STERILIZATION
Prof. Dr. Gülgün Yener
Glutaraldehyde
Concentration: Glutaraldehyde is
commercially available as 2% w/v aqueous
solution which must be made alkaline to
"activate" (e.g. by addition of 0.3% sodium
bicarbonate). A 2% glutaraldehyde
solution, for at least 10 hours, can be used
to sterilize heat labile items.
Glutaraldehyde
Spectrum: Active against vegetative
bacteria, spores, fungi and many viruses.
Note: Also available in stable
glycocomplexed form which does not
require addition of alkaline buffer.
Glutaraldehyde
. Use: for instruments such ascystoscopes,
bronchoscopes, corrugated rubber
anesthetictubes, face masks, endotracheal
tubes, metalinstruments, polythene
tubing.
Glutaraldehyde
Caution: Glutaraldehyde is known to
cause dermatitis and asthma. Less
irritating than formaldehyde.
Glutaraldehyde should not be used in an
area with little or no ventilation. Eye
protection, a plastic apron, and gloves
must be worn
Glutaraldehyde
Should be stored away from heat sources
and in containers with close-fitting lids.
The length of time that glutaraldehyde
solutions can be used varies but they are
usually good for up to 14 days. Solutions
should be replaced any time they become
cloudy.
Glutaraldehyde
Advantages Numerous use studies
published Relatively inexpensive
Excellent materials compatibility
Disadvantages: Pungent and irritating
odor Relatively slow mycobactericidal
activity Coagulate blood and fix tissues to
surfaces
Hydrogen peroxide
A concentration of 3% w/v generally used
for disinfection. Active against a range of
microorganisms. Fungi, spores and enteric
viruses require higher concentration. No
toxic end-products of decomposition.
Hydrogen peroxide
Caution: Do not use on aluminium,
copper, zinc or brass. Mechanism: H2O2
on decomposition liberates free hydroxyl
radical which is the active ingredient in the
process. Use: H2O2 is applied to disinfect
plastic implants, contact lenses, and
surgical prostheses.
Ethylene oxide
It is an alkylating agent. Exerts lethal
effect on proteins of bacteria. It is gas at
ordinary room temperature and active
against all types of bacteria and spores. It
has got a good degree of penetration
power, even through plastics.
ETO Machine
Uses:
Plastic goods,
polythene tube,
artery and bone grafts,
cystoscopes,
vaccines and culture media can be
sterilized by ethylene oxide.
ETO Machine
These objects are kept in a cabinet from
which air is removed by a vacuum pump
and then a mixture of ethylene oxide and
carbon dioxide is introduced in the
cabinet.
Ideal Chemical Disinfectant
An ideal chemical antiseptic or
disinfectant should have the following
properties:
Wide spectrum of activity
Active in the presence of organic matter
Effective in acid as well as alkaline
media
Speedy action
Ideal Chemical Disinfectant
High penetrating power
Stable
Compatible with other antiseptics and
disinfectants
Not corrode metals
Ideal Chemical Disinfectant
Not cause local irritation or sensitization
Not interfere with healing
Not be toxic if absorbed into circulation
Cheap and easily available
Safe and easy to use
Modes of Action of Chemical
Disinfectants
Protein coagulation
Disruption of cell membrane resulting in
exposure, damage or loss of the contents.
Removal of free sulphydryl groups
essential for the functioning of the
enzymes.
Modes of Action of Chemical
Disinfectants
Substrate competition – a compound
resembling the essential substrate of the
enzyme diverts or misleads the enzymes
necessary for the metabolism of the cell
and causes cell death.
3. MECHANİCAL METHOD
FILTRATION METHOD
STERIL FILTRATION
It removes
microorganisms from
liquid medium but does
not kill
Sterile Room
Laminar Flow
Cabinet
Membrane Filter
STERILE ROOM
Preparation
Air to be sent to
the room
Room humidity
Room interior
surfaces
Staff
PHYSICAL METHODS
STERILIZATION WITH HEAT
Basic Principle
It is due to the degradation of albumin in
the bacterial cell and the collapse of the
membrane and plasma
Effect of heat depends on
Degree
Time
Moisture
PHYSICAL METHODS
STERILIZATION WITH HEAT
Heat is the most rapid and best method of
sterilization. It is the method of choice
that the material to be sterilized is stable
enough to withstand the required
temperature necessary to kill the
microbes.
PHYSICAL METHODS
STERILIZATION WITH HEAT
The time needed for sterilization depends
on the initial number of organisms
present, type of materials to be sterilized
(hence washed and cleaned items are
easier to sterilize than dirty ones) and also
on the temperature used.
Spores need higher temperatures while
vegetative bacteria can be destroyed at
lower temperatures.
PHYSICAL METHODS
STERILIZATION WITH HEAT
Heat resistance varies among different
microorganisms. These differences can be
expressed in terms of thermal death
point.
Thermal Death Point (TDP) is the
lowest temperature at which all the
microorganisms in a particular liquid
suspension will be killed in 10 minutes.
PHYSICAL METHODS
STERILIZATION WITH HEAT
Another factor to be considered in
sterilization is the duration of time
required. This is expressed
as Thermal Death Time (TDT). TDT is
the minimal time required for all
microorganism in a particular liquid culture
to be killed at a given temperature.
PHYSICAL METHODS
STERILIZATION WITH HEAT
Both TDP and TDT are useful guidelines
that indicate the degree of treatment
required to kill a given population of
bacteria.
PHYSICAL METHODS
STERILIZATION WITH HEAT
Decimal Reduction Time
(DRT) is related to bacterial heat
resistance. DRT is the time, in minutes, in
which 90% of a population of
microorganism at a given temperature will
be killed.
Heat is employed either as dry heat or
moist heat.
DRY HEAT
OBJECTIVE: To destroy the chemical
activity of pyrogen and endotoxins
together with microorganisms
EFFICIENCY: The heat penetrates to
every point of the material in the specified
time depends on placement
DRY HEAT
For lethal effect
Time and temperature are inversely
proportional
1 hour at 170C –
3 hours at 140C
The total time
Lag time
Time required for sterilization
DRY HEAT APPLICATION
Non-degradable materials at
temperatures above 140 0C can be used
Glassware (ampoules, vials, bottles)
Sterilize metal goods, oils, chemicals
Cellulosic materials such as paper and
fabric start to deteriorate at 160 0C,
rubber rapidly oxidizes
DRY HEAT INSTRUMENTS
Hot Air Oven
-Strong air circulation equipment
System used: The material to be sterilized
is placed in such a way that it gives
homogeneous heat. Temperature control
is performed with sensitive chemicals
HOT AIR OVEN
This is the most widely used method of
sterilization using dry heat. The oven is
usually heated by electricity and it has a
thermostat that maintains the chamber air
constantly at the chosen temperature.
It has a fan or turbo-blower to assist the
circulation of air and to ensure rapid,
uniform heating of the load. In Hot Air
Oven, the air is heated at a temperature
HOT AIR OVEN
STERILIZATION
1400C 120 minutes
1600C 120 –180 minutes
1700C 90 –120 minutes
1800C 45 – 60 minutes
DEPYROGENIZATION
2300C 60 – 90 minutes
2500C 30 – 60 minutes
HOT AIR OVEN
HOT AIR OVEN
This is the best method of sterilizing dry
glass ware such as test tubes, petri
dishes, flasks, pipettes and instruments
such as forceps, scalpels and scissors. It is
also used to sterilize some pharmaceutical
products such as liquid paraffin, dusting
powder, fats and grease.
HOT AIR OVEN
Quality control of dry heat
sterilization:
The spores of a nontoxigenic strain
of Clostridium tetani are used to test
the efficiency of dry heat sterlization.
DRY HEAT STERILIZER
Highly used in
industry
Heated by
convective heat
principle
With two doors
Internal
pressure
monitored
DRY AIR TUNNEL
(CONTINUOUS TUNNEL)
The only system in the industry that is
widely used for continuous sterilization
Drying / sterilization / pyrogen-free / cooling
tunnel
In the dry air tunnel, the temperature is
shorter and the processing time is shorter
DURATION: 300 0C 3 - 4 minutes
DRY AIR TUNNEL (CONTINUOUS
TUNNEL)
STERILIZATION WITH
MOISTURE HEAT
Precipitates bacterial cell proteins at lower
temperatures than dry heat
Water Bath
Pressured Steam (AUTOCLAVE)
STERILIZATION IN WATER BATH
Sterilization at and below
100 ° C
Time : 15 minutes – 1 hour
Boiling water should be without lime
For applications above 100 0C, salt is
added to the water
STERILIZATION WITH
MOISTURE HEAT
Moist heat kills microorganisms primarily by the
coagulation of proteins (denaturation), which is caused
by breakage of the hydrogen bonds that hold the
proteins in three dimensional structure.
There are three methods employed in moist heat
sterilization.
· Temperature below 100°C.
· Temperature at 100°C.
· Temperature above 100°C.
STERILIZATION WITH MOISTURE HEAT
Temperature below 100°C:
Pasteurization
The process of heating a liquid food or beverage
either at 62.8°C for 30 minutes or 72°C for 15
seconds to enhance their shelf life and destroy
harmful microorganisms. It should be noted that
pasteurization process kills only vegetative cells
but not the spores. Pasteurization named in
honour of its developer Louis Pasteur.
Comparison between
Sterilization and Pasteurization
Pasteurization
Pasteurization can be done in the following methods,
Low Temperature Holding Method (LTH)
In this method milk, beer and fruit juices are maintained
at 62.8°C for 30 minutes.
High Temperature Short Time Method (HTST)
Products are held at 72°C for 15 seconds.
Ultra High Temperature (UHT)
Milk can be treated at 141°C for 2 seconds (This method
employ temperature above 100°C).
Pasteurization
Basic principle:
Heat briefly below 100ºC and 10-12 0C
and cooling
Milk
610C 30 minutes
710C 15 seconds
Higher pasteurization : 80-850C
instantaneous heating
Ultra pasteurization (UHP) : 1350C 5
seconds
Temperature at 100°C:
i) Water at 100°C (Boiling):
Boiling is one of the moist heat sterilization
methods. It kills vegetative forms of bacterial
pathogens, almost all viruses and fungi
(including their spores) within 10 minutes,
usually much faster.
· Most vegetative bacteria will die in 5-10
minutes when immersed in boiling water, but
some spores will survive at this temperature for
several hours.
· Articles sterilized by this method cannot
be stored for a long time.
Tyndallization
Fractional sterilization
70 ° C 3 days 1 hour heating
Storage at room temperature
Removed from pharmacopoeias
Tyndallization
It is a process discovered by John Tyndall in
19th century for sterilizing substances to kill the spores
of bacteria. The process of exposure of materials to
steam at 100°C for 20 min for three consecutive days is
known as tyndallization. First exposure kills all the
vegetative forms and in the intervals between heating,
the remaining spores germinate into vegetative forms
which are killed on subsequent heating. Tyndallization is
also called fractional sterilization or intermittent boiling.