This document discusses various physical sterilization methods including heat, radiation, and filtration. It focuses on moist heat sterilization methods such as autoclaving, which uses steam under pressure at 121°C for 15 minutes to reliably kill microorganisms. Proper sterilization requires validation using biological indicators like Bacillus stearothermophilus spores to confirm sterilization conditions achieve a 6 log reduction in microorganisms. Physical sterilization methods are effective at eliminating pathogens but can damage heat or moisture sensitive materials so the appropriate method must be selected.

1. STERILIZATION – PHYSICAL METHODS
(Definitions, Physical Agents, Radiation)
BY
DR.T.UMA ARASU
ASSISTANT PROFESSOR
DEPT. OF MICROBIOLOGY
SVMCH & RI

2. Sterilization
• Process by which all living microorganisms
including viable spores, are either destroyed or
removed from an article, surface or medium.
• Results in reduction of ≥106 log CFU of
microorganisms and their spores.

3. Disinfection
• Process that destroys or removes most if not all
pathogenic organisms but may or may not destroy
bacterial spores.
• Results in reduction of ≥103 log CFU of most
microorganism but not spores.
• Achieved by a physical agent or a chemical agent.
• Used only on inanimate objects, not on body surfaces.

4. Cleaning (Decontamination)
• Refers to the reduction in the pathogenic
microbial population to a level at which items
are considered as safe without protective attire.
• Results in reduction of at least ≥1 log CFU of
most of the microorganism but not spores.

5. Central Sterile Supply Department
(CSSD)
• Performs sterilization of medical devices,
equipment and consumables
• Processing area of CSSD - four unidirectional
zones starting from an unsterile area to a sterile
area separated by physical barrier. Decontamination
area, Packaging area, Sterilization area & Sterile storage
area.

6. METHODS OF STERILISATION
Physical methods:
1. Sunlight
2. Heat (a) Dry heat & (b) Moist heat
3. Filtration
4. Radiation

7. A. Physical Methods
1. Sunlight: It has an active germicidal effect due to its content of
ultraviolet rays. It is a natural method of sterilisation in cases of
water in tanks, rivers and lakes.
2. Heat: It is the most reliable and commonly employed method of
sterilisation. Two types of heat are used, dry heat and moist heat.
Principle
(i) Dry heat kills the organisms by denaturation of bacterial protein,
oxidative damage and by the toxic effect of elevated levels of
electrolytes.
(ii) Moist heat kills the microorganisms by denaturation and
coagulation of proteins.

8. DRY HEAT STERILISATION
• Red heat: Inoculating wires or loops, tips of forceps and needles are
held in the flame of the bunsen burner till they become red hot.
• Flaming: Glass slides, scalpels, mouth of culture tubes are passed
through bunsen flame without allowing them to become red hot.
• Incineration: Instrument incinerator may be used for this purpose.
Incineration is used for the treatment of biomedical waste materials
(soiled dressings, animal carcasses, bedding and pathological
materials). It burns the waste by providing a very high temperature
870–1,200°C and thereby converting the waste into ash, flue gas and
heat.
• Hot Air Oven: It is the most widely used method of sterilisation by
dry heat. The oven is electrically heated and is fitted with a fan to
ensure adequate and even distribution of hot air in the chamber. It is
fitted with a thermostat that maintains the chamber air at a chosen
temperature.

9. DRY HEAT STERILISATION

10. DRY HEAT STERILISATION
• Temperature and time: 160° for 2hrs (holding time) is required
for sterilisation (previously it was 160°C for one hour).
• Uses: It is used for the sterilization of:
• Glasswares like glass syringes, petridishes, flask, pipettes, and test
tubes.
• Surgical instruments like scalpels, scissors, forceps etc.,
• Chemicals such as liquid paraffins, fats, sulphonamide powders etc.,
• Precautions: Should not be overloaded.
• The material should be arranged in a manner which allows free
circulation of air.
• Material to be sterilised should be perfectly dry.
• The oven must be allowed to cool for 2 hrs before opening the
doors.

11. DRY HEAT STERILISATION
• Sterilisation control: (i) The spores of Bacillus subtilis subsp.
niger (NCTC 10075 or ATCC 9372) or Spores of Bacillus
atrophaeus are kept inside the oven. These spores should be
destroyed if the sterilisation is proper.
• (ii) Thermocouples may also be used. (iii) Browne‘s tube with
green spot is available. After proper sterilisation green colour
is produced after 2 hrs.

12. DRY HEAT STERILISATION
Advantages:
• It is non-toxic and does not harm environment
• Low operating costs
• Penetrates well into materials
• Noncorrosive for metals.
Disadvantages:
• High temperatures are not suitable for most materials.

13. MOIST HEAT STERILISATION
• 3 types: (i) At a temperature below 100°C (ii) At a temperature
of 100°C (iii) At a temperature above 100°C.
• (i) At a temperature below 100°C
• Pasteurisation of milk: Two types of method- holder method
(63°C for 30 minutes) and flash method (72°C for 20 seconds
followed by cooling quickly to l3°C or lower) are used. All
non-sporing pathogens such as Mycobacteria, Brucellae and
Salmonellae are killed except Coxiella burnetii.
• Inspissation: Some serum or egg media, such as Lowenstein-
Jensen's and Loeffler's serum, are rendered sterile by heating at
80-85°C temperature for half an hour daily on three
consecutive days. This process of sterilisation is called
inspissation used is called inspissator.

14. MOIST HEAT STERILISATION
• Vaccine bath: Bacterial vaccines are sterilised in special
vaccine baths at 60◦C for 1hr. Serum or body fluids can be
sterilised by heating for 1hr at 56◦C in water on several
successive days.
• Low temperature steam formaldehyde ( LTSF)
sterilisation: Items which cannot withstand the temperature of
100°C may be sterilised by a method known as low
temperature steam-formaldehyde (LTSF) sterilisation. In this
method steam at subatmospheric pressure at the temperature of
75°C with formaldehyde vapour is used. Bacillus
stearothermophilus has been used as biological control to test
the efficacy of LTSF sterilisers.

15. MOIST HEAT STERILISATION
• (ii) At a Temperature of 100°C
• Boiling: Boiling for 10 to 30 minutes may kill most of the vegetative
forms but many spores withstand boiling for a considerable time. When
better methods are not available, boiling may be used for glass syringes
and rubber stopper . It is not recommended for the sterilisation of
instruments for surgical procedures.
• Tyndallisation: Steam at 100◦C for 20mins on three successive days is
known as tyndallisation or intermittent sterilisation. The principle is
that the first exposure kills all the vegetative forms, and in the intervals
between the heatings the remaining spores germinate into vegetative
forms which are killed on subsequent heating. It is used for sterilisation
of egg, serum or sugar containing media which are damaged at higher
temperature of autoclave.

16. MOIST HEAT STERILISATION
• Steam steriliser at 100°C for 90 mins: Koch's or Arnold's steam
steriliser usually used for 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 at
atmospheric pressure for 90 minutes. Most of the vegetative forms
are killed by this method.
(iii) At a Temperature above 100°C (under pressure):
• Steam sterilizer 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.
• When the atmospheric pressure is raised, the boiling temperature is also
raised. ™
At normal pressure, water boils at 100°C but when the pressure
inside a closed vessel increases, the temperature at which water boils also
increases.

17. MOIST HEAT STERILISATION
• Mechanism of action: Moist heat destroys microorganisms by
irreversible coagulation, denaturation of enzymes and structural
proteins.
• Components of Steam Sterilizer (Autoclave):
• Steam sterilizer is a pressure chamber; consists of a cylinder, a lid
and an electrical heater. ™
• Pressure chamber: It consists of: „
A large cylinder (vertical or
horizontal) made up of gunmetal or stainless steel, in which the
materials to be sterilized are placed „
A steam jacket (water
compartment).
• ™
Lid: It bears the following: „
A discharge tap for the passage of air
and steam „
A pressure gauge (sets the pressure at a particular level) „
A safety valve (to remove the excess steam). ™
• Electrical heater: It is attached to the jacket; that heats the water to
produce steam.

18. Autoclave

19. Autoclave
• Procedure: The materials to be sterilized are placed
inside the cylinder. The steam jacket is filled with
sufficient water, lid is closed and the electrical heater
is put on. The sterilization process can be divided into
three phases. ™
• Conditioning phase: Air is displaced by steam, at 15
pounds per sq inch, safety valve opens and excess
steam escapes.
• Exposure phase: Holding period 15mins.
• ™
Exhaust phase: Electric heater is switched off and
allowed to cool.

20. Sterilization Conditions
• Cycle duration varies (3 to 18 min) depending on the
sterilization temperature (121°C–135°C)
• Most commonly used sterilization condition is 121°C for 15
min at pressure of 15 pounds (lbs) per square inch (psi).

21. Uses of Steam Sterilizer (Autoclave):
• All critical and semi-critical items that are heat
and moisture resistant (e.g. surgical instruments,
anesthetic equipment, dental instruments etc).
• Culture media preparation.
• Biomedical waste treatment of waste and sharps.

22. Precautions
• It should not be used for sterilizing waterproof
materials such as oil and grease or dry
materials such as glove powder.
• Chamber should not be overfilled
• Separate steam sterilizers should be used for
treatment of biomedical waste.

23. Types of Steam Sterilizer
• Steam sterilizers are available in various sizes and dimensions.
 Horizontal type (large volume capacity)
 Vertical type (small volume capacity)

24. Advantages
• Low cost than ETO and plasma sterilizer.
• Sterilization cycles are fast compared to ETO.
• It is non-toxic and leaves no by-product behind
(unlike ETO).

25. Disadvantages
• Heat can damage acrylics and styrene, PVC
material and corrode some metals.
• Higher temperature for prolonged time can
harm or shorten the life of instruments.
• Moisture also can adversely affect electronics
and can cloud the sensitive materials or leave
water mark stains on them.

26. Sterilization Control
• Biological indicator: Spores of Geobacillus
stearothermophilus are the best indicator
(killed in 12 minutes at 121°C).
• Chemical indicators: External pack control,
e.g. autoclave tape; Bowie-Dick test and
Internal pack control.
• Physical indicators: Digital displays on the
equipment displaying temperature, time and
pressure.

27. Flash Sterilization
• Modification of conventional steam sterilization, designed to be
used at emergency or during unplanned procedures.
• Involves fast sterilization (134°C for 3-10 minutes) of surgical
instruments in an unwrapped condition in steam sterilizers located
close to the operation theatre This practice should only be restricted
for emergency situations.
• Not suitable for porous and cannulated instruments, implants and
suction tubing
• As the instruments are not packed, they remain wet following
sterilization - there is a high-risk of recontamination.

28. 3. Filtration
• Filtration acts by removing microorganisms,
not by killing.
• CDC considers filtration is a sterilization
method, although some authors arguably
disregard this as it does not kill the
microorganisms, rather only filters them out.

29. Filtration
• There are two types of filters:
 Depth Filters- used as drinking water purifiers.
 Membrane filters – widely used in hospitals (bacterial filters –
0.22µm pore size).

30. Filtration of Air
• Surgical (3-ply) mask and respirators: They are simplest
examples of filters being used for purification of air. They
remove microorganisms based on their pore size. These
filters are made up of flat, non-woven fibers.
• HEPA filters (High-efficiency particulate air filters): „HEPA
filter removes 99.97% of particles that have a size of 0.3 μm
or more. HEPA filters in hospitals are used in biological
safety cabinets, airflow system, operation theatre, and
isolation rooms.
• ™
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 0.12 μm or larger

31. Filtration of Liquid
• Used for bacteriological examination of water in hospital settings -
dialysis water.
• To remove bacteria from pharmaceutical fluids that are heat labile and
cannot be purified by any other means.
• Sterilization control of membrane filters -Brevundimonas diminuta and
Serratia marcescens.

32. 4. Radiation
• Ionizing Radiation: It include cobalt 60 gamma rays or electron
accelerators. ™
• Use: It is a low-temperature sterilization method that has been used
for a number of medical products (e.g. tissue for transplantation,
pharmaceuticals, medical devices).
• ™
Mechanism: It causes ionization of the molecules in organisms
leading to breakage of DNA.
• Advantages of ionizing radiation—(1) high penetrating power, (2)
rapidity of action, and (3) temperature is not raised (hence this
method is also called as cold sterilization).
• ™
Disadvantages: High sterilization costs and may have deleterious
effects on the equipment made up of polyethylene. ™
• Sterilization control: Efficacy of ionizing radiation is tested by
using Bacillus pumilus.

33. Non-ionizing Radiation
• Infrared radiation technology - alternate
method of sterilization for selected heat
resistant instrument.
• Ultraviolet radiation does not achieve
sterilization, described under intermediate-
level disinfectant.

34. Answers Please???
1. Who introduced sterilisation techniques and the development of steam
steriliser, autoclave?
a) Louis Pasteur b) Robert Koch c) Paul Ehrlich d) Edward Jenner
2. CSSD stands for
a) Central Sanitization Supplying Department b) Central Sterile Supplies
Department c) Central Sterile Storage Department d) None of the above
3. Cold sterilisation is achieved by ------ rays to sterilize disposable rubber,
plastic syringes.
a) Ionising rays b) Non-ionising radiation c) Both d) None
4. Biological indicators used as sterilsiation controls for hot air oven ------
a) Aspergillus sps b) Clostridium sps c) Geobacillus stearothermophillus
d) Bacillus atrophaeus
5. Moist heat method of sterilsation below 100◦C used to sterilize LJ medium
is
a) Autoclave b) Pasteurization c) Inspissation d) Tyndallisation

35. Answers Please???
6. Holder method and flash process are the subtypes of ----- method of
sterilisation
a) Autoclave b) Pasteurization c) Inspissation d) Tyndallisation
7. Inspissation is otherwise known as
a) Pasteurization b) Fractional sterilsation c) Both d) None
8. Sterilisation by gamma rays is known as
a) Cold sterilisation b) Ionising radiation c) Both d) None
9. Geobacillus stearothermophillus is used as an indicator for efficacy of
a) Hot Air oven b) Autoclave c) Filtration d) Ultra violet rays
10. Which of the following is most resistant to sterilization
a) Tubercle bacillus b) Viruses c) Spores d) Prions

36. Answers
1. a) Louis Pasteur
2. b) Central Sterile Supplies Department
3. a) Ionising rays
4. d)Bacillus atrophaeus
5. c) Inspissation
6. b) Pasteurization
7. b) Fractional sterilisation
8. a) Cold sterilisation
9. b) Autoclave
10. d) Prions

37. Expected Questions
1) Seitz filter (5)
2) Tyndallisation (5)
3) Sterilisation by autoclave (5)
4) Bacterial filters (5)/ Sterilization by Filtration methods.
5) Dry heat sterilization (5)
6) Hot air oven (5)
7) Enumerate the methods of sterilization. Describe moist-heat methods. (15)
8) Gaseous method of sterilization (5)
9) Discuss the various methods of sterilisation including Hepa filters. (5)
10) Sterilisation by radiation (5)
11) Pasteurisation. (5)
13) Define Sterilization. Discuss the methods of sterilization by Heat. (15)
14) Cold sterilization. (5)
15) Radiation sterilization and gas sterilization. (5)
16) Define sterilization. Write in detail about CSSD. (5)
17) Explain plasma sterilization and its uses. (5)

38. Expected Questions
18) A tertiary care hospital is used to organize its regular board meetings related to
infection control on 4th Saturday of every month. Due to an increase in number of post-
surgical complications and outbreak of MRSA, the chairperson of infection control
committee suggested the lead microbiologist for the proper sterilisation methods to be
implemented. A training program is organised by the infection control team for the
same. Assuming that one group of the training team are 2nd year UGs, answer the
following questions related to sterilisation? (15)
a) Definition of sterilisation
b) Classify the methods of sterilisation
c) Describe the principles, uses, and sterilisation controls used in hospital for
sterilisation of different materials used for surgery and lab ware.
d) Expand CSSD. Mention the various compartments and functions of CSSD.
19) Draw a neat, labelled diagram of an autoclave and enumerate its uses in a CSSD.
(5)
20) Enumerate the applications of sterilisation process in a Microbilogy lab. (5)
21) Prions are resistant structures to routine methods of sterilisation. State true or false.
Justify your answer. (5)

39. Expected Questions
22) Describe in detail about the physical methods of sterilsation under the following
headings. (15)
a) Classification of physical methods of sterilisation with appropriate examples.
b) Uses of cold sterilisation.
c) Mention the specific surgical instruments sterilised in autoclave and hot air oven.
d) Importance of CSSD unit in a tertiary care hospital.
23) Describe the process and mention the culture media sterilsied by Tyndallisation. (5)