The document provides a comprehensive overview of sterilization techniques, including definitions of key terms such as disinfection, antisepsis, and methods of sterilization which include physical and chemical processes. It outlines various methods like heat, pasteurization, autoclaving, and ultraviolet radiation, detailing operational principles, advantages, and applications for each. Additionally, the document addresses specific factors influencing sterilization efficacy and the limitations of each method.

1. STERILIZATION
DR.N.C.J.PACKIA LEKSHMI
NICHE

2. INTRODUCTION
• Sterilization is making a substance free from all
micro organisms both in vegetative and sporing
states.
• Spore is a reproductive structure that is adapted
for dispersal and surviving for extended periods
of time in unfavourable conditions.
• Spores form part of the lifecycles of many
bacteria, plants, algae , fungi and some protozoa.

3. Terms in Sterilization
• Disinfection: The destruction or removal of all pathogenic
organisms capable of giving rise to infection. Disinfection
does not affect spore state organisms.
• Antisepsis: The term is used to indicate the prevention of
infection, usually by inhibiting the growth of bacteria in
wounds or tissues.
• This is done by the antiseptics: Chemicals or disinfectants
which can be safely applied on skin or mucous membrane
to prevent infection by inhibiting the growth of bacteria.
• Bactericidal agents / germicides: Those which able to kill
bacteria.

4. Cont…
• Bacteriostatic agents: Only prevent multiplication
of bacteria, but they remain alive.
• Cleaning: Important preparatory step before
sterilization or disinfection, by removing soil and
other dirt.
• Decontamination: The process of rendering an
article or area free of contaminants, including
microbial, chemical, radioactive and other
hazards.

5. Methods of Sterilization
1. Physical
2. Chemical
• Physical agents: Sunlight
• Drying
• Heat - Dry heat: flaming, incineration, hot air,
Moist heat: pasteurization, boiling, steam under
pressure.
• Filtration: candles, asbestos pads, membranes
• Radiation
• Ultrasonic and sonic vibrations.

6. Physical methods of sterilization
• Sunlight: Action primarily due to UV rays
however, effects vary due to places Eg: In tropical
country, the germicidal effect is better than 4
seasoned countries. Bacteria in water are readily
destroyed by sunlight.
• Drying: Moisture is essential for growth of
bacteria.
• Drying in air has deleterious effect on many
bacteria.
• However, spores are unaffected. Therefore, it is
not really unreliable.

7. • Heat: Most reliable method of sterilization and should
be the method of choice.
Dry Heat & Moist Heat
The factors influencing sterilization by heat:
• Nature of heat-dry or moist
• Temperature and time
• Number of microorganisms present
• Characteristics of organisms –species, strain, sporing
capacity
• Type of material from which organism have to be
eliminated.
• Killing effect is due to protein denaturation, oxidative
damage and toxic effect of elevated level of
electrolytes.
• Killing effect of moist heat due to denaturation and
coagulation

8. • Thermal Death Time:TDT “Minimum time
required to kill a suspension of organisms at a
predetermined temperature in a specified
environment.
• Thermal death time is inversely proportional
to temperature.
• TDT is increased in presence of organic
substance, proteins, nucleic acid, starch,
gelatin , sugar , fats, oils.

9. • Dry heat: Flaming: Heating over fire, till they become
red hot. Flaming is done to inoculation loops and
straight-wires in microbiology labs for streaking.
• Leaving the loop in the flame of a Bunsen burner or
alcohol burner until it glows red ensures that any
infectious agent is inactivated.
• Instruments like: Point of Forceps, Spatulas, Inoculating
loops and Wires. (Inoculating loop is better dipped in
disinfectant first before flaming to prevent spattering)
• A variation on flaming is to dip the object in a 70% or
more concentrated solution of ethanol, then briefly
touch the object to a Bunsen burner flame. The
ethanol will ignite and burn off rapidly, leaving less
residue than a gas flame.

10. • Incineration: It is a process that involves the
combustion of organic substances contained
in waste materials. Items: contaminated cloth,
animal carcasses and pathological material.
PVC, polythene can be dealt. (However,
polystyrene will emit black smoke. Hence
should be autoclaved in appropriate
container.)

11. Hot air oven
• Hot air ovens are electrical devices used in sterilization.
• The oven uses dry heat to sterilize articles. Generally,
they can be operated from 50 to 300 C (122 to 572 F) .
• There is a thermostat controlling the temperature.
• This is the most widely used method of sterilization by
dry heat.
• Items: glassware, forceps, scissors, scalpels, all-glass
syringes, swabs, liquid paraffin, dusting powder, fats,
grease. (Materials should be properly arranged to allow
free circulation of air )

12. • They were originally developed by Pasteur.
• Generally, they use a thermostat to control the temperature.
• Their double walled insulation keeps the heat in and conserves
energy, the inner layer being a poor conductor and outer layer
being metallic.
• There is also an air filled space in between to aid insulation.
• An air circulating fan helps in uniform distribution of the heat.
These are fitted with the adjustable wire mesh plated trays or
aluminium trays and may have an on/off rocker switch, as well as
indicators and controls for temperature and holding time.
• The capacities of these ovens vary.
• Power supply needs vary from country to country, depending on
the voltage and frequency (hertz) used.
• Temperature sensitive tapes or biological indicators using bacterial
spores can be used as controls, to test for the efficacy of the device
during use.

14. Precautions
• Glass wares should be dry.
• Oven should not be over loaded.
• Articles are to be arranged in a manner to allow
free circular of air.
• Door of the Oven should be opened after it cools
down (2Hours).
• The standard settings for a hot air oven are:
1.5 to 2 hours at 160 °C (320 °F)
6 to 12 minutes at 190 °C (374 °F)

15. Advantages & Disadvantages
• They do not require water and there is not much
pressure build up within the oven, unlike an
autoclave, making them safer to work with.
• Suitable to be use in a laboratory environment.
They are much smaller than autoclaves but can
still be as effective.
• As they use dry heat instead of moist heat, some
organisms like prions, may not be killed by them
every time.

16. Moist heat Sterilization
• sterilization techniques that uses hot air that is
heavily laden with water vapor and where this
moisture plays the most important role in the
sterilization.
• Heating an article is one of the earliest forms of
sterilization practiced.
• The various procedures used to perform moist
heat sterilization process cause destruction of
micro-organisms by denaturation of
macromolecules.

17. Methods
Moist heat can be categorized into 3 groups:
• Temperature below 100°C
• Temperature at 100°C
• Temperature above 100°C

18. Temperature below 100o C
• Pasteurization is a process in which water and
certain packaged and non-packaged foods (such
as milk and fruit juice) are treated with mild heat,
usually to less than 100°C (212 °F), to eliminate
pathogens and extend shelf life.
• The process is intended to destroy or deactivate
organisms and enzymes that contribute to
spoilage or risk of disease, including vegetative
bacteria, but not bacterial spores.
• Since pasteurization is not sterilization, and does
not kill spores, a second "double" pasteurization
will extend the quality by killing spores that have
germinated.

19. Cont…
• The process was named after the French scientist
Louis Pasteur, whose research in the 1880s
demonstrated that thermal processing would
inactivate unwanted microorganisms in wine.
• Spoilage enzymes are also inactivated during
pasteurization.
• Today, pasteurization is used widely in the dairy
industry and other food processing industries to
achieve food preservation and food safety.

20. Pasteurization Process
• In acidic foods (pH <4.6), such as fruit juice and beer, the
heat treatments are designed to inactivate enzymes (pectin
methylesterase and polygalacturonase in fruit juices) and
destroy spoilage microbes (yeast and lactobacillus).
• Due to the low pH of acidic foods, pathogens are unable to
grow.
• The shelf-life is thereby extended several weeks. In less
acidic foods (pH >4.6), such as milk and liquid eggs, the
heat treatments are designed to destroy pathogens and
spoilage organisms (yeast and molds).
• Not all spoilage organisms are destroyed under
pasteurization parameters, thus subsequent refrigeration is
necessary.

21. Technique
• Food can be pasteurized in two ways: either before or after being
packaged into containers.
• When food is packaged in glass, hot water is used to lower the risk
of thermal shock.
• Plastics and metals are also used to package foods, and these are
generally pasteurized with steam or hot water since the risk of
thermal shock is low.
• Most liquid foods are pasteurized using continuous systems that
have a heating zone, hold tube, and cooling zone, after which the
product is filled into the package.
• Plate heat exchangers are used for low-viscosity products such as
animal milks, nut milks and juices.
• A plate heat exchanger is composed of many thin vertical stainless
steel plates which separate the liquid from the heating or cooling
medium.
• Scraped surface heat exchangers contain an inner rotating shaft in
the tube, and serve to scrape highly viscous material which might
accumulate on the wall of the tube.

23. Methods of Pasteurization
• Holding period: 63°C, 30 minutes (holder
method)
• 72°C, 15-20 minutes flash method - followed by
cooling quickly to 130°c or lower. Target: all
nonsporing pathogens Eg: Mycobacteria,
Brucellae, Salmonella. Coxiellaburnetti, relatively
heat resistant, may survive the holder method.
• More than 100oC for 2 seconds – ultra
pasteurization

24. Temperature at 100°C
• Boiling Not recommended for sterilising but used for
disinfection.
• sterilization may be promoted by addition of 2% sodium
bicarbonate to the water.
• Holding period: 10-30 minutes.
• Steam at atmospheric pressure (100°C) Used to sterilize
culture media.
• This is an Inexpensive method Holding period: 100°C, 20
minutes on three succesive days (intermittent sterilization).
• Principle: first exposure kills vegetative bacteria and then
the next exposure will kill vegetative bacteria that matures
from the spore.

25. Temperature above 100oC
• Steam under pressure Autoclave/steam sterilizer--:
autoclave is a device that uses steam to sterilize equipment
and other objects.
• This means that all bacteria, viruses, fungi, and spores are
inactivated.
• However, prions may not be destroyed by autoclaving at
the typical 134°C for 3 minutes or 121°C for 15 minutes.
• Principle: Water boils when its vapour pressure equals the
surrounding atmosphere. Thus, when pressure inside
closed vessels increases, the temperature at which water
boils increases too.
• Holding period: varies. Temperature: between 108°C and
147°C. Items: dressings, instruments, laboratory ware,
media and pharmaceutical products.

26. Types of steam sterilizer
• Laboratory autoclaves
• Hospital dressings sterilizers
• Bowl and instrument sterilizers
• Rapid cooling sterilizers
• domestic pressure cooker can be used as a
steriliser. Recommended temperature and
duration: Temperature(oc) Duration(min)
121oC 15 lbs for 15 minutes

27. Horizontal autoclave
Vertical Autoclave

28. Tyndallization
• Tyndallization is a process dating from the nineteenth century for
sterilizing substances, usually food, named after its inventor, scientist John
Tyndall, that can be used to kill heat-resistant endospores. Although
considered old-fashioned, it is still occasionally used.
• A simple and effective sterilizing method commonly used today is
autoclaving: heating the substance being sterilized to 121 °C for 15
minutes in a pressured system.
• If autoclaving is not possible because of lack of equipment, or the need to
sterilize something that will not withstand the higher temperature,
unpressurized heating for a prolonged period at a temperature of up to
100 °C, the boiling point of water, may be used.
• The heat will kill the bacterial cells; however, bacterial spores capable of
later germinating into bacterial cells may survive. Tyndallization can be
used to destroy the spores.

29. Cont…
• Tyndallization essentially consists of heating the substance to
boiling point (or just a little below boiling point) and holding it there
for 15 minutes, three days in succession.
• After each heating, the resting period will allow spores that have
survived to germinate into bacterial cells; these cells will be killed
by the next day's heating.
• During the resting periods the substance being sterilized is kept in a
moist environment at a warm room temperature, conducive to
germination of the spores.
• When the environment is favourable for bacteria, it is conducive to
the germination of cells from spores, and spores do not form from
cells in this environment (see bacterial spores).
• The Tyndallization process is usually effective in practice. But it is
not considered totally reliable—some spores may survive and later
germinate and multiply.
• It is not often used today, but is used for sterilizing some things that
cannot withstand pressurized heating, such as plant seeds.

30. Filtration
• Fluids that would be damaged by heat, irradiation or chemical
sterilization, such as drug solution, can be sterilized by microfiltration
using membrane filters.
• This method is commonly used for heat labile pharmaceuticals and
protein solutions in medicinal drug processing. A microfilter with pore size
of usually 0.22 µm will usually effectively remove microorganisms.
• Some staphylococcal species have, however, been shown to be flexible
enough to pass through 0.22 µm filters.
• In the processing of biologics, viruses must be removed or inactivated,
requiring the use of nanofilters with a smaller pore size (20–50 nm) are
used.
• Smaller pore sizes lower the flow rate, so in order to achieve higher total
throughput or to avoid premature blockage, pre-filters might be used to
protect small pore membrane filters. Tangential flow filtration (TFF) and
alternating tangential flow (ATF) systems also reduce particulate
accumulation and blockage.

31. Cont…
• Membrane filters used in production processes are
commonly made from materials such as mixed cellulose
ester or polyethersulfone (PES).
• The filtration equipment and the filters themselves may be
purchased as pre-sterilized 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.
• To ensure proper functioning of the filter, the membrane
filters are integrity tested post-use and sometimes before
use. The nondestructive integrity test assures the filter is
undamaged and is a regulatory requirement.
• Typically, terminal pharmaceutical sterile filtration is
performed inside of a cleanroom to prevent contamination.

32. Radiation
• Sterilization can be achieved using
electromagnetic radiation, such as electron
beams, X-rays, gamma rays, or irradiation by
subatomic particles.
• Electromagnetic or particulate radiation can
be energetic enough to ionize atoms or
molecules (ionizing radiation), or less
energetic (non-ionizing radiation).

33. Non Ionizing radiation
• Ultraviolet light irradiation (UV, from a germicidal
lamp) is useful for sterilization of surfaces and some
transparent objects.
• Many objects that are transparent to visible light
absorb UV.
• UV irradiation is routinely used to sterilize the interiors
of biological safety cabinets between uses, but is
ineffective in shaded areas, including areas under dirt
(which may become polymerized after prolonged
irradiation, so that it is very difficult to remove).
• It also damages some plastics, such as polystyrene
foam if exposed for prolonged periods of time.

34. Ionizing Radiation
• Gamma radiation is very penetrating, and is commonly used for
sterilization of disposable medical equipment, such as syringes,
needles, cannulas and IV sets, and food.
• It is emitted by a radioisotope, usually cobalt-60 (60Co) or caesium-
137 (137Cs), which have photon energies of up to 1.3 and 0.66
MeV, respectively.
• Electron beam processing is also commonly used for sterilization.
Electron beams use an on-off technology and provide a much
higher dosing rate than gamma or X-rays.
• Due to the higher dose rate, less exposure time is needed and
thereby any potential degradation to polymers is reduced. Because
electrons carry a charge, electron beams are less penetrating than
both gamma and X-rays.
• Facilities rely on substantial concrete shields to protect workers and
the environment from radiation exposure.

35. Cont…
• High-energy X-rays allow irradiation of large packages and
pallet loads of medical devices.
• They are sufficiently penetrating to treat multiple pallet
loads of low-density packages with very good dose
uniformity ratios.
• X-ray sterilization does not require chemical or radioactive
material: high-energy X-rays are generated at high intensity
by an X-ray generator that does not require shielding when
not in use.
• X-rays are generated by bombarding a dense material
(target) such as tantalum or tungsten with high-energy
electrons, in a process known as bremsstrahlung
conversion.

36. Cont…
• Irradiation with X-rays, gamma rays, or electrons
does not make materials radioactive, because the
energy used is too low. Generally an energy of at
least 10 MeV is needed to induce radioactivity in
a material.
• Neutrons and very high-energy particles can
make materials radioactive, but have good
penetration, whereas lower energy particles
(other than neutrons) cannot make materials
radioactive, but have poorer penetration.

37. Sonication
• Ultrasonic cleaning is a process that uses ultrasound (usually from
20–40 kHz) to agitate a fluid.
• The ultrasound can be used with just water, but use of a solvent
appropriate for the item to be cleaned and the type of soiling
present enhances the effect.
• Cleaning normally lasts between three and six minutes, but can also
exceed 20 minutes, depending on the object to be cleaned.
• Ultrasonic cleaners are used to clean many different types of
objects, including jewelry, lenses and other optical parts, watches,
dental and surgical instruments, tools, coins, fountain pens, golf
clubs, fishing reels, window blinds, firearms, car fuel injectors,
musical instruments, gramophone records, industrial parts and
electronic equipment.

38. Fumigation
• Fumigation is a process of gaseous sterilisation
which is used for killing of micro-organisms
and prevention of microbial growth in air,
surface of wall or floor.
• It is generally used in the pharmaceuticals,
operation theatres, hospitals, hotels and
offices and wherever required.
• Method of fumigation completely fills an area
with gaseous fumigants to suffocate or poison
the microbes & pests within.

39. Widely used fumigants include:
• Formaldehyde
• phosphine
• 1,3-dichloropropene
• chloropicrin
• methyl isocyanate
• hydrogen cyanide
• sulfuryl fluoride
• Iodoform
• Methyl bromide Fumigation equipment

40. Formaldehyde
• A gas at room temperature, formaldehyde is
colorless and has a characteristic pungent,
irritating odor.
• When dissolved in water, formaldehyde forms a
hydrate methanediol.
• A saturated water solution, that contains about
40% formaldehyde by volume or 37% by mass, is
called "100% formalin".
• A typical commercial grade formalin may contain
10– 12% methanol polymerization. added to
suppress oxidation

41. Cont…
• Formaldehyde at approximately 5% in a solution
with water is used as a fumigant and disinfectant
in hospitals.
• Formoldehyde kills microbes by alkylating the
amino acids and sulfydral group of proteins and
purine bases.
• In order to be effective, the gas has to dissolve in
the film of moisture surrounding the bacteria, for
this reason relative humidities in the order of
75% RH and temperature above 22°C.

42. Methods
• Electric Boiler Fumigation Method: For Each 1000 cu.ft
500ml of formaldehyde added in 1000ml of water in an
electric boiler. Switch on the boiler, leave the room and
seal the door. After 45 minutes switch off the boiler
without entering in to the room.
• Potassium Permanganate Method: For every 1000
cubic feet add 450gm of Potassium permanganate
(KMnO4) to 500 ml of formaldehyde. Take about 5 to 8
bowels with equally divided parts of formaldehyde and
add equally divided KMnO4 to each bowel. This will
cause auto boiling and generate fume.

43. Neutralisation
• After the initiation of formaldehyde vapour
immediately leave the room and seal it for at
least 48 hours
• Neutralise residual formalin gas with
Ammonia by exposing 250ml of Ammonia per
litre of Formaldehyde used.
• Place the Ammonia solution in the centre of
the room and leave it for 3 hours to neutralise
formalin vapour.

44. Phosphine
• Phosphine is the compound with the chemical formula PH3.
• It is a colorless, flammable, toxic gas. Pure phosphine is odorless,
but technical grade samples have a highly unpleasant odor.
• Phosphine-producing materials have become the predominant
fumigants used for the fumigation of OT & bulk-stored grain
throughout the world.
• It is available in solid formulations phosphide or magnesium
phosphide of aluminum.
• When exposed to heat and moisture the formulations release
phosphine, a highly toxic gas.
• The time required for release of phosphine will vary with
temperature and formulation.
• Phosphine is very toxic to all forms of animal life, hence exposure of
human beings even to small amounts should be avoided.
• Phosphine can be produced via tablets or phosphine producing
equipment.
• Phosphine is generaly used for fumigating huge quantity of food
grain than operation theatre

45. Methyl Bromide
• Methyl Bromide is most widely used fumigant
for quarantine purposes
• It is a preferred fumigant because of its good
penetrating ability, rapid action, high toxicity
to a broad spectrum of micro organisms and
pests.
• The effectiveness of methyl bromide is based
on the following: • Dosage of the fumigant •
Duration of exposure • Temperature

46. Preparation for fumigation
• First the area intended to be fumigated is usually covered
to create a sealed environment.
• The fumigant is released into the space to be fumigated.
• The space is held for a set period while the fumigant gas
percolates through the space and acts on and kills any
infestation.
• The space is neutralised & ventilated so that the poisonous
gases are allowed to escape from the space, and render it
safe for humans to enter.
• Windows, doors should be closed. AC and AHU should be
switched off before starting fumigation.
• Area under fumigation, do not enter status label should be
displayed on either side of the entrance.
• Fumigants are irritating and toxic to human eye, nose and
throat. Therefore use of nose mask and goggles while doing
fumigation is advisable.

47. • Under certain conditions formaldehyde can react with
hydrochloric acid and chlorine containing disinfectants
such as hypochlorites to form chloromethyl a potential
lung carcinogen.
• HCl and chlorine containing disinfectants must
therefore be removed from areas before fumigation.
• Check levels of residual fumigant in the room with
suitable air monitoring equipment(formaldameter or
air sampling tubes).
• The procedure is best carried out overnight.
• After a period of not less than 12 hours the room must
be well ventilated.
• OT is usable only when the level of formaldehyde is
less than 2ppm.

48. Personal care while Fumigation
• Adequate care must be taken by wearing cap,
mask,foot cover, spectacles.
• Formaldehyde is irritant to eye & nose; and it
has been recognized as potential carcinogen.
• Immediately leave the room after the
initiation of fumigants.
• So the fumigating employee must be provided
with personal protective equipment.

49. Advantages of Fumigation
• Fumigants are toxic to all forms of life. Therefore,
it is possible to control all life stages of Micro
organisms.
• Fumigation is often the quickest way of
controlling an infestation, saving time and money.
• Fumigants can reach where sprays, dusts,
aerosols etc cannot reach.
• Reduced residue problems in treated areas.
• Fumigants are used where standards call for "zero
Microbial tolerance" in products or living
environments.

50. Disadvantages of Fumigation
• Generally Formaldehyde is used for OT fumigation.
• Based on the available evidence in OSHA’s record on
Formaldehyde, it determines formaldehyde as is
genotoxic, showing properties of both a cancer initiator
and promoter.
• In humans formaldehyde exposure has been associated
with cancers of the lung, nasopharynx and nasal
passages.
• Symptoms of excessive exposure include respiratory
irritation, itchy eyes, runny or stuffy nose, sore throat
and headache.