STERILIZATION BY

PHYSICAL METHODSDr.T.V.Rao MD

04/11/2023 Dr.T.V.Rao MD 1

Why we need Sterilization• Microorganisms capable of causing

infection are constantly present in the external environment and on the human body.

• Microorganisms are responsible for contamination and infection.

• The aim of sterilisation is to remove or destroy them from materials or from surfaces.04/11/2023 Dr.T.V.Rao MD 2

How can microorganisms be killed?

1 Denaturation of proteins (e.g. wet heat, ethylene oxide)

2 Oxidation (e.g. dry heat, hydrogen peroxide)3 Filtration4 Interruption of DNA synthesis/repair (e.g.

radiation)5 Interference with protein synthesis (e.g. bleach)6 Disruption of cell membranes (e.g. phenols)

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Classification There are two types of sterilization: physical and chemical.

1. Physical sterilization includes:

heat radiation filtration

2. Chemical sterilization includes:

Alcohols Aldehydes Phenolics Oxidizing agents Quaternary ammonium co

mpounds

ethylene oxide gas Others

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Definitions: Sterilisation :

– It is a process by which an article, surface or medium is made free of all microorganisms either in vegetative or spore form.

Disinfection :– Destruction of all pathogens or organisms capable of

producing infections but not necessarily spores.– All organisms may not be killed but the number is

reduced to a level that is no longer harmful to health.

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Antiseptics :

Antiseptics :– Chemical disinfectants which can safely

applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms.

Asepsis :– Technique by which the occurrence of

infection into an uninfected tissue is prevented.

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Factors that influence efficacy of disinfection/sterilization

1 Contact time2 Physico-chemical environment (e.g. pH)3 Presence of organic material4 Temperature5 Type of microorganism6 Number of microorganisms7 Material composition

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Uses of sterilisation:1. Sterilisation of materials,

instruments used in surgical and diagnostic procedures.

2. Sterilisation of Media and reagents used in the microbiology laboratory.

3. Food and drug manufacturing to ensure safety from contaminating organisms.

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Understanding the Terminology• a suffix indicating that the antimicrobial agent will kill or • destroy a certain group of microorganism • suffix “cide” – meaning to kill• viricide – destroys virus• fungicide – destroys fungi• bactericide – destroys bacteria•

• Suffix “static/stasis” – meaning to stand still • a suffix indicating that the agent will prevent the growth or • multiplication of the type of organism but are not killed outright •

• bacteriostatic - prevents the growth of bacteria• fungi static – prevents the growth of fungi

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Relative Resistance of Microbial Forms

Highest resistance Moderate resistance Least resistance

bacterial endospore (Bacillus & Clostridium)

protozoan cystsome fungal sporessome naked virusvegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas)

most bacterial vegetative cellsordinary fungal spores & hypaeenveloped virusYeastsTrophozoites

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What to sterilize?• It is mandatory to sterilize :

– all instruments that penetrate soft tissues and bone.– Instruments that are not intended to penetrate the

tissues, but that may come into contact with oral tissues.

• If the sterilization procedure may damage the instruments, then, sterilization can be replaced by Disinfection procedure

Ideal sterilization/disinfection process

• Highly efficacious• Fast• Good penetrability• Compatible with all materials• Non-toxic• Effective despite presence of organic material• Difficult to make significant mistakes in process• Easily monitored

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Control of Microbial Growth:Rate of Microbial Death

When bacterial populations are heated or treated antimicrobial chemicals, they usually die at a

constant rate.

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Figure 9.1 A plot of microbial death rate

90% die

1 min

90% die

1 min

Constant percentageof the extant populationis killed each minute

Time (min)

Nu

mb

er

of

livin

g m

icro

bes

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Methods

1.Physical methods

2.Chemical methods

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Physical methods:• Physical

methods:1.Sunlight 2.Heat

1.Dry heat2.Moist heat

3.Filtration 4.Radiation

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Chemical methods• Chemical methods:

1. Alcohols2. Aldehydes3. Phenols4. Halogens5. Oxidizing agents6. Salts7. Surface active agents8. Dyes9. Vapor phase disinfectants

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Physical Methods

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Materials Method

1 Inoculating wires and loops Red heat

2 Glass ware- syringes, petridishes, testtubes, flasks etc.

Hot –air oven

3 Disposable syringes, and other disposable items

Gamma radiation

4 Culture media Autoclaving

5 Culture media containing serum and egg Tyndallisation

6 Toxin , serum, sugar, and antibiotic solutions

Filtration

7 Cystoscope and endoscope Glutaraldehyde

8 Infected soiled dressings Incineration

9 Skin Iodine, alcohol

10 Milk Pasteurisation

How to Sterilize

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Physical Methods of Microbial ControlDry Heat:

Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow.

Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste.

Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat.

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Physical Methods of Microbial Control

• Heat-Related Methods– Moist heat

• Pasteurization– Used for milk, ice cream, yogurt, and fruit juices– Not sterilization

» Heat-tolerant microbes survive– Pasteurization of milk

» Batch method» Flash pasteurization» Ultrahigh-temperature pasteurization

© 2012 Pearson Education Inc.04/11/2023 Dr.T.V.Rao MD 21

Physical Methods of Microbial Control

Moist Heat (Continued): Pasteurization: Developed by Louis Pasteur to

prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. Classic Method of Pasteurization: Milk was

exposed to 65oC for 30 minutes. High Temperature Short Time Pasteurization

(HTST): Used today. Milk is exposed to 72oC for 15 seconds.

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Inspissation:

1. Inspissation: Heating at 80-85°C for half an hour daily on

three consecutive days Serum or egg media are sterilised

2. Vaccine bath: Heating at 60°C for an hour daily in vaccine

bath for several successive days. Serum or body fluids can be sterilised by

heating at 56°C for an hour daily for several successive days.

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Sun light:

• Sun light:– Active germicidal

effect due to its content of ultraviolet rays .

– Natural method of sterilisation of water in tanks, rivers and lakes.

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Heat :

• Factors influencing:• Nature of heat• Temperature and duration• Characteristic of organism and

spores• Type of material

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Heat effectively kills Majority of Microbes

Heat :• Principle:

– Dry heat kills the organism by• denaturation of the bacterial proteins, • oxidative damage• toxic effect of elevated levels of

electrolytes.

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Heat :• Dry heat:

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

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Dry-Heat Sterilization• Involves heating at atmospheric pressure

and often use a fan to obtain uniform temperature by circulation.

• Heat at 180º for half hour , 170º for 1 hr., or 160º C for 2 hrs.

• Times are the periods during which object is maintained at the respective temp.

Dry heat:• Dry heat:

1. Red heat: Materials are held in the flame of a bunsen burner till they become red hot.» Inoculating

wires or loops» Tips of forceps» Needles

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Dry heat:

• Dry heat:2. Flaming: Materials are

passed through the flame of a bunsen burner without allowing them to become red hot.

» Glass slides» scalpels» Mouths of culture

tubes

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Incineration: • Materials are

reduced to ashes by burning.

• Instrument used was incinerator.

• Soiled dressings• Animal carcasses• Bedding • Pathological material

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Dry-Heat Sterilization Disadvantages

• Disadvantages:–Less reliable than autoclaving–Large temp difference may arise within

device.–sharp instruments get dulled–Many materials do not tolerate dry heat

Hot air oven: • Most widely used method• Electrically heated and fitted with a fan to

even distribution of air in the chamber.• Fitted with a thermostat that maintains

the chamber air at a chosen temperature.• Temperature and time:

» 160 C for 2 hours.» 170 C for 1 hour» 180 C for 30 minutes.

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Uses of Hot Air Oven

– Sterilisation of 1.Glassware like glass syringes, petri

dishes, pipettes and test tubes. 2.Surgical instruments like scalpels,

scissors, forceps etc.3.Chemicals like liquid paraffin, fats

etc.

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– Precautions :1. Should not be overloaded 2. Arranged in a manner which allows free

circulation of air3. Material to be sterilized should be perfectly dry.4. Test tubes, flasks etc. should be fitted with

cotton plugs.5. petridishes and pipetts should be wrapped in

paper.6. Rubber materials and inflammable materials

should not be kept inside.7. The oven must be allowed to cool for two hours

before opening, since glass ware may crack by sudden cooling.04/11/2023 Dr.T.V.Rao MD 35

Sterilisation controls :

• Sterilisation controls

1.Spores of Bacillus subtilis subsp. niger

2. Thermocouples

3.Browne’s tube

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Sterilizing below100°C

1. temperature below 100° Pasteurization of milk

1.Inspissation2.Vaccine bath

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Principle of Pasteurization

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A temperature at 100°C

II. A temperature at 100°C

1. Boiling 2. Tyndallisation

3. Steam sterilisation

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Boiling :1 Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling.

2. Tyndallisation : Steam at 100C for 20 minutes on three

successive days Used for egg , serum and sugar containing

media.3. Steam sterilizer :

Steam at 100°C for 90 minutes. Used for media which are decomposed at high

temperature.04/11/2023 Dr.T.V.Rao MD 40

Temperatures above 100°CIII. A temperature

above 100°CAutoclave : -Steam above

100°C has a better killing power than dry heat.

-Bacteria are more susceptible to moist heat.

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Components of autoclave:

• Components of autoclave:– Consists of vertical or horizontal cylinder of

gunmetal or stainless steel.– Lid is fastened by screw clamps and

rendered air tight by an asbestos washer.– Lid bears a discharge tap for air and steam,

a pressure gauge and a safety valve.

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Figure 9.6 Autoclave-overview

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Autoclave: Closed Chamber with High Temperature and Pressure

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Sterilisation conditions• Sterilisation conditions:

–Temperature – 121 °C–Chamber pressure -15 lb per square

inch.–Holding time – 15 minutes–Others :

• 126°C for 10 minutes• 133°C for 3 minutes

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Sterilization – instrument Packing

• Often instruments are packed for sterilization to be stored and handled without being contaminated.

• Packing depend on the intended shelf life after sterilization.

• The available packing options are:– Textile has shelf life of 1 month– Paper has shelf life of 1 – 6 months– Nylon, glass, and metal have shelf life of 1 year if

tightly closed

Uses of Autoclaves:

• Uses :1. Useful for

materials which can not withstand high temp.

2. To sterilize culture media, rubber material, gowns, dressings, gloves etc.

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Sterilisation controls:

• Sterilisation controls:1. Thermocouples2. Bacterial spores-

Bacillus stearothermophilus

3. Browne’s tube4. Autoclave tapes

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Cap that allowssteam to penetrate

Flexible plasticvial

Crushable glassampule

Nutrient mediumcontaining pHcolor indicator

Endospore strip

Incubation

After autoclaving, flexiblevial is squeezed to breakampule and releasemedium onto spore strip.

Yellow mediummeans spores areviable; autoclavedobjects are notsterile.

Red mediummeans spores werekilled; autoclavedobjects aresterile.

Sterility Controls

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Filtration:• . Filtration:

• Useful for substances which get damaged by heat.

• To sterilize sera, sugars and antibiotic solutions.

• To obtain bacteria free filtrates of clinical samples.

• Purification of water.04/11/2023 Dr.T.V.Rao MD 51

FILTRATION STERILIZATION This method is commonly used for

sensitive pharmaceuticals and protein solutions in biological research.

A filter with pore size 0.2 µm will effectively remove bacteria.

If viruses must also be removed, a much smaller pore size around 20 nm is needed.

Prions are not removed by filtration. The filtration equipment and the

filters themselves may be purchased as presterilized disposable units in sealed packaging,

or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes.

To ensure sterility, the filtration system must be tested to ensure that the membranes have not been punctured prior to or during use.

To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air (HEPA filtration) or in a laminar flow cabinet or "flowbox", a device which produces a laminar stream of HEPA filtered air.

HEPA filters are critical in the prevention of the spread of airborne bacterial and viral organisms and, therefore, infection. Typically, medical-use HEPA filtration systems also incorporate high-energy ultra-violet light units to kill off the live bacteria and viruses trapped by the filter media.

Several Types of Filters• Types of filters:

1. Candle filters2. Asbestos disc filters3. Sintered glass filters4. Membrane filters5. Air filters6. Syringe filters

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Filtration Sterilize solutions

that may be damaged or denatured by high temperatures or chemical agents.

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The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 μm (filtration membranes are most popular for this purpose).

The filtering Depends on Pore Size

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Candle filters

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The roles of HEPA filters in biological flow safety cabinets

Exhaust HEPAfilter

Blower

Supply HEPAfilter

Light

High-velocityair barrier

Safety glassviewscreen

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Radiations :• Radiations :

•Ionizing radiations

•Non - Ionizing radiations

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Ionising radiations:

– Ionizing radiations:1. X rays2. Gamma rays3. Cosmic rays

• Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation)

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Physical Methods of Microbial Control

• Radiation– Nonionizing radiation

• Wavelengths greater than 1 nm• Excites electrons, causing them to make new covalent

bonds– Affects 3-D structure of proteins and nucleic acids

• UV light causes pyrimidine dimers in DNA• UV light does not penetrate well• Suitable for disinfecting air, transparent fluids, and

surfaces of objects

© 2012 Pearson Education Inc.04/11/2023 Dr.T.V.Rao MD 60

Forms of Radiation

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Physical Methods of Microbial Control:

Radiation: Three types of radiation kill microbes: Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.

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Non-Ionising radiation:

1. Infra red rays2. Ultraviolet (UV) rays

– Infra red is used for rapid mass sterilisation of syringes and catheters.

– Ultraviolet radiation is used for disinfecting enclosed areas such as bacterial laboratory, inoculation hood, laminar flow and operation theatres.

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Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students

Emaildoctortvrao@gmail.com

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