1.sterilization

sterilization

Shilpa.K

Microbiology Tutor

AIMSRC

‘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.

METHODS

Physical Agent

◊ Heat - dry heat

- moist heat

◊ Radiation

◊ Filtration

Chemical Agents

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.

METHODS OF STERILIZATION

Dry Heat

Moist Heat

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

Methods:

Red heat

Flaming

Incineration

Infra red rays

Hot air oven

RED HEAT

Sterilization of non-

inflammable materials

o Inoculating loops

or wires

o Tips of forceps

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

INCINERATOR

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.

Methods :

Low temperature steam with formaldehyde

Autoclave

Ultra high temperature

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

Laboratory Autoclave

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

Bowie-Dick Tape

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.