STERILIZATION BY

PHYSICAL METHODS
Prof. Clark Joy L. Barias

12/2/2012 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.
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HOW CAN MICROORGANISMS
BE KILLED?
• Denaturation of proteins (e.g. wet heat,
ethylene oxide)
• Oxidation (e.g. dry heat, hydrogen
peroxide) Filtration
• Interruption of DNA synthesis/repair
(e.g. radiation)
• Interference with protein synthesis (e.g.
bleach) Disruption of cell membranes (e.g.
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phenols) 3
 CLASSIFICATION
There are two types of • 2. Chemical sterilization
sterilization: physical and includes:
chemical. Alcohols
1. Physical sterilization Aldehydes
includes:
Phenolics
 heat
Oxidizing agents
 radiation Quaternary ammonium
 filtration
• compounds
ethylene oxide gas
Others
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4
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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5
 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
Contact time
Physico-chemical environment (e.g. pH)
3 Presence of organic material
4 Temperature
5 Type of microorganism
6Number of microorganisms
7 Material composition
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7
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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8
 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 protozoan cyst most bacterial vegetative cells

(Bacillus & Clostridium) some fungal spores ordinary fungal spores & hypae
some naked virus enveloped virus
vegetative bacteria that Yeasts
have higher resistance Trophozoites
( M. tuberculosis, S.aureus,
Pseudomonas)

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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

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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 deathrate

Number of living microbes

CONSTANT
90% die PERCENTAGE
OF THE EXTANT
1 min POPULATION IS
KILLED EACH MINUTE

90% die

1 min

12/2/2012 Time (min) Dr.T.V.Rao MD 14

 Methods
1.Physical
methods
2.Chemical
methods
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 PHYSICAL METHODS:
• Physical
methods:
1.Sunlight
2. Heat
1.Dry heat
2.Moist heat
3. Filtration
4.Radiation
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 CHEMICAL
METHODS
• Chemical methods:
1. Alcohols
2. Aldehydes
3. Phenols
4. Halogens
5. Oxidizing agents
6. Salts
7. Surface active agents
8. Dyes
9. Vapor phase disinfectants
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 HOW TO STERILIZE
Materials Method
1 Inoculating wires and loops Red heat
2 Glass ware- syringes, petridishes, testtubes, flasks Hot –airoven
etc.
3 Disposable syringes, and otherdisposable items Gamma radiation

4 Culture media Autoclaving

5 Culture media containing serum and egg Tyndallisation
6 Toxin , serum, sugar, and antibioticsolutions Filtration
7 Cystoscope and endoscope Glutaraldehyde

8 Infected soiled dressings Incineration

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9 Skin Iodine, alcohol
10 Milk Pasteurisation
PHYSICAL METHODS OF MICROBIAL
CONTROL DRY 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
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moist heat.
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
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© 2012 Pearson EducationInc.
 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
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seconds.
 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
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successive days.
 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 heat
2.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.
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 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
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31
 DRY-HEAT STERILIZATION
• Disadvantages:
–Less reliable than autoclaving
–Large temp difference may arise within
device.
–sharp instruments get dulled
–Many materials do not tolerate dry heat

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 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
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» 180 C for 30 minutes.
 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 air
3. 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
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STERILISATION CONTROLS
:
• Sterilisation controls
1.Spores of
Bacillus
subtilis
subsp. niger
2. Thermocouples
3.Browne’s
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tube Dr.T.V.Rao MD 36
 STERILIZING BELOW100°C

1.temperature
below 100°
Pasteurization of
milk
1.Inspissation
2.Vaccine bath

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PRINCIPLE OF
PASTEURIZATION

12/2/2012 Dr.T.V.Rao MD 38
 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
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temperature.
 TEMPERATURES ABOVE 100°C
III. A temperature
above 100°C
Autoclave :
-Steam above
100°C has a
better killing
power than
dry heat.
-Bacteria are
more
susceptible to
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moist heat.
 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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Dr.T.V.Rao MD
46
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
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2 ightly 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. Thermocouples
2. Bacterial spores-
Bacillus
stearothermophilus
3. Browne’s tube
4. Autoclave tapes

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 STERILITY CONTROLS
Yellow medium
means spores are
Cap that allows viable; autoclaved
steam to penetrate objects are not
sterile.
Flexible plastic
vial
Crushable glass Incubation
ampule
Nutrient medium
containing pH
color indicator Red medium
means spores were
After autoclaving, flexible killed; autoclaved
Endospore strip
vial is squeezed to break objects are
ampule and release sterile.
medium onto spore strip.

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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.
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 FILTRATION STERILIZATION
 This method is commonly used for  To ensure sterility, the filtration
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.
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 filters
2. Asbestos disc filters
3. Sintered glass filters
4. Membrane filters
5. Air filters
6. Syringe filters

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

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THE FILTERING DEPENDS ON PORE
SIZE
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).

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 Candle filters
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 Exhaust HEPA viewscreen
filter safety cabinets
Blower

Supply HEPA
filter

Light

High-velocity
air barrier

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 RADIATIONS :
• Radiations :
•Ionizing
radiations
•Non -
Ionizing
radiations
12/2/2012 Dr.T.V.Rao MD 58
 IONISING RADIATIONS:

– Ionizing radiations:
1. X rays
2. Gamma rays
3. 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,
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© 2012 Pearson EducationInc.
 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 rays

2. 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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