STERILIZATION&

DISINFECTION…

Prepared by:Maulik Chaudhari

DEFINITIONSterilization ‘Sterilization is a process by which an article,

surface, or medium is freed of all living microorganisms either in vegetative or spore state.

DEFINITIONDisinfection It is a process of destruction of pathogenic

organisms capable of giving rise to infection, but not necessarily spores.

Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination.

Asepsis: Absence of significant contamination.

Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery.

Bacteriostatic Agent:

An agent that inhibits the growth of bacteria, but does not necessarily kill them.

Bactericide: An agent that kills bacteria. Most do not kill

Endospores.

Sporicide: An agent that kills spores.

USES OF STERILIZATION/ DISINFECTION. Sterilization of of materials, instruments

used in surgical and diagnostic procedures.

For media and reagents used in the microbiology laboratory.

METHODS OF STERILISATION

Microbial Control

Methods

Physical METHODS

Chemical METHODS

METHODS….. Physical methods. 1) Sunlight 2) Heat - dry heat - moist heat 3) Radiation 4) Filtration

Chemical methods 1. Alcohol 2. Aldehyde 3. Phenols 4. Halogens 5. Oxidising agens 6. Dyes 7. Gases.

PHYSICAL METHODS

PHYSICAL METHODS… 1. SUNLIGHT Sunlight has an active germicidal effect

due to its content of ultraviolet rays. It is a natural method of sterilization in

cases of water in tanks, rivers and lakes.

2. HEAT Heat is the most reliable and commonly

employed method of sterilization. Two types of heat are used, dry heat and moist

heat. Principles:1) Dry heat kills the organisms by denaturation of bacterial protein, toxic effect or elevate levels of electrolyte.2) Moist heat kills the microorganisms by denaturation and coagulation of bacterial proteins.

Heat method

Dry heat Moist heat

The following procedures are used for sterilization by dry heat.

1. Red heat2. Flaming 3. Incineration 4. Hot air oven

Dry heat

RFIH

Red heat: Inoculating wires or loops, tips of forceps

and needles are held in the flame of a bunsen burner till they become red hot.

Flaming Glass slides, scalpels, and mouths of

culture tubes are passed through bunsen flame without allowing them to become red hot.

Incineration: By this method, infective material is

reduced to ashes by burning. Instrument named incinerator may be used for this purpose. Soiled dressing, animal carcasses, bedding and pathological materials are delt with this method.

Hot air oven: It is the most widely used method of

sterilization by dry heat. The oven is electrically heated and is

fitted with a fan to ensure adequate and even distribution of hot air in the chamber

It is also fitted with the thermostat that maintains the chamber air at a chosen temperature.

This method of sterilization may be used at different temperatures as follows.

1. At temperature below 100 C 2. At temperature of 100 C 3. At temperature above 100 C

Moist heat

1) at temperature below 100 c 1) Pasteurization of milk: Two types of methods are used are 1-Holder method= (63 C for 30

minutes ) 2-Flash method= (72 C for 20 seconds

followed by cooling) 2) inspissation: Some serum or egg media are sterilize

by this method The instruments used for sterilization is

called inspissator.

2) at a temperature of 100 c1) Boiling: Boiling for 10-30 minutes may kill most of

the vegetative forms but not spore forms When other method is not available then

boiling is effective in sterilization It is used for glass syringes and rubber

stoppers.

2) vaccine bath: It is used for killing non sporing bacteria

which may be present in vaccine. In vaccine bath the vaccine is treated

with moist heat for 1 hour at 60 C.

3) temperature above 100 c

AUTOCLAVE

AUTOCLAVE Autoclave is a

equipment used to remove microorganisms

( viruse,bacteria,fungus) And spores using high

pressure and high temperature steam sterilization

BRIEF HISTORY Denis Papin French-born British physicist invented a precursor known as

the steam digester in 1679. was essentially a cooking instrument.

Role of Chamberland Autoclave was invented by Charles

Chamberland in 1879. Colleague of Pasteur Building up on Papin’s marmite. began work on reforming the steam

digester. credited for starting the research

project. 

DEFINITION Autoclave is a pressurized device

designed to heat aqueous solutions above their boiling point at normal atmospheric pressure to achieve sterilization.

Auto= self Clavis= self locking device

Autoclave Machine

Principle of working Autoclave: Autoclave and pressure cooker are working

on a same principles. Water boils when its vapour pressure equals

to the surrounding atmosphere. When the atmospheric pressure is raised

then the boiling temperature is also raised. At normal pressure water boils at 100 C but

when pressure inside a closed vessel increases, the temperature at which water boils also increases.

This principle is applied in autoclave…..

Physical structure: Autoclave is a modified pressure cooker

or boiler. It consist of a vertical or horizontal

cylinder of gunmetal or stainless steel in a supporting iron case.

The lid is fastened by screw clamps and rendered air tight by an asbestos washer.

The lid having discharge tap for air and steam.

A pressure gauze and safety valve.

Heating is generally done by electricity The steam circulate within the jacket

and is supplied under high pressure to the inner chamber where materials are kept for sterilization.

Sterilization condition: temperature 121 C Chamber pressure-15 pounds (lbs) per

square inch. Holding time -15 minutes.

USES..1) To sterilize culture media, rubber

materials, gowns, dressing, gloves etc.2) It is particularly useful for materials

which can not be withstand the higher temperature of hot air oven.

3) For all glass syringes, hot air oven is a better sterilizing method.

PRECAUTION. The air must be allowed to escape from

the chamber as temperature of air steam mixture is lower than that of pure steam.

Materials should be arranged in such a manner to ensure free circulation of steam inside the chamber.

Sterilization indicator: Thermocouple- it is to record the

temperature directly by a potentiometer.

Bacterial spores- spores of bacillus stearothermophilus are used as a test organism. The spore of this organism are killed at a temperature 121 C for 12 minutes

an envelop containing a filter paper strip impregnated with 10 spores is placed inside the autoclave during sterilization.

Chemical indicator:Browne’s tube contains red solution which turns green when exposed to temperature of 121 C for 15 minutes.

FILTRATION This method of sterilization is useful for

substances which get damaged by heat process

E.g sera, sugars, antibiotics solutions.etc.

Principle: as viruses pass through the ordinary filters, filtration

can be used to obtain bacteria-free filtrates of clinical samples for virus isolation.

Types of filters: Earthenware candles Asbestos disk filters Sintered glass filters Membrane filters

Candle filter Types-

Unglazed ceramic filters Diatomaceous earth filters

Asbestos filters Disposable, single-used disc Usage is discouraged because of its carcinogenic property. Eg: Seitz and Sterimat filters

Sintered glass filters has low absorptive properties Brittle and expensive

Membrane filters Made of cellulose esters or other polymers Usually used for water purification and analysis,

sterilization and sterility testing and preparation of solutions for parenteral use.

RADIATION Two types of radiation method are used Ionizing Non-ionizing Ionizing radiations: ionizing rays including

gamma rays , X-rays and cosmic rays

Non-ionizing radiations: these include infrared and ultraviolet (UV) radiations.

Both methods work by damage to DNA of a bacteria.

CHEMICAL METHODS

Chemical methods 1. Alcohol 2. Aldehyde 3. Phenols 4. Halogens 5. Oxidising agens 6. Dyes 7. Gases.

Factors determine the potency of disinfectants:Concentration of the substanceTime of actionpH of the mediumTemperatureNature of the organismsPresence of extraneous material

Alcohol Frequently used:

Ethyl alcohol Isopropyl alcohol

Must be used at concentration 60-90% Isopropyl alcohol used in disinfection of

clinical thermometer. Methyl acohol is effective against fungal

spores, treating cabinets and incubators.

Methyl alcohol is also toxic and inflammable.

Aldehyde Formaldehyde:

Bactericidal, sporicidal and has lethal effect on viruses.

Used to preserve anatomical specimens, destroying anthrax spores on hair and wool.

Glutaldehyde: Effective against tubercle bacilli, fungi, viruses. Less toxic and irritant to eyes, skin Used to treat corrugated rubber anaesthetic

rubber, face masks, plastic endotracheal tubes, metal instruments and polythene tubing.

Dyes 2 groups of dyes:

Aniline dyeAcridine dye

Both are bacteriostatic in high dilution but are of low bactericidal activity.

Aniline dye is more active against gram +ve than gram-ve organisms.

Used in microbiology labs as selective agents in culture media.

Halogens Iodine

Skin disinfectantActive bactericidal, moderate action on

spores.

Chlorine Water supplies, swimming pools and food

and dairy industries.Along with hypochlorides are bactericidal.

Also act on viruses.

Phenols Obtained from distillation of coal tar

between 170-270°C.

Lethal effect: Capacity to cause cell membrane damage,

releasing cell contents and causing lysis.

Low concentration will precipitate proteins.

Gases Types of gases

Ethylene oxide Formaldehyde gas Beta propiolactone (BPL)

Ethylene oxide Action is due to its alkylating the amino,

carboxyl, hydroxyl and sulphydryl groups in protein molecules. Also on DNA and RNA.

Items: heart-lung machines, respirators, sutures, dental equipment, books, clothing.

Formaldehyde gasEmployed for fumigation of OT and other

rooms.After fumigation, the doors should be sealed

and left unopened for 48 hours.

BPLProduct of ketane and formaldehyde with a

boiling point of 163°C.Rapid biocidal activity but carcinogenic.Capable of killing all microorganisms and is

very active against viruses.

surface-active agents Def substance that alter the energy relationship at

interfaces, producing a reduction of surface or interfacial tension.

Widely used as wetting agents, detergents and emulsifiers.

4 main groups: anionic Cationic Nonionic amphoteric

metallic salts Salts of heavy metals have a greater

action.Eg: salts of silver, copper and mercury

Protein coagulant and have capacity to combine with free sulfhydryl group of cell enzymes.