STERILIZATION AND

DISINFECTION-I

Dr Harender
MAMC, AGROHA
 INTRODUCTION

2/13/2020 9:58 AM
 Sterilization
A physical or chemical process that completely destroys or
removes all microbial life, including spores from an article,
surface or medium.

 Disinfection
It is killing or removing of harmful microorganisms but not
necessarily spores. Or their number is reduced to a level that
is no longer harmful to health

 Antiseptic
2
These are chemical disinfectants that destroys or inhibits
the growth of microorganisms in or on living tissue.
 2/13/2020 9:58 AM
 Disinfectant
Products used to kill microorganisms on
inanimate objects or surfaces. Disinfectants are
not necessarily sporicidal, but may be sporostatic,
inhibiting germination or outgrowth.

 Asepsis
The technique by which , the occurrence of
infection into an uninfected tissue is prevented.

3
 Why do we need
to sterilize?

2/13/2020 9:58 AM
 Briefly,
to get rid of microbes!
 Application in
 Microbiology
 Surgery
DRAFT
 Food, drug industries

 Many other situations

4
 Methods of sterilization
PHYSICAL CHEMICAL

2/13/2020 9:58 AM
 Sunlight  Alcohols
 Drying  Aldehydes
 Heat  Gases
 Dry  Heavy Metal Salts
 Moist
DRAFT  Halogens
 Filtration  Dyes
 UV & Sonic waves  Surfactants
 Radiations  Phenols
5
 SUNLIGHT

2/13/2020 9:58 AM
 Active germicidal effect due to UV rays.
 Natural method of sterilization of water in
reservoir tanks, rivers and lakes

6
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
 Sterilization by Heat: Most common
and most reliable method
Dry Heat:
 Principle- denaturation of bacterial
proteins, oxidative damage & toxic
effect of elevated levels of electrolytes.

Factors influencing:
 1. Nature of heat: dry/ moist

 2. Temp and Duration

 3. Characteristics of microorganisms
and spores
 4. Types of material
7
 DRY HEAT

2/13/2020 9:58 AM
 Flaming : Simplest method is exposing the item
to be sterilized to the naked flame e.g. Bunsen
burner- for glass slides, scalpels, mouths of culture
tubes

 Red heat: sterilizing bacteriological loops, tip of

forceps and needles are held in the flame of Bunsen
burner till they become red hot.

8
 INCINERATION

2/13/2020 9:58 AM
 Infective material are reduced to ashes by
burning.
 2 steps burning (- 850°C and followed by 1000°C)

 Rapid and terminal destruction of soiled waste.

Incinerator

DRAFT

9/20/2011 8 Kher
DRY HEAT ……..HOT AIR OVEN
 Hot air oven expose items to 160°C for 2 hour. It

2/13/2020 9:58 AM
has electric element in the chamber as source of
heat plus a fan to circulate air for even distribution
of heat in chamber.
 Oven without fan is dangerous.
 Temperature controlled by thermostat
 Good penetration and non- corrosive

 Used for:
 1. Glassware ( glass syringes, petridishes, flask,
pipettes, test tube, Dry swab in test tube- plugged
with non- absorbable cotton wool) ,
 2. Surgical instruments: like scalpels, scissors,
forceps etc.
 3. Ointment, Oils, Waxes, Powders i.e. items that
10
are lacking water
 170 °C for 1 hr, 180 °C for 30 minutes
PRECAUTIONS:

2/13/2020 9:58 AM
 Should not be overloaded.
 Arrange the item to allow free circulation of air

 Item should be perfectly dry

 Test tubes, flask should be fitted with cotton

plugs.
 Petridishes and pipettes should be wrapped in
brown kraft paper or aluminium foil.
 Oven must be allowed to cool x 2 hrs before
opening the doors (temp < 400C).

 Sterilization control:
 1. Spores of Bacillus atropheaus
11
 2. Thermocouples

 3. Browne's tubes with green spot

2/13/2020 9:58 AM
12
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
Moist Heat: Uses hot water.
Principle: Moist heat kills microorganisms by
denaturation and coagulation of proteins.
i.) At a temperature of 100 0C:

A) Boiling – x 10-30 min. quite common especially in

domestic circumstances.

B) Tyndallization named after John Tyndall

 Lengthy process designed to reduce the level of
activity of sporulating bacteria that are left by a
13
simple boiling water method.
Boiling Sterilizer

2/13/2020 9:58 AM
DRAFT

14
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
Moist heat:
Tyndallization / Intermittant sterilisation
 The process involves steaming at 100 0C for 20
min., at atmospheric pressure on 3 successive days.

 The three incubation periods are to allow heat-

resistant spores surviving the previous exposure
period to germinate to form the heat-sensitive
vegetative (growing) stage, which can be killed by
the next exposure step.

 Uses : sterilization of egg, serum or sugar

containing media 15

 Instrument: Koch’s or Arnold’s steam steriliser

METHODS OF STERILIZATION

2/13/2020 9:58 AM
Moist heat: ii) at a temperature below 100°C
1. Pasteurization
 It aims to reduce the number of viable pathogens in
liquids so they are unlikely to cause disease.
 It uses heat at temperatures sufficient to inactivate
harmful organism in milk. Does not achieve
sterilization.
 1. Holder method: 63°C for 30 minutes.

 2. Flash method (high temperature short time

(HTST) : 72°C for 15-20 seconds .
 3. Ultra-high temperature (UHT): Temperature
may be 138°C for a fraction of a second. 16
 Followed by sudden cooling to ≤13°C in all methods.
2. INSPISSATION:

2/13/2020 9:58 AM
 Heating at 80-85 0C temperature for 30 min. on 3
consecutive days.
 Used for sterilising serum containing media
(LSS),
 Egg containing media (Lowenstein- Jensen’s
media)
 Instrument used: Inspissator

17
3. VACCINE BATH:

2/13/2020 9:58 AM
 Bacterial vaccines are sterilised in a special
vaccine bath at 60 0C for 1 hr.
 Serum or body fluid can be sterilised by heating
at 56 0C for 1 hr in a water bath on several
successive days.

4. LOW TEMPERATURE STEAM

FORMALDEHYDE (LTSF) Sterilisation : for
items which can not withstand 100 0C.
18
 Steam at sub-atmospheric pressure at
temperature of 75 0C with formaldehyde vapours.
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
Moist heat: iii) a temperature above 100°C
Autoclaving – Standard sterilization method
in hospitals.

 The Autoclave works under the same principle

as the pressure cooker where water boils at
increased atmospheric pressure i.e. because of
increased pressure the boiling point of water is
>100°C.

 The autoclave is a tough double walled chamber

in which air is replaced by pure saturated 19
steam under pressure.
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
 The air in the chamber is evacuated and
filled with saturated steam. The chamber is
closed tightly, the steam keeps on filling into
it and the pressure gradually increases.

 The items to be sterilized get completely

surrounded by saturated steam (moist heat)
which on contact with the surface of
material to be sterilized condenses to release
its latent heat of condensation which adds to
already raised temperature of steam so that
eventually all the microorganisms in what
ever form –are killed.

 The usual temperature achieved is 121 °C at

a pressure of 15 psi. at exposure time of only
15-20 mins. By increasing the temperature,
the time for sterilizing is further reduced. 20
TYPES OF AUTOCLAVE

2/13/2020 9:58 AM
21
 METHODS OF STERILIZATION

2/13/2020 9:58 AM
Advantages of Autoclave
 Temperature is > 100°C therefore spores are killed.

 Condensation of steam generates extra heat (latent

heat of condensation).

 The condensation also allows the steam to penetrate

rapidly into porous materials.

 Note: that autoclavable items must be steam

permeable. Can not be used for items that are lacking
22
water.
2/13/2020 9:58 AM
23
 MONITORING OF AUTOCLAVES
STERILIZATION

2/13/2020 9:58 AM
 Physical- use of thermocouple to
measure accurately the temperature.

 Chemical- it consists of heat sensitive

chemical that changes color at the
right temperature and exposure time.
 Autoclave tape
 Browne’s tube.

 Biological – where a spore-bearing

organism( Bacillus
stearothermophilus) is added during
the sterilization process and then
cultured later to ensure that it has 24

been killed.
 2/13/2020 9:58 AM
BOWIE DICK TEST PACKET

25
2/13/2020 9:58 AM
26
 2/13/2020 9:58 AM
CHEMICAL INDICATOR STRIP

27
 METHODS OF STERILIZATION
Radiation: cold sterilization

2/13/2020 9:58 AM
 Non ionizing : Infra red & U.V. light-
Has limited sterilizing power because of
poor penetration into most materials.
Generally used in irradiation of air in
certain areas eg. Operating Rooms and
T.B. laboratories, laminar flow. Spores are
resistant to UV..

 Infrared : rapid mass sterilisation of syringes and

catheters.
28
 Principle: Acts by denaturation of bacterial
proteins and interference with DNA replication
IONIZING RADIATION-

2/13/2020 9:58 AM
 e.g. Gamma rays, X rays, cosmic rays:
 High penetrating power, highly lethal to all cells .

 Damage DNA by various mechanism.

 Source Cobalt60 has greater energy than U.V. light,

therefore more effective.
 Used mainly in industrial facilities.

 sterilization of :

 1. Disposable plastic syringes,

 2. Gloves,

 3. Specimens containers

 4. Petri Dishes,
29
 5. Cannulas,

 6. Catheters etc.
 Filtration

2/13/2020 9:58 AM
Methods of Sterilization
 May be done under either negative or positive
pressure.
 Best known example is the membrane filter
made from cellulose acetate. Generally
removes most bacteria but viruses and some
small bacteria e.g. Chlamydias &
Mycoplasmas may pass through.
 Thus filtration does not technically sterilize
items but it is adequate for circumstances
under which it is used.

 Uses: 1. to sterilise heat labile substances

e.g. sera, Sugars,antibiotics solutions etc
 2. sterilisation of hydatid fluid.
 3. separation of toxins and bacteriophages
from bacteria. 30
 Filtration

2/13/2020 9:58 AM
Basically used for heat labile liquids

 Candle filters –
 Unglazed ceramic e.g. Chamberland
 Diatomaceous earth e. g. Berkfield
 Asbestos filter
 Seitz filter
 Sintered glass filter DRAFT
 Membrane filters –
 Cellulose acetate
 Cellulose nitrate
31
Membrane filter

2/13/2020 9:58 AM
DRAFT

32
Methods of sterilization / Disinfection of important materials
Material Method

2/13/2020 9:58 AM
1 Inoculating wires & Loops Red Heat
2 Glassware – Syringes, petridishes, test tubes, Flasks, Hot air oven
Universal container, Oily fluids, powders
3 Disposable syringes & other disposable items Ethylene Oxide / Gamma
irraditaion
4 Culture Media Autoclaving
5 Culture Media containing serum, eggs Inspissation (Tyndallization)

6 Toxins, sera, sugars, antibiotic solutions Filtration

7 Milk, Milk products Pasteurization
8
9
Cystoscope & Endoscopes
Operation Theatre (Fumigation)
DRAFT Glutaraldehyde
Formaldehyde
10 Infective material like soiled dressings, beddings, animal Incineration
carcasses
11 Skin Tincture iodine, 70%
ethanol, savlon
12 Aprons, gloves, catheters, surgical instruments except Autoclaving
sharps 33

13 Sharp instruments 5% cresol

14 Rubber, plastic & polythene tubes Glutaraldehyde
 HAND HYGIENE

2/13/2020 9:58 AM
THANK YOU VERY MUCH 34