Semester 5 sterilization practical

Dr. fatima fasihResearch / Teaching AssociateDUHS

Objectives

What are various methods of disinfection & sterilization?

What is the difference between disinfection & sterilization?

What are different instruments used for sterilization?

What precautions are necessary for sterilization?

Sterile

Free of living micro-organisms

Sterilization Defined as a process by which articles are

made free from all viable micro-organisms and their products like spores.

Physical method Chemical method

Physical MethodThese are of following types:Heat Radiation Filtration

All objects should be physically cleaned before the process of sterilization.

Heat Sterilization Heat kills by denaturing proteins but

membrane damage and enzymatic cleavage of DNA may also be involved.

It is of two types:

Dry Heat

Moist Heat

Dry Heat

Methods include

Incineration

Red Heat

Flaming

Hot Air Oven

Incineration :

This means burning to ashes.

Red Heat

In this method articles are sterilized by holding them on flame until they become red-hot e.g. wire loop, neck of glass tube, flask etc.

Flaming

Articles are sterilized by passing them through flame without becoming red hot e.g. needles, glass slides.

Hot Air Oven

It is an oven heated by electricity and has regulatory thermostat that maintains the temperature of the chamber at the desired level.

It is used to sterilize glass ware. Temperature is kept at 160- 170oC for two hour.

Precautions:

Glassware must be dry.

Oven must not be overloaded.

Oven is allowed to cool for two hours approximately before the door is opened, otherwise the glassware would break.

Cotton stoppers must plug test tubes & flasks.

Petri dishes should be wrapped in paper before putting inside oven.

Moist HeatThis method could be used at different

temperature for different time periods.

Temperature below 100oCTemperature at 100oCTemperature above 100oC

Temperature below 100oC:

Pasteurization: e.g. milk is pasteurized by being held in water at 63oC for 30 minutes & cooled suddenly.

Tyndallization: Heating at 56oC for 60 minutes for three successive days is known as tyndallization

During this spores that survive the 1st episode may start to germinate & would be killed in the remaining two episodes. It is used to sterilize media, especially those containing spores.

Temperature at 100oC:

Boiling: In this method boiling at 100oC for 5 minutes is sufficient to kill all non-spore forming bacteria e.g. Syringes, glass needles, surgical instruments. Boiling at 100oC also kills resistant bacteria.

Steam at 100oC: This is obtained in a closed drum like vessel in which the water boils at 100oC and steam formed is passed through the articles , which are to be sterilized.

Done in a steamer. Usually culture media and linen are sterilized.

To ensure the destruction of spores of microorganisms by moist heat temperature above 100oC is required .

Temperature above 100oC:

Autoclave: Autoclaving is carried out by obtaining

temperature up to 121oC in a closed container.

When water is boiled within a closed vessel, steam collects and pressure inside increases above that of normal outside pressure. When the pressure is maintained at 15 lbs/sq. inch above the atmospheric pressure water boils at 121oC.

Autoclave is used to sterilize culture media , destruction of live cultures, surgical supplies, and linen etc. Both vegetative & spore forming organisms are killed.

Precautions:

At start exhaust valve must be kept open to release air.

After sterilization autoclave lid should only be opened till the pressure gauge returns to zero.

Exhaust valve is opened to eliminate steam.

Lid is opened after 10 -15 minutes.

Radiation

Ultra violet light inhibits DNA replication.

It is a useful sterilization of the atmospheric air e.g. operation theatre.

Ionizing RadiationInclude X-rays, Particle Radiations.

These produce free radicals, which break the covalent bond of DNA and thus kill the organisms.

They are specially used for sterilization of large quantities of commercial products e.g. disposable syringes, rubber gloves, transfusion sets etc.

FiltrationThis is used for sterilizing solution containing

sera and amino acids that would be denatured by heating and water.

Types of filters are cellulose membrane, earthenware candles, asbestos, sintered glass.

PERSONAL UNIVERSAL PRECAUTION

HAND WASHING GLOVES

MASK

CleaningPhysical removal of foreign material, e.g.

dust, soil, organic material such as blood, secretions, excretions and microorganisms.

Cleaning generally removes rather than kills microorganisms. It is accomplished with water, detergents and mechanical action.

Decontamination

Removal of disease-producing microorganisms to leave an item safe for further handling

Chemical Methods:

These are used either as antiseptics or disinfectant.

Those chemicals that can kill spores are called sterilants. This method is used when heat sensitive instruments are to be sterilized.

Antiseptics

These are applied to the surface of body.

They are also applied to open wounds or abraded tissue surfaces or mucous membrane

Examples:Volatile: e.g. alcohol, chloroform.

Phenols: e.g. Lysol, cresol, dettol

Metallic salts: e.g. Mercuric chloride (mentholated)

Detergents: e.g. soaps.

Hand SanitizerHand sanitizer contain high concentration of alcohol.

However, it is not suitable for cleaning all types of dirt or soil.

Alcohol, kills most bacteria, and hand sanitizer contains a high amount of alcohol. When it is rubbed on the hands for about 30 seconds, it eliminates many forms of bacteria and viruses.

It has been proven very effective against some dangerous forms of bacteria, like methicillin-resistant staphylococcus aureus (MRSA)

Disinfectants

They are used to disinfect non-living objects.

Examples:

Formaldehyde: Used as vapor for sterilization of rooms, furniture and blankets and as solutions 10% for washing floors and walls

Ethylene oxide: Gaseous disinfectant kills bacteria and spores . Ethylene is used to sterilize plastic, rubber, blankets, machine and pharmaceutical products.

Antiseptics versus Disinfectants

Antiseptics are used on skin and mucous membranes to kill microorganisms. Not for use on inanimate objects

Disinfectants are used to kill microorganisms on inanimate objects. Not for use on skin or mucous membranes.

Questions?Write about Autoclave machine and its

working

What is a Difference between disinfectant and antiseptic.

What are different methods of dry heat sterilization describe each one.

What is Pasteurization and tyndallization.

Thank you!