7.11.

2019

Sterilization – physical agents

By:
Dr. Abirami devi
1st year PG
Department of orthodontics
WHY sterilization?
 Sterilization
Sterilization [Latin sterilis, unable to
produce offspring or barren] is defined as the
process by which an article, surface, or medium is
freed of all living microorganisms either in the
vegetative or spore state.
Disinfection
Disinfection is the killing, inhibition, or removal of
microorganisms that may cause disease.

Antiseptics
Antiseptics are chemical agents applied to tissue to
prevent infection by killing or inhibiting pathogen
growth; they also reduce the total microbial
population.
 History

• The Egyptians used fire to sterilize infectious

material and disinfectants to embalm bodies
• The Greeks burned sulfur to fumigate
buildings.
• Mosaic law commanded the Hebrews to burn
any clothing suspected of being contaminated
with the leprosy bacterium.
 METHODS OF STERILIZATION AND
DISINFECTION

A. Physical agents
B. Chemical agents.
 Physical agents
a. Dry heat
1. Sunlight Incineration
2. Drying Red heat
3. Heat Flaming
Hot air sterilizer
4. Filtration Microwave ovens.
5. Radiation b. Moist heat
6. Ultrasonic and sonic Pasteurization
vibrations. Boiling
Steam under normal
pressure
Steam under pressure.
 Sunlight

• Bactericidal activity
• Disinfectant action is due
primarily to its content of
ultraviolet rays.
• Natural method of
sterilization in cases of water
in tanks, rivers and lakes.
 Drying
• Water constitutes 4/5th of the weight of the
bacterial cell and is essential for the growth of
bacteria.
• Spores are unaffected by drying.
 Heat
• The most reliable and universally applicable
method of sterilization and, Applied only in
thermostable products but can be used for
moisture sensitive products.
• Either dry or moist heat may be applied.

Materials that may be damaged

by heat can be sterilized at
lower temperature, for longer
periods or by repeated cycles.
Factors influencing sterilization by heat

1. Nature of heat
2. Temperature and time
3. The number of microbes
4. Characteristics of the organisms
5. The nature of contaminated material
 Mechanisms behind

Dry heat Moist heat

• Protein denaturation,
• Coagulation
• Oxidative damage,
• Denaturation of their
• Toxic effects of
enzymes and
elevated levels of
structural proteins.
electrolytes.
 Dry Heat Sterilization

i. Red heat
ii. Flaming
iii. Incineration
iv. Hot air sterilizer
v. Microwave ovens
 Red Heat
• Inoculating wires loops and points of forceps
• The points of forceps and the surface of searing spatulae
may also be heated until red.
 Flaming
Scalpel blades, glass slides, mouth of
culture tubes and bottles are exposed to a flame
for a few seconds
 Incineration

• Efficient method for the sterilization and disposal

of contaminated materials at a high temperature.
• Pathological waste materials, surgical dressings,
contaminated material, animal carcasses and other
clinical wastes
 Hot Air Oven

• Fitted with a fan to

provide forced air
circulation throughout
the oven chamber,
• A temperature
indicator, a control
thermostat and timer,
open mesh shelving and
adequate wall
insulation.
 Preparation of Load

No overloading
Articles-clean and dry
Glassware
Test tubes , pipetts and flasks-wrapped
No Rubber materials
Heat-sensitive materials-not suitable(p)
 Sterilizing Cycle

i. The sterilization hold time

160ºC for 2 hours or 170ºC for 1 hour, or
180ºC for 30 minutes
ii. Cutting instruments
Such as those used in ophthalmic surgery,
should ideally be sterilized at 150°C for two hours.
iii. Oils, glycerol and dusting powder
The British Pharmacopoeia recommends a
holding time of one hour at 150°C for oils, glycerol
and dusting powder
 Cooling

Cooling may take up to several hours

and, therefore do not attempt to open the
chamber door until the chamber and load have
cooled below 80ºC.
Glassware is liable to crack if cold air is admitted
suddenly while it is still very hot.
 Uses of Hot Air Oven
It is a method of choice for sterilization of:
1. Glassware: Such as tubes, flasks, measuring
cylinders, all-glass syringes, glass petri dishes
and glass pipettes.
2. Metal instruments: Such as forceps, scissors
and scalpels.
3. Nonaqueous materials and powders, oils and
greases in sealed containers and swab sticks
packed in test tubes, parraffin and fats.
 Sterilization Controls
Biological control Chemical indicator
Bacillus subtilis subsp niger Browne’s tubes No. 3
 Microwave Ovens

In microwave ovens, the heating effect

is not uniform and not reliable for sterilization
process

A specialised microwave oven become available that can be used to

sterilize media in 10mins . It has 12 pressure vessels, each of which
holds 100 ml of medium.
 Moist heat

Moist heat is divided into three forms:

A. At temperature below 100ºC

B. At a temperature of 100ºC
C. At temperature above 100ºC.
 At a Temperature Below 100°C
• 56ºC for 1 hour.
• Such treatment is sufficient to kill vegetative
bacteria
It includes:

Pasteurization of milk
Vaccine preparation
Inspissation
Water bath
 Pasteurization of milk
• 63ºC for 30 minutes or 72ºC for 15-20 seconds
followed by rapid cooling to 13ºC or lower.
• ultrahigh-temperature (UHT) sterilization :
140 to 150ºC for 1 to 3 seconds.
 Vaccine preparation
Vaccines prepared from
nonsporing bacteria may be
inactivated in a water bath at
60ºC for 1hr
1hr at 56ºC in a water
bath on several successive
days.
 Inspissation

Medias are rendered sterile by heating

at 80 to 85°C for ½ hr on 3 successive days
(fractional sterilization).
 Water bath
Washing or rinsing laundry or heating
utensils in water bath at 70 to 80ºC for few
minutes will kill most non sporing
microorganisms present.
 Low Temperature Steam
Formaldehyde (LTSF) Sterilization
• 75°C with formaldehyde vapor is used.
• Mixture of steam & formaldehyde vapour-
sporicidal
• Rubber goods, plastic, linen & metals
• Bactericidal & viricidal
 Temperature at 100°C
Boiling
100ºC for 10 to 30 mins
Sporing bacteria require
prolonged periods of boiling.
Addition of 2 % sodium
bicarbonate may promote
sterilization.
Uses of boiling at 100°celcius:
i. For the disinfection of surgical instruments
prior to processing.
ii. For the disinfection of medical and surgical
equipment—when sterility is not essential in
emergency or under field conditions
 Steam at atmospheric pressure at
100°C for 90 minutes
• To disinfect selective heat-labile culture media
in the laboratory which may decompose if
subjected to higher temperatures. This can be
provided by the traditional Koch and Arnold
steamer (or by the multipurpose autoclave).
Koch and Arnold Steamer
 Tyndallization intermittent sterilization

100ºC for 20 mins on 3 successive days is called

tyndallization.

Uses
sterilizing heat-sensitive culture media
containing such materials as carbohydrates, egg or
serum , which are damaged by higher temperature
of autoclave.
 Moist Heat at Temperatures Above
100°C
Steam Under Pressure
Steam above 100ºC or saturated steam is more
efficient sterilizing agent than hot air because:
1. It provides greater lethal action of moist heat.
2. It is quicker in heating up articles to be sterilized.
3. It can penetrate easily porous material such as
cotton wool stoppers, paper and cloth wrappers,
bundles of surgical linen and hollow apparatus.
 Autoclave

Autoclaving is the process of sterilization

by saturated steam under high pressure above
100°C. Steam sterilization is carried out in a
pressure chamber called an autoclave
 Various Components of Autoclave
Simplest form
The laboratory autoclave consists of
a vertical or horizontal cylinder of
gunmetal or stainless steel, in a
supporting sheet iron case.
The lid or door is fastened by screw
clamps and made airtight by a suitable
washer.
Discharge tap for air and steam, a
pressure gauge and a safety valve that can
be set to blow off at any desired pressure.
Heating - gas or electricity
Principle of Autoclave
 Precautions

i. Air escape from the chamber

ii. Arrangement of the materials
 Uses
i. culture media and other laboratory supplies,
aqueous solutions, rubber material, dressing
materials, gowns, linen, gloves, instruments and
pharmaceutical products.
ii. For all materials that are water containing,
permeable or wettable and not liable to be
damaged by the process.
iii. Particularly useful for materials which cannot
withstand the higher temperature of hot air
oven.
 Sterilization Controls
A. Biological control (Bacterial spores):
spores of Bacillus stearothermophilus
B. Chemical control: A Browne’s tube containing
red solution changes to green when exposed to
temperature of 121ºC for 15 minutes in
autoclave. It indicates proper sterilization.
C. Autoclave tapes.
D. Thermocouples: May also be used which
records the temperature by a potentiometer.
 Filtration

Filtration is the principle method used

in the laboratory for the sterilization of heat
labile materials
e.g. sera, solutions of sugars or antibiotics used
for preparation of culture media.
 Uses of Filtration

1. Heat sensitive solutions

2. For separation of bacteriophages and
bacterial toxins from bacteria.
3. Isolation of organisms which are scanty in
fluids.
4. Concentration of bacteria from liquids
5. For virus isolation
 Types of Filters

i. Earthware filters
ii. Asbestos filters
iii. Sintered glass filters
iv. Membrane filters
v. Syringe filters
vi. Vacuum filters
vii. Pressure filtration
viii. Air filters
 Earthware filters
The fluid to be sterilized is forced by suction or
pressure from inside to outside or vice versa.
After use they can be sterilized by scrubbing
with stiff brush followed by boiling and
autoclaving.
They are of two types:
a. Unglazed ceramic filters (Chamberland)
b. Compressed diatomaceous earth filters(the
Mandler filters)
 Asbestos filters (Seitz filter):
• It is then fitted on to a sterile flask through a
silicone rubber bung.
• Sterilized fluid is collected from the flask and
filter disk is discarded after use.
• Examples: Seitz filter, Carlson and Sterimat
filters.
 Membrane filters
Polymeric materials such as cellulose nitrate, cellulose
diacetate, polycarbonate and polyester.
They are manufactured as disks from 13 to 293 mm
diameter and with porosities from 0.015 to 12 mm
Average pore diameters - 0.22 mm size being most
widely used for sterilization because the pore size is smaller
than that of bacteria.

Uses
1. They are used routinely in water analysis and
purification.
2. Sterilization and sterility testing.
3. For preparing sterile solutions for parenteral use.
4. Bacterial counts of water
 Radiation
Two types of radiations are used:
I. Nonionizing
II. Ionizing
 Nonionizing (ir,uv)

• The effectiveness of UV light as a lethal and mutagenic

agent is closely correlated with its wavelength. The
wavelength 240- 280 most effectively absorbed by DNA
and this infers with DNA replication.
• Ultraviolet radiation can be produced artificially by
mercury vapor lamps.
• Energy its power of penetration is poor. UV radiation
around 260 nm is quite lethal but does not penetrate
glass, dirt films, water and other substances very
effectively.
Microbial Sensitivity to UV Radiation
i. Bacterial spores are generally more resistant
ii. Viruses are inactivated
iii. HIV is not inactivated by UV radiation.
Practical Applications of UV Radiation
1. To disinfect drinking water.
2. Disinfection of enclosed areas
 Precautions
Because UV radiation burns skin and damages eyes,
people working in such areas must be certain that the
UV lamps are off when the areas are in use.
 Ionizing Radiation (X, γ, cosmic rays)

Very high penetrative power - highly lethal to all

cells including bacteria.
Damages DNA structural defects in microbial
DNA synthesis, leading to cell death.
Spores are among the most radiation resistant
microorganisms known.
Applications
i. For sterilization in pharmacy and medicine.
ii. Sterilization of packaged disposable articles -
plastic syringes, intravenous lines, catheters and
gloves
 Cold Sterilization

Since there is no appreciable increase

in temperature in this method it is known as
cold sterilization. Large commercial plants use
gamma radiation emitted from a radioactive
element, usually cobalt 60 for this type of
sterilization.
(Used for antibiotics, hormones, sutures, and
vaccines and to prevent food spoilage. )
 ULTRASONIC & SONIC VIBRATION
• These waves are credited with bactericidal
powers , but the results have been variable
• Microorganisms vary in their sensitivity to
them and survivors have been found after
such treatment
• Hence this method is of no practical value in
sterilization and disinfection.
 CONCLUSION
Standard of care that everyone has a right to
expect from their dental treatment . Infection
control is the discipline concerned with preventing
nosocomial or healthcare associated infection.
Thanku