenic- Capable of producing disease

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Terminology … cont.

 TDT is the time required to kill a known population of microorganisms in a


specific suspension at a particular temperature

 Increasing temperature decreases TDT

 Lowering the temperature increases TDT

 Acidic or basic pHs decrease TDT

 Fats and oils slow penetration and increase TDT

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Terminology … cont.

• Spore- Certain microorganisms which are capable of forming a thick wall


around themselves enabling them to survive in adverse conditions. It is
highly resistant to heat
• Sterile- Completely devoid of all living microorganisms
• Thermal equilibrium- A condition where all parts of a system have reached
the same temperature; in a steam autoclave, when the temperature
throughout the entire load is the same

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Action (mechanism) of Steam

 Steam contacts a cooler surface, condenses, causing a


huge decrease in volume and setting up a negative
pressure that draws more steam
 Condensation occurs as long as there is a
temperature differential
 Action of steam ensures: Surface heating,
penetration, and protein coagulation

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Advantages of Steam Sterilization

• Stainless steel instruments withstand repeated processing without damage and


no residue
• Steam kills at lower temperatures than dry heat
• Easiest, safest, and surest method of on-site sterilization
• Fastest method, total cycle time is shortest
• Least expensive and most easily supplied agent-piped in from the facility’s
boiler room
• Most sterilizers have automatic controls and recording devices to eliminate
human error from the process

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Disadvantages of Steam Sterilization

• Preparing, packaging, loading and operating the sterilizer. Also, drying time
• Instruments need to be free from grease and oil and not be sensitive with heat
• All items must have direct contact with steam and must be able to penetrate
packaging material
• Timing is adjusted for the differences in load and material and subject to
human error
• The steam may not be pure. Refers to the amount of solid, vapor, or liquid
contamination in the steam

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Autoclave

Autoclave:
An apparatus for
sterilization by steam under
pressure, usually at
temperatures of 250 degrees
to 270 degrees F

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Principles

Two important factors should be controlled:

1- Application of pressure:
Because it is not possible to raise the temperature of the steam above
100 °C under atmospheric conditions, pressure is employed to achieve
higher temperature (it should be recognized that the temperature, not the
pressure is destructive to the microorganism and that the application of
pressure solely for the purpose of increasing the temperature of the
system.

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Principles … cont.
2- Application of time:
Time is another important factor in the destruction of microorganisms by
heat. Most modern autoclaves have gauges to indicate to the operator
the internal conditions of temperature and pressure and timing device to
permit the desired exposure time for the load. The usual conditions
(pressure/temperature/time), are as follows:

Pressure Temperature Time


10 pounds 115.5 °C 30 minutes
15 pounds 121.5 °C 20 minutes
20 Pounds 126.5 °C 15 minutes
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Standard Temperature and Pressure

250 °F (121 °C) and 15 p.s.i.

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How Long to Autoclave

Dry goods @ 121 °C Time (min)

 Glassware, empty, inverted 15

 Instruments, wrapped 30

 Utensils, wrapped 30

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How Long to Autoclave … cont.

Biohazardous waste bags, @ 121 °C, loosely


tied
Time (min)
• 2 or more bags 100 +
• 1 bag, full 90
• 1 bag, half full 60

Testing available for individual situations

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How Long to Autoclave … cont.

Liquids, in bottles with vented caps


Size (ml) Time (minutes)
75 25
250 30
500 40
1000 45
1500 50
2000 55

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Autoclave applicable:

A. Applicable for pharmaceuticals preparations and materials that can


withstand the required temperature and are penetrated but not adversely
affected by, moisture.
B. In sterilizing aqueous solutions, the moisture is already present, and all
that is required is the elevation of the temperature of the solution for the
prescribed period of time. Thus solutions packaged in sealed container as
ampoules, are readily sterilized by this method.
C. Also applicable to bulk solutions, glassware, surgical dressings, and
instruments.

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Autoclave not applicable:

A. The sterilization of oils, fats, or any oleaginous preparations.


B. Other preparations not penetrated by the moisture.
C. Sterilization of exposed powders that may be damaged by
condensed moisture.

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Types of steam sterilizers

1. Gravity displacement

Definition: Gravity pushes air through the


packages and down through the drain.
Sterilization begins when steam passes the
thermometer and reaches the desired
temperature.

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Gravity Displacement Uses

1. Metalware
2. Glassware
3. Thermoplastics
4. Linens
5. Rubber

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Types of steam sterilizers … cont.

2. Pre-vacuum (high temperature)


Sterilizer
Definition: Air is completely evacuated from the
chamber by a vacuum. The steam-injector helps
eliminate the air out of packages. Steam then
penetrates the packages on all surfaces.

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Pre-vacuum Sterilizer Uses:

Metalware
Rubber
Thermoplastics

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Types of steam sterilizers … cont.

3. High Pressure (flash) Sterilizer


Definition: Another sterilizer that can be used in
either gravity displacement or pre-vacuum. Gravity
displacement is the most common used. Steam in
the jacket should be maintained at all times.

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High Pressure Flash Uses

Urgent items: (dropped items for which no


method of sterilization exists) Items that are
dropped, or forgotten

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Types of steam sterilizers … cont.

4. Washer-sterilizer
Definition: Wash and sterilize instruments. The
water floods the chamber with cold water to
prevent proteins from setting on the instrument.
The water is then heated with steam to agitate and
clean the instruments.

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Washer-sterilizer Uses:

to wash and terminally sterilize items which are


non-heat sensitive, immediately after operative
procedures.

NO GLASS, SHARPS OR DELICATE INSTRUMENTS


GO THROUGH THIS STERILIZER. BREAKAGE
COULD OCCUR!!!

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Biological Indicator Controls/Spore
Tests
Bacillus stearothermophilus: is used strictly as biological
indicator of effective heat sterilization (steam and dry heat
sterilizers) by including filter paper strips carrying
a saturated number of spores into the autoclave cycle.
The strips are then incubated to attempt to recover viable
organism. The usual autoclave cycle of 121 °C for
15 minutes is adequate to kill Bacillus stearothermophilus
with a high margin of safety.

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Biological Indicator Control/Spore
Tests…cont.
When Used:
Upon installation of new system
After major repairs
Routine quality assurance.
With all implants

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Biological Indicator Controls/Spore
Tests
• Record results:
• Negative- no color change from original.

• Positive- color changed is usually amber in color.


• Take sterilizer out of service
• Report to supervisor
• Recall all items sterilized in sterilizer for
last 24 hour period

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STERILIZATION by FILTRATION
Filtration
- It depends upon the physical removal of
microorganisms by adsorption on the
filter medium or by sieving mechanisms.
- It is used for sterilization of heat-
sensitive solutions.
- Medicinal preparations sterilized by this
method are required to undergo severe
validation and monitoring since the
effectiveness of the filtered product can
be greatly influenced by the microbial
load in the solution being filtered.
- It removes, but does not destroy
M.O.
- Commercially available filters are
produced with a variety of pore-size
specifications (e.g. Millipore filters).

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Filtration …cont.
Most filters consist of:
1- diatomaceous earth
2- cellulose acetate or
3- nitrocellulose

0.45 µm filters removes most


bacteria

0.20 µm more inclusive,


viruses need 0.01 µm

Useful for sterilizing liquids


(enzymes, vaccines) that are
destroyed by heat

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Filtration …cont.
Factors affecting removal of M.O.:
1- Pore size of filter
2- Electrical charge of the filter
and that of the M.O.
3- pH of the solution
4- T, P, and Vacuum applied

Advantages:
1- Speed
2- Good for thermolabile
materials
3- Inexpensive
4- The complete removal of living
and dead M.O. as well as
other particulate matter from
the solution

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Filtration …cont.
Disadvantages:

1- The membrane is fragile and because


of that, it is essential to determine that
the assembly was properly made and the
membrane was not ruptured

2- Filtration of large volumes of liquids


would require more time (particularly if
the liquids were viscous)

3- Useful when heat cannot be used and


small volumes of liquids.

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OTHER TECHNIQUES
Ultrasonic vibrations

– High frequency sound waves


– Know as Sonicator, untrasonic machines
– Killing by –shock waves that disintegrates cell wall

and membranes. 45
PYROGENS AND PYROGEN
TESTING
PYROGEN

- A pyrogen is a material which when injected into


a patient will cause a rise in body temperature
(pyrexia).
- The lipopolysacchride that comprise a a major
part of the cell wall of gram-negative bacteria are
called endotoxins, and it is these that are the
most commonly encountered pyrogens.
- Bacterial cells may be pyrogenic even when they
are dead and when they are fragmented, and so
a solution or material that passes a test for
sterility will not necessarily pass a pyrogen test.

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PYROGEN… cont.
Two main procedures are used for the detection of pyrogens.

A. The traditional method:


It requires the administration of the sample to laboratory rabbits whose
body temperature is monitored for a period of time thereafter.

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PYROGEN… cont.

Steps:
1- Render the syringes, needles, and glasswares free form pyrogens by
heating at 250 °C for not less than 30 minutes.
2- Warm the product to be tested to 37 °C ± 2 °C
3- Inject into an ear vein of each of three rabbits 10 ml of the product per
kg of body weight.
4- Record the temperature at 30-minute intervals between 1 and 3 hours
subsequent to the injection
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PYROGEN… cont.

Steps:
5- If no rabbit shows an individual rise in temperature of 0.5 °C or more
above its respective control temperature, the product meets the
requirements for the absence of pyrogens.
6- If any rabbit shows an individual temperature rise of 0.5 °C or more,
continue the test using five other rabbits.

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PYROGEN… cont.

Steps:
7- If no more than three of the eight rabbits show individual rise in
temperature of 0.5 °C or more and if the sum of the eight individual
maximum temperatures rises does not exceed 3.3°, the material under
examination meets the requirements for the absence of pyrogens.

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PYROGEN… cont.

B. Limulus Amoebicyte Lyste Test (LAL)

In recent years, it has been shown that an extract from the blood cells of
the horseshoe crab (Limulus polyphemus) contains an enzyme and
protein system that coagulates in the presence of low levels of
lipopolysaccharides.

The discovery has led to the development of the Limulus Amoebicyte


Lyste Test (LAL) for the presence of bacterial endotoxins.

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PYROGEN… cont.

B. Limulus Amoebicyte Lyste Test (LAL) … cont.

Proclotting enzyme

Endotoxin

Activated clotting enzyme

Coagulation Clottable protein GEL


The Lysate Clotting Mechanism 53
PYROGEN… cont.
B. Limulus Amoebicyte Lyste Test (LAL) … cont.

In the test procedure, the lysate is mixed with equal volume of the test
solution in a depyrogenated container, such as a glass tube. The tube is
then incubated undisturbed at 37 °C for a period of about 60 minutes.
The test is a pass or fail test. The end point is identified by gently inverting
the glass tube. A positive result is indicated by the formation of a solid
clot. The clot doesn’t disintegrate when the tube is inverted. A negative
result is indicated if no gel clot has been formed.

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