PRINCIPLES OF STERILISATION AND

DISINFECTION

OBJECTIVES

The objectives of this lecture are

to
Define Sterilization, Disinfection and
Antisepsis.
Describe the physical and chemical methods
of sterilization and disinfection commonly used
in the microbiology laboratory and the hospital.
Explain the importance of Quality control.

INTRODUCTION
Microorganisms cause contamination, infection
and decay, it becomes necessary to remove or
destroy them from materials or from areas. This
is the object of Sterilization.
STERILIZATION:Sterilization is define as the process by which
an article, surface or medium is free of all living
microorganisms either in the vegetative or spore
state.

 DISINFECTION:-

Disinfection means the destruction or removal of all

pathogenic organisms or organisms capable of
giving rise to infection. Disinfection can be done by
chemicals.
ANTISEPSIS:- Reduction of or inhibition of
microbes found on living tissue.
ANTISEPTICS:-

Chemical disinfectants which can be safely applied

to the skin or mucous membrane and are used to
prevent infection by inhibiting the growth of bacteria
are called antiseptics.

DIFFERENCES BETWEEN ANTISEPTICS

AND DISINFECTION
ANTISEPTICS

DISINFECTION

1. All antiseptics are

All disinfectants are not

antiseptics.

2. It can be apply on the

It can be applied on
inanimate object.

3. Eg:- Skin or mucous

Eg:- Surface, lab working

tables, floor or material.

disinfectants.
live tissues.
membrane.

METHODS OF STERILISATION AND

DISINFECTION

The following methods are commonly used in

the microbiology laboratory and hospital.
(I) PHYSICAL METHOD

Sunlight, Drying,
Dry heat: Red heat, Flaming, Incineration, Hot air
oven.
Moist heat: Pasteurization, Boiling, Autoclave (Steam
under pressure).
Filtration and Radiation etc.

(II) CHEMICAL METHOD

Alcohols, Aldehydes, Halogens (Iodine, Chlorine etc),
Phenols, Gases and Surface - active agents (soap,
detergent) etc.

HOT AIR OVEN

This is the most widely used method of
Sterilization by Dry heat.
A holding period of 160C for one hour is
used to sterilize glassware, forceps, all-glass
syringes, swabs etc.
Glassware should be perfectly dry before being
placed in the oven. Test tubes and flasks should
be wrapped in paper.
The material should arranged so as to allow free
circulation of air in between the objects.

Hot air oven continued..

It should not be overloaded.
It must be fitted with a fan to ensure even
distribution of air.
The oven is usually heated by electricity with
heating elements in the wall of the chamber.
The oven must be allowed to cool slowly for
about two hours before the door is opened,
since the glassware may crack due to sudden or
uneven cooling.

HOT AIR OVEN

STERILIZATION BY MOIST HEAT

Moist Heat is divided into 3 methods;

1)

Temperature bellow 100C

Eg: Pasteurisation of milk.

2)

Temperature at 100C
Eg: Boiling of water.

3)

Temperature above 100C (Steam under

pressure)
Eg: Autoclave.

AUTOCLAVE
In its simplest form, the laboratory autoclave consists of a
vertical or horizontal cylinder of gunmetal or stainless
steel.
The lid or door is made airtight.
The autoclave has on its lid or upper side a discharge tap
for air and a safety valve that can be set to blow off at any
desired pressure.
Heating is by gas or electricity.
Sufficient water is put in the cylinder, the material to be
sterilized is placed on the tray and the autoclave is
heated.
The lid is screwed tight.

Autoclave continued ..
The steam pressure rises inside and when it reaches the desired
set level, the safety valve opens and the excess steam escapes.
From this point, the holding period is calculated. The holding
period is 121 C at 15 pound pressure for 15 minutes.
When the holding period is over, the heater is turned off and the
autoclave allowed to cool till the pressure inside is equal to the
atmospheric pressure.
The discharge tap is opened slowly and air is let into the
autoclave.
The domestic pressure cooker serves as a miniature autoclave
and may be used for sterilizing small articles in clinics.

AUTOCLAVE

FILTRATION

Membrane filters are widely used which is made of

cellulose esters or other polymers. Their average pore
diameters (APD) is 0.22 mm size.
Filtration helps to remove bacteria from liquids such as
sera and solutions of sugars or antibiotics used for
preparation of culture
media.
Membrane

filters

TwoRADIATION
types of radiation are used for Sterilization
Nonionizing & Ionizing.
X-rays, Gamma rays &
Cosmic rays
are highly
IONISING

Infrared and
NONIONISING
RADIATION
lethal to all cells including
Ultraviolet
RADIATION rays are of
bacteria.

the nonionizing low

energy type.
Applications:-

Clean surfaces like

inoculation hoods,
microbiology & other
laboratories, hospital
wards and operation

They have very high

penetrative power. They
damage DNA by various
mechanisms.

Applications:Commercial plants use

gamma radiation for
sterilization of disposable
items such as plastics

(II) CHEMICAL AGENTS

Several chemical agents are used as antiseptics and
disinfectants used in the microbiology laboratory and hospital.
However little is known about the mechanism of action of
many of these agents.

An ideal antiseptic or disinfectant should be:Have a wide spectrum of activity and must be effective
against all microorganisms, that is, bacteria including spores,
viruses, protozoa and fungi;
Have speedy action; Have high penetrating power; Be stable.
Not corrode metals; Not cause local irritation or sensitization;
Not interfere with healing;
circulation;

Not be toxic if absorbed into

Be cheap and easily available;

Be safe and easy to use.

ALCOHOLS
Ethyl alcohol (ethanol), Isopropyl alcohol and Methyl alcohol
are the most frequently used.
They are used as Disinfectants & antiseptics. Their mode of
action is denaturing bacterial proteins.
Methyl alcohol is effective against fungal spores and is used
for treating cabinets and incubators affected by them.

ALDEHYDES

Formaldehyde is active against the amino group in protein

molecule.
It is used to preserve anatomical specimens.
Surfaces which have disinfected by this agent may give off
an irritant vapour for some time after disinfection.

HALOGENS
IODINE

Iodine is widely used as a skin disinfectant (antiseptic).

It is active against bacteria, fungi and viruses etc.
For eg: Betadine.
CHLORINE
Chlorine is used as disinfectants in microbiology
laboratories, hospital, water supplies & swimming
pools etc.
Chlorine is commonly used as hypochlorites.
Chlorine and hypochlorites are markedly bactericidal.
They have a wide spectrum of action against viruses.

PHENOL

Phenol (carbolic acid) is a powerful and good general

disinfectants.
Phenol is widely used as disinfectants for various
purposes in microbiology laboratories & hospitals. Lysol
and cresol are active against a wide range of organisms.
GASES
FORMALDEHYDE GAS
This is widely employed for fumigation of operation
theatres and other rooms. After sealing the windows and
other outlets, formaldehyde gas is generated and left
unopened for 48 hours.

Commonly used Chemical Agents in

Microbiology laboratory and Hospital
ALCOHOL
FORMALDEHYDE

IODINE

LYSOL

PHENOL

CHLORINE

DETERGENTS

Importance of quality control

continued.
Example of Quality control:Sterilization control for Hot air oven
The spores of Clostridium tetani are used as test of
dry heat efficiency.
Paper strips impregnated with
spores are placed
in envelopes and inserted into suitable packs.
After sterilization, the strips are removed and
inoculated into thioglycollate or cooked meat media
and incubated for sterility test under strict
anaerobic conditions for five days at 37C. If there is
no growth in the culture media, the hot air oven
works properly.

LEARNING OUTCOMES
At the end of the lecture, student should be
able to:
Define Sterilization, Disinfection and Antisepsis.
Describe the various methods of physical and
chemical methods of sterilization.
Select the ideal method of sterilization for different
types of culture media, laboratory equipments and
surgical instruments.
Explain with suitable examples the importance of
quality control for sterilization and disinfection.

BOOKS FOR REFERENCES

Text book of Microbiology by
Ananthanarayan and Panikers.
A Text book of Microbiology by
P.Chakraborty.
Medical Microbiology by Jawetz,
Melnick, & Adelberg's.
Medical Microbiology by Murray.