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‘Sterilization is a process by which the articles are freed from
microorganisms both in vegetative form and spore state.’
APPLICATIONS
Sterilization is required for culture media,
suspending fluids, reagents, containers and
equipments used in microbiology.
In surgery, for medical and surgical
instruments.
Food and drug manufacture.
PHYSICAL AGENTS
HEATFactors Influencing Sterilization By Heat
Temperature And Time
Time required for sterilization is inversely
proportional to temperature of exposure
PROCESS TEMPERATURE (⁰C)
HOLDING TIME (MIN)
Dry Heat 160170180
1206030
Moist Heat 121126134
151003
Nature Of Heat
Number Of Microorganisms
Higher the number of microorganisms, longer
exposure time is required to sterilize the article.
Characteristics Of Microorganisms
Presence Of Organic Material
Organic material protects the microorganisms by
preventing full contact between object and agent.
Nature Of Article To Be Sterilized
DECREASING ORDER OF RESISTANCE OF MICROORGANISMS TO STERILIZATION
Prions
Bacterial spores
Mycobacteria
Non enveloped viruses
Fungi
Vegetative bacteria
Enveloped viruses
Certain terms to remember
¤ Thermal Death Point (TDP)
It is the minimum temperature required to kill all microorganisms in an aqueous suspension within 10 minutes.
¤ Thermal Death Time (TDT)
It is the minimum temperature required to kill an aqueous suspension of microorganisms at predetermined temperature in a specified environment.
¤ Decimal Reduction Time(D-value)
Time in ( in minutes) required at a particular temperature to reduce number of viable organisms by 90%.
¤ Z-value
Number of degree centigrade required to produce a 10-fold change in D-value.
Mechanism :
Dry heat kills microorganisms by
Denaturation of their proteins
Oxidizing their chemical constituents ( oxidative
damage).
Toxic effects of elevated levels of electrolytes.
DRY HEAT METHOD OF STERILIZATION
FLAMING
INCINERATION
Direct burning of materials at high temperature (800⁰- 1000⁰C)
Used for destroying materials :
o Soiled dressings
o Bedding of patient
o Animal carcasses
o Pathological materials
Drawbacks
Method is destructive
Toxic air emission and heavy metals in ash
INFRA-RED RAYS
HOT AIR OVEN
It has a 2-walled chamber
Inner wall of copper
vessel.
Outer wall of asbestos
to reduce radiation of
heat.
The chamber is divided into small
compartments with the help of
removable racks.
Thermometer is inserted at one of
the corners.
Heated with heating elements.
Fan is fitted at the back of the
chamber.
Procedure :
Holding temperature and time generally used is 160⁰C for 2 hours.
Cover all glass wares to be sterilized with craft paper and keep
in the chamber in a manner, which allows free circulation of air in
between the objects.
Adjust the temperature at the required level.
Record the temperature with the help of thermometer and
maintain at 160⁰C for 2 hours.
After this specific time, cut off the electricity and allow the material
to cool.
Open the oven when the temperature inside the oven equals
atmospheric temperature.
Precautions :
Oven should not be overloaded.
Glassware must be free from water traces and properly covered with
craft paper.
The material should be properly arranged to allow free circulation of
air.
Rubber material or any inflammable material should not be kept
inside the oven.
After use it should be allowed to cool for 2 hours before opening the
door to avoid cracking of glasswares by sudden cooling.
Uses :
Sterilization of
Glass wares such as flasks, pipettes, test tubes,
petriplates, glass bottles, etc.
Forceps, non-sharp surgical instruments.
Swabs
Pharmaceutical products such as liquid paraffin,
sulphonamides, dusting powder, fats, grease, etc.
Advantages :
Sterilization can be done in controlled manner.
Articles are obtained dry after sterilization.
Limitations :
Can not be used for sterilization of liquids.
Dry heat has low penetration power so it requires
higher temperature for longer duration.
Time consuming and less efficient compared to moist
heat.
TESTS FOR EFFICACY
Physical Control : Thermocouples
Chemical Control : Browne’s tube type 3
Biological Control: Bacillus subtilis var niger (ATCC 9372)
MOIST HEAT METHODS OF STERILIZATION
Mechanism :
Kills microorganisms by coagulating and
denaturing their enzymes and structural proteins.
Water participates in this process.
LOW TEMPERATURE STEAM WITH FORMALDEHYDE
Principle -
Steam at sub-atmospheric pressure kills the spores of
thermophile bacteria very slowly. Formaldehyde by itself is
sporicidal, but only at high concentration and in the presence
of moisture. Together steam and formaldehyde are synergistic.
Working- Temperature : 73⁰C
Pressure :263 mmHg
Time :1 hour
Uses-
Sterilization of heat sensitive equipments
Used in the sterilization of endoscopes, catheters and face
masks.
Tests For Efficacy-
Biological Indicator :Spores of Bacillus stearothermophilus (ATCC 7953)
Disadvantage :
Formaldehyde concentration above 2 ppm is
toxic and irritates the eyes and respiratory tract.
AUTOCLAVE
Principle-
Water boils at 100⁰C at atmospheric pressure. When we increase
the pressure,the steam is at higher temperature than 100⁰C. When
this steam condenses on surfaces of articles it releases the latent
heat which is very high and lethal to the microorganism.
Condensation has 3 effects:
Heats up the items in the load
Condensed water provide condition essential for killing of
microorganisms,
Condensed steam causes significant reduction of volume
drawing more steam to the site.
Saturated steam has better penetrating power and
microorganisms are more susceptible to moist heat.
For effective sterilization, steam should be
Saturated
Dry
Pure
Pressure -temperature ratio should be maintained to maintain the
efficiency.
Structure of Autoclave
o It consists of vertical or horizontal cylinder of stainless steel
or gun metal, supported by a case made of iron sheet.
o Lid is fastened by screw clamps and made air tight by asbestos
washer.
o Lid consists of air and steam discharge tap
pressure gauge
safety valve
Working
Place the sufficient water in the autoclave chamber.
Place the articles to be sterilized on the perforated tray .
Screw down the lid to close the chamber. Open the discharge
tap and turn on the source of heat.
When water boils, allow steam air mixture to escape freely
through the discharge tap till all the air displaces.
Close the discharge tap and allow the pressure to rise until the
required level is reached. Safety valve opens and closes as the
pressure rises above and below the level.
Allow the autoclaving to continue. Holding temperature and
time commonly used is 121°C for 15 minutes at 15lb/in²
pressure.
Turn off the heater and allow the autoclave to cool slowly until
the gauge indicates that chamber is at atmospheric pressure.
At this point, open discharge tap slowly to allow air to enter
the chamber.
Open the lid and take out the material for use.
Uses: For sterilization of
Liquids like water and saline.
Media (except media containing sugar, blood, and
serum)
Dressings, instruments and lab wares
Pharmaceutical products.
Rubber teats
For disposal of culture
Limitations :
Insufficient air discharge results in incomplete
sterilization.
No facility for drying the load after sterilization.
Heat labile substances cannot be sterilized.
TYPES OF AUTOCLAVE
Steam-Jacketed Autoclave
oA Supply of steam from the
external source .
oA steam jacket that heats the
side walls independently of the
presence of steam in the
chamber and so facilitates
drying of the load.
Downward displacement Autoclave
oAir is displaced downwards from the chamber.
oVenturi pump or condenser assist drying of the load.
Uses :
• Sterilization of bottled fluids, bulky packages of surgical dressing.
Disadvantages :
• No vacuum assistance for air removal.
•Time consuming.
Transportable bench-top Autoclave
Turbulent air displacement.
Uses:
• Only for unpackaged, unwrapped and non-porous items.
Disadvantages :
•No vacuum assisted air removal facility.
•Lacks means for drying the load after sterilization.
•Cannot be used for liquid loads, porous loads, discard loads and
packaged items.
Multipurpose Autoclave for porous load
o Vacuum assisted air removal stage prior to steam sterilization.
Uses:
• Sterilization of packaged and wrapped goods.
• Sterilization of Linen and instruments with lumens.
Advantage:
Produces high vacuum to dry the load.
High security Autoclave
o It sterilizes the effluent steam and condensate and modifies the
exhaust air.
o Discards are placed in portex nylon bags, which allow free
penetration of steam and egress of air.
TESTS FOR EFFICACY
Physical Control: Recording Thermometer
Produces a graphic time record of change in
temperature.
Thermocouple
A device that detects heating-up time
required.
Chemical Control: Browne’s control tube (type-2) shows change
in color from red to green.
Bowie-Dick tape
Biological Control: Bacillus stearothermophilus
ULTRA HIGH TEMPERATURE (UHT)
Milk and milk products are sterilized by exposure at
140°C for 1-3 seconds.
UHT-processed milk can be stored at room temperature
for 2 to 4 months without change in flavor.
RADIATIONThe time required for destruction of microorganisms depends upon:
Intensity of radiation
Duration of exposure
Distance of source of illumination
Nature of medium in which the organisms are exposed.
Ionizing radiation : It includes α rays, X-rays and high speed
electrons.
They have high penetrating power
Highly lethal to DNA and other vital cell constituents
There is no appreciable increase in temperature so
method is known as COLD STERILIZATION.
Uses:
For sterilization of Disposable plastic syringes, swabs,
catheters, surgical gloves, rubber, fabrics etc.
For sterilization of oils and grease, food products,
pharmaceutical products etc.
Demerits:
Harmful to skin and eyes
Expensive machinery
Test for efficacy:
Biological indicator: Bacillus pumilis
CHEMICAL AGENTS
Ethylene Oxide
Low Temperature Steam –Formaldehyde
Gluteraldehyde
Hydrogen peroxide gas plasma
Peracetic acid
ETHYLENE OXIDE
Highly penetrative, non-corrosive, microbicidal gas used in
industry for sterilization of single use, heat sensitive medical
devices.
To ensure sterility materials should be exposed to :
Gas concentration : 700-1000mg/L
Temperature : 45°-60°C
Relative humidity : .70%
Duration : 2 hours
It is rendered non explosive by mixing with inert gases
at 10% concentration.
Mode of Action
Microbicidal activity of Ethylene oxide is due to the result of
Alkylation of protein, DNA and RNA
Replacement of a hydrogen atom with an alkyl group
Prevents cellular metabolism and replication of cell
Advantages
• Broad spectrum activity.
• Effective for heat and moisture sensitive materials.
Disadvantages
• It requires aeration time to remove ethylene oxide residues.
• Highly inflammable and explosive.
• Vapors are irritant to eyes and mucous membrane
• Mutagenic and Carcinogenic.
• Chronic inhalation can cause neurologic dysfunction.
TESTS FOR EFFICACY: Bacillus subtillis var niger
GLUTERALDEHYDE
Gluteraldehyde is a colorless oily chemical sterilant.
Gluteraldehyde is sporicidal only in alkaline condition with
shelf life of 14 days.
Mode of Action:
Inactivation of DNA,RNA,and Protein synthesis through
alkylation of sulfhydryl, hydroxyl, carboxyl and amino
groups of microorganisms.
Microbial Activity:
A standard 2% aqueous solution of Gluteraldehyde buffered
to pH 7.5 to 8.5 is Bactericidal, Tuberculocidal, Sporicidal,
Fungicidal, and Virucidal.
Spores of Bacillus and Clostridium spp are destroyed by 2%
Gluteraldehyde in 3 hours.
Variables:
•pH and temperature
•Gluteraldehyde concentration at the time of use
•Presence of organic ions
•Age of the solution
Uses:
Sterilization of
oBronchoscopes, cystoscopes, Transducers,
oPlastic endotrachial tubes, facemasks.
oMetal instruments and polythene tubing.
oLensed, Rubber Plastic and sharp instruments.
Advantages:
Excellent biocidal property even in presence of organic
matter.
Non corrosive.
It does not harm the lens.
Disadvantages:
Extremely susceptible to pH changes because its active only
in alkaline environment.
Expensive.
HYDROGEN PEROXIDE GAS PLASMA
Principle:
Gas plasmas are generated in an enclosed chamber under
deep vacuum. Radio frequency is used to excite the gas
molecules and produce charged particles in the form of free
radicals that are capable of interacting with essential cell
components and thereby disrupt the metabolism of
microorganisms.
Mode of action
Inactivates microorganisms by combined use of hydrogen
peroxide gas and generation of free radicals during plasma
phase of the cycle.
Operating temperature : 37°-44°C
Cycle time : 28-38 minutes
Uses:
Sterilization of materials and devices that cannot tolerate
high temperatures and humidity such as plastics, electrical
devices and corrosion susceptible metal alloys.
TESTS FOR EFFICACY
Biological indicator: Bacillus subtilis
PERACETIC ACID
Working:
Supply the 35% peracetic acid and an anticorrosive agent in a single-dose container.
Puncture the container immediately prior to closing the lid and initiating the cycle.
Dilute the peracetic acid to 0.2% with filtered water at 50°C and circulate within the chamber of the machine and pump through the channels of endoscope for 12minutes.
Mode of Action:
•It denaturates proteins
•Disrupts cell wall permeability
•Oxidizes sulfhydryl and sulfur bonds in proteins, enzymes and other metabolites.
Microbial Activity of peracetic acid:
Uses:
Sterilization of Gastrointestinal endoscopes and surgical instruments.
TESTS FOR EFFICACY
Biological Control: Bacillus stearothermophillus
TIME CONCENTRATION (ppm)
Bacteria, fungi, yeasts <5 minutes 100
Bacteria, fungi, yeasts in presence of organic matter
<5 minutes 200-500
Viruses 15 minutes 1500-2250
Bacterial spores 15 seconds- 30 minutes
500-10,000
CENTRAL STERILE SUPPLY DEPARTMENT
(CSSD)
Objective:
To make reliably sterilized articles available at the required
time and place for any purpose in the hospital as
economically as possible.
Advantages:
It frees nurses from cleaning syringes, packing swabs etc.
Expensive automatically controlled equipment can be used
intelligently and economically to ensure proper sterilization.
Requirement of number of autoclave can be reduced by
great potential high pre-vacuum sterilizer.
It provides number of dressings and instruments in separate
packs.
REFRENCES
Ananthanarayan and Paniker’s.Text book of
Microbiology;7th edn.
Greenwood. Medical microbiology;16th edn.
Mackie and McCartney. Practical Medical
Microbiology;13th and 14th edn.
Robert Cruickshank. Medical Microbiology;vol 1;12th edn.
Topley and Wilson’s. Bacteriology;vol 1;10th edn.
CDC Guidelines for Disinfection and Sterilization in Health
care Facilities;2008.
Bailey and Scott’s. Diagnostic Microbiology;12th edn.