Dr. Swapnaneel Pradhan's document discusses sterilization and related terms. It defines sterilization as removing all microorganisms from an object through physical or chemical processes. It distinguishes sterilization from disinfection and asepsis. Various sterilization methods are described, including heat (dry and moist), filtration, radiation, and chemicals. Moist heat sterilization using an autoclave at 121°C for 15 minutes is commonly used. Proper loading and cycle parameters are important for effective sterilization.

1. Sterilization
Presented by
Dr. Swapnaneel Pradhan

2. INTRODUCTION
 Microorganisms are ubiquitous. They are found in the surroundings, on
inanimate objects and on the surface of the human body. Since they cause
contamination, infection and decay, it becomes necessary to remove
and/or destroy them. This is the objective of sterilization.
 Sterilization 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.

3. IMPORTANT TERMINOLOGY
Sterilization
Steri1ization is a process
by which all Living
microorganisms, including
viable spores, are either
destroyed or removed from
all articles body surface or
medium.
Disinfection
It refers to a process that
destroys or removes most if
not all pathogenic
organisms but not bacterial
spores.
Asepsis
It is a process where the
chemical agents (called
antiseptics) are applied on
to the body surfaces (skin),
which kill or inhibit the
microorganisms present on
skin.
Decontamination (or
Sanitization)
It is the process of
rendering an article or area
free of contaminants,
including all chemical,
radioactive and other
hazardous materials from
an object or body surface.

4. Sterilization
• It results in reduction of at
least 106 log colony-forming
units of microorganisms and
their spores.
• It can be achieved by a
physical agent or a chemical
agent (called chemical
sterilant).

5. Disinfection
• It results in reduction of at
least 103 log colony-forming
units of microorganism, but
not spores.
• The primary goal in
disinfection is to destroy
potential pathogens, but it
also substantially reduces the
total microbial population.
Agents: Disinfectants can also
be achieved by a physical agent
or a chemical agent (called
disinfectant) and they are
normally used only on
inanimate objects, nor on body
surfaces.

6. Asepsis
• Antiseptics are agents that
can be safely applied on the
skin or mucous membrane to
prevent infection by
inhibiting the growth of
bacteria.
• They prevent the entry of
pathogens into sterile tissues
and thus prevents infection
or sepsis.
• However, they are generally
not as toxic as disinfectants
as they must not destroy too
much of host tissue.

7. Decontamination
• It results in reduction of at
least 1 log colony-forming
unit of most of the
microorganisms, but not
spores.
• Examples include manual or
mechanical cleaning by soap
and detergents to eliminate
debris or organic matter
from the medical devices.

8. The agent used for sterilization/disinfection can also be named with suffix based
on whether they kill or inhibit the microorganisms.
◦ Suffix 'cide' is used for the agents that can kill microorganisms. Examples
include bactericide, virucide and fungicide.
◦ Suffix 'static' is used for the agents that do not kill; but inhibit the microbial
growth. If these agents are removed, growth will resume- for example,
bacteriostatic and fungistatic.
Although these agents have been described in terms of their effects on pathogens, it should be noted that
they also kill or inhibit the growth of nonpathogens as well.

9. Factors Influencing Efficacy of Sterilant/Disinfectant
◦ Organism load: Larger microbial population requires a longer time to die than a smaller one.
◦ Nature of organisms: It greatly influences the efficacy of the disinfectants or sterilizing agents.
◦ Concentration of the chemical agent or the temperature of heat sterilization.
◦ Nature of the Sterilant/ disinfectant such as its:
◦ Microbicidal ability
◦ Rapidity of action
◦ Ability to act in presence of organic matter
◦ Residual activity
◦ Duration of exposure: More is the exposure time to sterilant/disinfectant, better is the efficacy.
◦ Temperature: An increase in the temperature at which a chemical acts often enhances its activity. A
lower concentration of disinfectant or sterilizing agent can be used at a higher temperature.
◦ Local environment: The microorganisms to be controlled are not isolated but surrounded by
environmental factors that may either offer protection or aid in their destruction, e.g. pH, organic matter,
biofilm etc.

10. Classification of sterilization/disinfection methods
I. Physical Methods
i. Drying
ii. Heat
a. Dry Heat
◦ Flaming
◦ Incineration
◦ Hot air oven
b. Moist heat
◦ Temperature below 100°C, e.g. pasteurization, water
bath and inspissation
◦ Temperature at 100°C, e.g. boiling, steaming and
tyndalllization
◦ Temperature above 100°C, e.g. autoclave
iii. Filtration: Depth filters and membrane filters
iv. Radiation
a) Ionizing radiation: y rays, X-rays and cosmic rays
b) Non ionizing radiation: Ultraviolet (UV) and infrared rays
v. Ultrasonic vibration
I. Chemical Methods
i. Alcohol: Ethyl alcohol, isopropyl alcohol
ii. Aldehydes: Formaldehyde, glutaraldehyde,
Orthophthaladehyde
iii. Phenolic: compounds: Cresol, Lysol chlorhexidine,
chloroxylenol, hexachlorophene
iv. Halogens: Chlorine, Iodine, iodophors
v. Oxidizing agents: Hydrogen peroxide, peracetic
acid
vi. Salts: Mercuric chloride, copper salts
vii. Surface active agents: Quaternary ammonium
compounds and soaps
viii. Dyes: Aniline dyes and acridine dyes
ix. Gas sterilization:
a) low temperature steam formaldehyde
b) Ethylene oxide (ETO}
c) Betapropiolactone (BPL)
d) Plasma sterilization

11. PHYSICAL AGENTS OF
STERILIZATION

12. Drying
Moisture is essential for the
growth of bacteria. 70-80% of
the weight of the bacterial cell
is due to water. Drying,
therefore has a deleterious
effect on many bacteria.
Microwave drying machine
Both drying and sunlight
are not reliable. They do not
affect many microbes, including
spores.

13. Heat
Heat is the most reliable and commonly employed method of sterilization/ disinfection. It should be considered as the method of
choice unless contraindicated. two types of heat are used, dry heat and moist heat.
Mechanism of action heat:
◦ Dry heat kills the organisms by charring, denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated
levels of electrolytes.
◦ Moist heat kills the microorganisms by denaturation and coagulation of proteins.
Materials containing organic substances require more time for sterilization/ disinfection.

14. DRY HEAT

15. Flaming
Items are held in the flame of a
Bunsen burner either for a long time
or short time.
• longer time exposure in flame till
they become red hot: this is done
for inoculating wires or loops, tips
of forceps, etc.
• For shorter period of time without
allowing the items to become red
hot: this is done for fragile items,
e.g. mouth of test lubes.

16. Incineration
Incineration is used for the
disposal of biomedical waste
materials. It burns (sterilizes)
the anatomical waste and
microbiology waste by
providing a very high
temperature 870 - 1,200°C and
thereby converting the waste
into ash, flue gas and heat.

17. Precautions: The following precautions should be taken while using hot air oven.
• Overloading of hot air oven should be avoided.
• The material should be arranged in a manner so that free circulation of air is
maintained.
• Material to be sterilized should be dried completely.
• Cotton plugs should be used to close the mouths of test tubes, flasks, etc.
• Paper wrapping of the items should be done.
• Any inflammable material like rubber (except silicone rubber) should not be kept
inside the oven.
• the oven must be allowed to cool for two hours before opening the doors, since
the glassware may crack by sudden cooling.
Sterilization control: the effectiveness of the sterilization done by hot air oven can
be monitored by:
• Biological indicator: Spores (106) of nontoxigenic strains of Clostridium tetani or
Bacillus subtilis subspecies niger are used to check the effectiveness of
sterilization by hot air oven. These spores should be destroyed if the sterilization
is done properly.
• Thermocouples: It is a temperature measuring device that records the
temperature by a potentiometer.
• Browne's tube: It contains a heat sensitive red dye which turns green after being
exposed to certain temperature for a definite period of lime. It was invented by
Albert Browne in 1930.
Hot air oven is the most widely used method of
sterilization by dry heat. It is electrically heated and is
fitted with a fan to ensure adequate and even
distribution of hot air in the chamber. It is also fitted
with a thermostat which maintains the chamber air at a
chosen temperature.
Temperature: A holding temperature and time of 160°C
for 2 hours is required for sterilization in hot air oven.
Materials sterilized: Hot air oven is the best method for
sterilization of:
• Glassware like glass syringes, petri dishes, flasks,
• pipettes and test tubes.
• Surgical instruments like scalpels, forceps, etc.
• Chemicals such as liquid paraffin, fats, glycerol, and
glove powder, etc.

18. MOIST HEAT

19. At temperature below
100°C

20. Pasteurization
It is used for control of
microorganisms from beverages like
fruit and vegetables, juices, beer, and
dairy products, such as milk.
Two methods are available: Holder
method (63°C for 30 minutes) and
Flash method (72°C for 20 seconds
followed by rapid cooling to 13°C or
lower).
All nonsporing pathogens, including
mycobacteria, brucellae and
salmonellae are killed except Coxiella
burnetii which being relatively heat
resistant may survive in Holder
method.

21. Water Bath
It is used for disinfection of
serum, body fluids and
vaccines.
• Bacterial vaccines are
disinfected at 60°C for 1 hour.
• Serum or heat labile body
fluids can be disinfected at
56°C for one hour.

22. Inspissation (fractional sterilization)
It is a process of heating an article on 3 successive days
at 80-85°C for 30 minutes by a special instrument called
inspissator.
Working principle: In inspissator, the first exposure kills all the
vegetative forms, and in the intervals between the heating; the
remaining spores germinate into vegetative forms which are
then killed on subsequent heating.
Uses: Inspissation is useful for sterilization of egg and serum
based media which generally get destroyed at higher
temperatures:
Egg based media: e.g. Lowenstein-Jensen medium and
Dorset's egg medium.
Serum based media: e.g. Loeffler's serum slope.

23. At temperature 100°C

24. Boiling
Boiling of the items in water for
15 minutes may kill most of the
vegetative forms but not the
spores, hence not suitable for
sterilization of surgical
instruments.
Though boiling is a simple, easily
available option to most people,
however, boiling can be
hazardous and not effective;
hence should not be used if better
methods are feasible.

25. Steaming
Koch's or Arnold's steam
sterilizer are useful for those
media which are decomposed at
high temperature of autoclave.
The articles are kept on a
perforated tray through which
steam can pass.
They are exposed to steam
(100°C) at atmospheric pressure
for 90 minutes.
Most of the vegetative forms are
killed by this method except
thermophiles and spores.

26. Tyndallization
(intermittent
sterilization)
Named after John Tyndall, it
involves steaming at 100°C for
20 minutes for 3 consecutive
days.
The principle is similar to that of
inspissation, except that here, the
temperature provided is 100°C,
instead of 80°C .
It is used for sterilization of
gelatin and egg, serum or sugar
containing media, which are
damaged at higher temperature of
autoclave.

27. At temperatures above
100°C

28. Autoclave
PRINCIPLE OF AUTOCLAVE
Autoclave functions similar to a pressure
cooker and follows the general laws of gas:
Water boils when its vapor pressure equals
that of the surrounding atmosphere. So, when
the atmospheric pressure is raised, the boiling
temperature is also raised.
At normal pressure, water boils at 100°C but
when pressure inside a closed vessel
increases, the temperature at which water
boils also increases.

29. Components of Autoclave
Autoclave comprises of three parts: a pressure chamber, a lid
and an electrical heater.
◦ Pressure chamber consists of-
◦ It is a large cylinder (vertical or horizontal) in which the
materials to be sterilized are placed. It is made up of
gunmetal or stainless steel and placed in a supporting iron
case
◦ A steam jacket (water compartment)
◦ The lid is fastened by screw clamps and rendered air tight by
an asbestose washer. The lid bears thee following:
◦ A discharge tap for air and steam discharge
◦ A pressure gauge (sets the pressure at a particular level)
◦ A safety valve (to remove the excess steam)
◦ An electrical heater is attached to the Jacket; that heats the
water to produce steam.

30. Procedure
1. The cylinder is filled with sufficient water and the material to be sterilized is placed inside the pressure chamber. The
lid is closed and the electrical heater is put on. The safety valve is adjusted to the required pressure. ·
After the water boils, the steam and air mixture is allowed to escape through the discharge tap till all the air has been
displaced.
2. This can be tested by passing the steam-air mixture liberated from the discharge tap into a pail of water through a
connecting rubber tube.
When the air bubbles stop coming in the pail, it indicates that all the air has been displaced by steam. The discharge
tap is then closed.
3. The steam pressure rises inside and when it reaches the desired set level (e.g. 15 lbs per square inch in most cases),
the safety valve opens and excess steam escapes out.
4. The holding period is counted from this point of time, which is about 15 minutes in most cases.
5. After the holding period, the electrical heater is stopped and the autoclave is allowed to cool till the pressure gauge
indicates that the pressure inside is equal to the atmospheric pressure.
6. The discharge tap is opened slowly and air is allowed to enter the autoclave. The lid is now opened and the
sterilized materials are removed.

31. Sterilization Conditions
121°C for 15 minutes at pressure of 15 pounds per square inch (psi): This is the most commonly
used sterilization condition for autoclave.
126°C for 10 minutes at pressure of 20 psi
133°C for 3 minutes at pressure of 30 psi

32. Uses of Autoclave
Autoclave is particularly useful for media containing water that cannot be sterilized by dry heat. It is the
method of choice for sterilizing the following:
Surgical instruments
Culture media
Autoclavable plastic containers
Plastic tubes and pipette tips
Solutions and water
Biohazardous waste
Glassware (autoclave resistible)

33. Types of Autoclave
There are different types of autoclaves available:
• Gravity displacement type autoclave: It is the most common type used in laboratories. They are
available in various sizes and dimensions.
• Vertical type (small volume capacity)
• Horizontal autoclave (Large volume capacity)
• Positive pressure displacement type autoclave
• Negative pressure (vacuum) displacement type.

34. DISADVANTAGES OF GRAVITY
DISPLACEMENT TYPE:
 The method of air discharge is inefficient, and it is difficult to
decide when the discharge is complete.
 Materials remain moist after removal from the autoclave.
ADVANTAGES OF VACUUM
DISPLACEMENT TYPE:
 They usually have automatic cycle control.
 The air is drawn out, whereby steam penetrates faster and the time
required for sterilization is shorter.
 Once the sterilization is over, post-cycle vacuum can be
programmed for quick drying.

35. Precautions
 Autoclave should not be used for sterilizing waterproof materials, such as oil and grease or dry materials,
such as glove powder.
 Materials are loaded in such a way that it allows efficient steam penetration (do not overfill the chamber).
 Material should not touch the sides or top of the chamber.
 The clean items and the wastes should be autoclaved separately.
 Polyethylene trays should not be used as they may melt and cause damage to the autoclave.

36. Sterilization Control
 Biological indicator: Spores of Geobacillus stearothermophilus (formerly called Bacillus
stearothermophilus) are the best indicator, because they are resistant to steaming. Their
spores are killed in 12minutes at 121°C.
 Other indicators such as: Browne's tube, and thermocouple.
 Autoclave tapes.

37. FILTRATION

38. Filtration
Filtration is an excellent way to
remove the microbial population
from solutions of heat labile
materials like vaccine, antibiotics,
toxin, serum and sugar solution as
well as for purification of air.
Filters
Depth filter
Membrane
filter

39. • They are porous filters that retain particles
throughout the depth of the filter, rather
than just on the surface.
• Depth fillers are composed of random mats
of metallic, polymeric, or inorganic materials.
• These fillers rely on the density and thickness
of the filter to trap particles rather than the
pore size.
• For example:
1. Candle filters made up of diatomaceous
earth (Berkefeld filters), unglazed porcelain
(Chamberland fillers)
2. Asbestos filters (Seitz and Sterimat filters)
3. Sintered glass filters.
Advantages
• They can retain a large mass of particles before
becoming clogged
• Flow rate of the fluid is high
• Low cost
Disadvantage
• As some of die particles still come out in the
filtrate; hence they are not suitable for
filtration of solution containing bacteria.
Uses
• Depth fillers are commonly used when the
fluid to be filtered contains a high load of
particles, e.g. industrial applications, such as
filtration of food, beverages and chemicals.

40. • They are the most widely used filters for bacterial
filtration. They are porous; retain all the particles on
the surface that are larger than their pore size.
• Membrane filters are made up of cellulose acetate,
cellulose nitrate, polycarbonate, polyvinylidene
fluoride, or other synthetic materials.
• Pore size: Most commonly used membrane fillers
have an average pore diameter of 0.22 μm which
removes most of the bacteria; allowing the viruses to
pass through
• Filters of 0.45 μm are used to retain coliform bacteria
in water microbiology
• 0.8 μm filters are used to remove airborne
microorganisms in clean rooms and for the
production of bacteria free gases.
The sterilization control of membrane filters includes Brevundimonas diminuta and Serratia
marcescens.

41. Filtration of
Water
 To sterilize sera, sugar and antibiotic
solutions.
 Separation of toxins and bacteriophages
from bacteria.
 To obtain bacteria free filtrates of clinical
samples for virus isolation.
 Purification of water- when water
samples pass through, the filter discs
retain the organisms which can then be
cultured, e.g. testing of water samples for
Vibrio cholerae or typhoid bacilli.

42. Filtration of
Air  Air fillers are used to deliver bacteria-free
air. Air can be filtered by various methods:
 Surgical masks (that allow air in but keep
microorganisms out) are the simplest
examples.
 There are two important air filters That are
used in biological safety cabinets and
laminar airflow systems:
 HEPA fillers (High-efficiency par1icula1e air
fillers): HEPA filter removes 99.97% of
particles that have a size of 0.3 μm or more.
 ULPA filters (Ultra-low particulate/
penetration air): An ULPA filter can remove
from the air at least 99.999% of dust, pollen,
mold, bacteria and any airborne particles
with a size of 0.12μm or larger.
HEPA ULPA

43. RADIATION
• Ionizing radiation • Non-ionizing radiation

44. Ionizing
Radiation
Advantages of ionizing radiation-
• high penetrating power,
• rapidity of action,
• temperature is not raised
• It destroys bacterial endospores and vegetative cells, both
eukaryotic and prokaryotic.
Ionizing radiations include, X-
rays, gamma rays (from
Cobalt 60 source), and cosmic
rays.
Mechanism: It causes breakage of
DNA without temperature rise
(hence this method is also called as
cold sterilization).
Disadvantages of ionizing radiation-
• Not always effective against viruses
Uses: Gamma radiation is used in the sterilization/disinfection
of-
• Disposable plastic supplies, such as disposable rubber or
plastic syringes, infusion sets and catheters.
• Catgut sutures, bone and tissue grafts and adhesive
dressings as well as antibiotics and hormones.
• Irradiation of food (permitted in some countries)
Sterilization/disinfection control: Bacillus pumilus.

45. Non-ionizing Radiation
• Examples of non-ionizing radiation include infrared and ultraviolet radiations.
• They are quite lethal but do not penetrate glass, dirt films, water; hence their use is
restricted.
• The recommended dose is 250-300 nm wavelength, given for 30 minutes.
• It is used for disinfection of clean surfaces in operation thearers, laminar flow
hoods as well as for water treatment.
• Because UV radiation burns the skin and damages eyes, hence the area should be
closed and UV lamps must be switched off immediately after use.

46. High-frequency ultrasonic and sonic sound
waves disrupt bacterial cells; but this method is
not reliable, hence is not used now a days.
ULTRASOUND (ULTRA SONIC) WAVES

47. Recent Advancements in Physical
Sterilizations
 Pulsed light sterilization
 Ultra high pressure sterilization
 Hydroclave
 Plasma Sterilization (STERRAD)

48. Pulsed light
sterilization
A non thermal
sterilization method
that destroys
microorganisms
using brief pulses of
white light
generated by Xenon
lamps.

49. Advantages
 Total DNA destruction
 Quick
 No chemicals used
 Safe and easy to use
 Minimum space required
Disadvantage
 Its is a surface treatment. Only areas receiving light are decontaminated.
Applications
 Sterilization of cups, caps, lids and packaging materials.
 Sterilization of food packaging.

50. Ultra high
pressure
sterilization
Also called high hydrostatic
pressure sterilization
(HHP)/pascalisation.
It is a cold sterilization
Technique in which, products
already sealed in its final
package are introduced into
a vessel and subjected to
high level of isostatic
pressure (200 – 600MPa)
transmitted by water, for few
seconds to few minutes.

51. Advantages
 Characteristics of fresh product are retained.
 Destroys pathogens
 Extends product self life.
 Avoids or reduces the need of food preservatives.
 Environment friendly.
Disadvantage
 For inactivation of bacterial endospores, synergistic action of very high pressure
(600MPa) and Temperature (>60°C) is required.
Applications
 Mainly used in food sterilization technology.

52. Hydroclave
Sterilizes the waste
utilizing steam, similar to
an autoclave, but with
much faster and much
more even heat
penetration.
It hydrolyses the organic
components of the waste
and dehydrates the waste.
It substantially reduces
the weight and volume of
waste.

53. Advantages
 Guaranteed high level of sterilization.
 Automatic operation and operator skill does not effect sterility.
 No infectious or harmful emissions.
 It is very economic to use.
 Dry waste regardless of original water content.
Applications
The Hydroclave can sterilize:
 Bagged waste in ordinary bags
 Sharp containers and needles.
 Metal objects, plastic, liquids etc.

54. Gas Plasma
Sterilization
(STERRAD)
Plasma is defined as an
ionized gas with equal
number of +ve and –ve
ions. It is the 4th state of
matter.
Gas plasma is generated
in an enclosed chamber
under deep vacuum using
radio or microwaves to
excite gas molecules
(hydrogen peroxide) to
produce ionized gas
particles.

55. Mechanism of action
 Free radical interaction
 UV/VUV radiation: It causes formation of thymine dimers in DNA, inhibiting
bacterial replication. Breakage of DNA strands.
 Volatilization: It is able to vaporize microbiological matter, causing physical
destruction of spores.
Applications
 STERRAD System enables sterilization of surgical instruments, rigid and flexible
endoscopes, cameras, catheters etc.

56. Advantages
 Process occurs at room temperature and hence no associated thermal dangers.
 Byproducts are generally water and oxygen which are harmless to the
 Fast treatment time (1 min or less)
Disadvantage
 Weak penetration of Plasma
 Complications arise in:
 Presence of organic residue
 Packaging material
 Complex geometries
 Bulk sterilization
 High power consumption
 Can corrode certain materials
Due to its small size, absence of
heating and fast sterilization times it
has the potential of becoming an
integral part of modern Dental
Clinics.

57. Thank you