The above PPT includes different methods of sterilization- Dry heat, Moist heat, Radiation and Chemical methods. It also includes principle and working of hot air oven and autoclave.

1. STERILIZATION

2. INTRODUCTION
 A physical or chemical process that completely destroys
or removes all microbial life, including spores present in
a specified region, such as a surface, a volume of fluid
or in a compound such as biological culture media
 Sterilization can be achieved with one or more of the
following:
 heat
 chemicals
 radiation
 high pressure and
 filtration
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3. 3

4. METHODS OF STERILIZATION:
PHYSICAL
 Sterilization by Heat: Most common
method
 Dry Heat
 Simplest method is exposing the item to be
sterilized to the naked flame e.g. Bunsen
burner- for sterilizing bacteriological loops,
knives, blades.
 Heat sterilization is the most widely used
and reliable method of sterilization,
involving destruction of enzymes and other
essential cell constituents.
 It employs higher temperatures in the
range of 160-180°C and requires exposures
time up to 2 hours, depending upon the
temperature employed.
 The benefit of dry heat includes good
penetrability and non-corrosive nature
which makes it applicable for sterilizing
glass-wares and metal surgical
instruments.
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5. METHODS OF STERILISATION
 It is also used for sterilizing non-aqueous
thermo-stable liquids and thermostable
powders.
 Dry heat destroys bacterial endotoxins (or
pyrogens) which are difficult to eliminate by
other means and this property makes it
applicable for sterilizing glass bottles which are
to be filled aseptically.
 Examples of Dry heat sterilization are:
1. Incineration
2. Red heat
3. Flaming
4. Hot air oven
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6. METHODS OF STERILIZATION
 Hot-air oven :
 Dry heat sterilization is usually
carried out in a hot air oven, which
consists of the following:
(i) An insulated chamber
surrounded by an outer case
containing electric heaters.
(ii) A fan
(iii) Shelves
(iv) Thermocouples
(v) Temperature sensor
(vi) Door locking controls.
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7. HOT AIR OVEN
 Operation
(i) Articles to be sterilized are first wrapped or
enclosed in containers of cardboard, paper or
aluminium.
(ii) Then, the materials are arranged to ensure
uninterrupted air flow.
(iii) Oven may be pre-heated for materials with poor
heat conductivity.
(iv) The temperature is allowed to fall to 40°C, prior
to removal of sterilized material.
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8. 8
HOT AIR OVEN
 Principle
 Sterilizing by dry heat is accomplished by conduction.
 The heat is absorbed by the outside surface of the item, then
passes towards the centre of the item, layer by layer.
 The entire item will eventually reach the temperature required
for sterilization to take place.
 Dry heat does most of the damage by oxidizing molecules.
 The essential cell constituents are destroyed and the organism
dies.
 The temperature is maintained for almost an hour to kill the
most difficult of the resistant spores.

9. FLAMING
 This is an emergency method, the forceps-tips,
the surfaces of the scalpels and the needles
 may be sterilized by holding the items directly in
the flame of a Bunsen burner.
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10.  Advantages of dry heat sterilization
 A dry heat cabinet is easy to install and has relatively low
operating costs;
 It penetrates materials
 It is nontoxic and does not harm the environment;
 And it is noncorrosive for metal and sharp instruments.
 Disadvantages for dry heat sterilization
 Time consuming method because of slow rate of heat
penetration and microbial killing.
 High temperatures are not suitable for most materials.
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11. METHODS OF STERILIZATIONMETHODS OF STERILIZATION
Moist Heat (or Steam):
 Uses hot water.
 Moist heat kills microorganisms by denaturing proteins.
 Moist heat may be used in three forms to achieve microbial
inactivation
1. Dry saturated steam – Autoclaving
2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point
4. Pasteurisation
 Moist heat sterilization involves the use of steam in the range
of 121-134°C.
 Steam under pressure is used to generate high temperature
needed for sterilization.
 Saturated steam acts as an effective sterilizing agent.
 Steam for sterilization can be either wet saturated steam
(containing entrained water droplets) or dry saturated steam
(no entrained water droplets).
11

12.  Boiling –
 quite common especially in domestic circumstances.
 Boiling is done for metallic devices like surgical
scissors, scalpels, needles etc like instruments. Here
substances are boiled to sterilize them.
 Pasteurisation
 Pasteurization is the process of heating the milk at a
temperature of 60 degrees or 72 degrees 3 to four
times.
 Here alternative heating and cooling kills all the
microbes and molds without boiling the milk.
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13. METHODS OF STERILIZATIONMETHODS OF STERILIZATION
Moist heat: Tyndallization
 The process involves boiling for a period
(typically 20 minutes) at atmospheric pressure,
cooling, incubating for a day, boiling, cooling,
incubating for a day, boiling, cooling,
incubating for a day, and finally boiling again.
 The three incubation periods are to allow heat-
resistant spores surviving the previous boiling
period to germinate to form the heat-sensitive
vegetative (growing) stage, which can be killed
by the next boiling step.
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14. METHODS OF STERILIZATIONMETHODS OF STERILIZATION
Moist heat: Autoclaving
 Standard sterilization method in
hospitals.
 The Autoclave works under the
same principle as the pressure
cooker where water boils at
increased atmospheric pressure
i.e. because of increased pressure
the boiling point of water is
>100°C.
 The autoclave is a tough double
walled chamber in which air is
replaced by pure saturated steam
under pressure.
 Before using the autoclave,
check the drain screen at the
bottom of the chamber and clean
if blocked. If the sieve is blocked
with debris, a layer of air may
form at the bottom of the
autoclave, preventing efficient
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15. AUTOCLAVEAUTOCLAVE
 The air in the chamber is flushed out and filled with
saturated steam.
 Water is boiled to produce steam, which is released
through the jacket and into the autoclave's chamber.
 Hot, saturated steam enters the chamber and the
desired temperature and pressure, usually 121°C.
 At this temperature saturated steam destroys all
vegetative cells and endospores.
 Moist heat is thought to kill so effectively by degrading
nucleic acids and by denaturing enzymes and other
essential proteins.
 It also may disrupt cell membranes. The chamber is
closed tightly the steam keeps on filling into it and the
pressure gradually increases.
 The items to be sterilized get completely surrounded by
saturated steam (moist heat) which on contact with the
surface of material to be sterilized condenses to release
its latent heat of condensation which adds to already
raised temperature of steam so that eventually all the
microorganisms in what ever form are killed.
 The usual temperature achieved is 121 °C at a pressure
of 15 pps.ie. at exposure time of only 15-20 mins. By
increasing the temperature, the time for sterilizing is
further reduced.
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16. METHODS OF STERILIZATIONMETHODS OF STERILIZATION
Advantages of Autoclave
 Temperature is > 100°C therefore spores are killed.
 Condensation of steam generates extra heat (latent
heat of condensation).
 The condensation also allows the steam to penetrate
rapidly into porous materials.
 Note: that autoclavable items must be steam
permeable. Can not be used for items that are
lacking water. 16

17. RADIATIONRADIATION
 Electromagnetic radiation
 Gamma rays
 UV rays
 Particulate radiation
 Accelerated electrons
 The major target for these radiation is microbial DNA.
 Gamma rays and electrons cause ionization and free radical
production while UV light causes excitation.
 U.V. light has limited sterilizing power because of poor
penetration into most materials. Generally used in irradiation
of air in certain areas eg. Operating Rooms and T.B.
laboratories.
 Ionizing radiation- e.g. Gamma radiation: Source Cobalt60
has
greater energy than U.V. light, therefore more effective. Used
mainly in industrial facilities e.g. sterilization of disposable
plastic syringes, gloves, specimens containers and Petri Dishes.
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18. RADIATION
 Gamma ray Sterilizer: Gamma rays for sterilization are
usually derived from cobalt-60 source, the isotope is held as
pellets packed in metal rods, each rod carefully arranged
within the source.
 This source is housed within a reinforced concrete building
with 2 m thick walls.
 Articles being sterilized are passed through the irradiation
chamber on a conveyor belt and move around the raised
source.
 Penetrates deep into objects and is an excellent sterilizing
agent.
 It destroys bacterial endospores and vegetative cells of
both prokaryotic and eukaryotic origin but not against
viruses.
 Gamma radiation from a cobalt 60 source is used in the
cold sterilization of antibiotics, hormones, sutures and
plastic disposable supplies such as syringes, and Petri
dishes.
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19. RADIATIONRADIATION
 Ultraviolet radiation: The optimum wavelength for
UV sterilization is 260 nm. A mercury lamp giving
peak emission at 254 nm is the suitable source of UV
light in this region.
 But this does not penetrate glass, dirt films, water
and other substances very effectively.
 UV radiation is used as a sterilizing agent only in a
few specific situations, like UV lamps are placed on
the ceilings of rooms or in biological safety cabinets to
sterilize air and other exposed surfaces.
 Commercial UV units are available for water
treatment. Pathogens and microorganisms are
destroyed when a thin layer of water is passed under
the lamps (water purifiers). 19

20. INFRARED RADIATION
 Infrared radiation (IR) is a thermal radiation, i.e. when
absorbed by some article its energy is converted to heat
and therefore it is often known as radiant energy.
 A tunnel having an IR source is used for this purpose.
 The instruments and glass wares are kept in trays are
passed through this tunnel keeping on the conveyor
belt, at a controlled speed exposing them to a
temperature of 1800
C for 17 minutes, thereby achieving
the sterility. 20

21.  This is a continuous process and is used in
hospitals for regular supply of sterile syringes
and other apparatus.
 Heating at or above 2000
C by IR in vacuum is
employed as a means of sterilizing surgical
instruments.
 Cooling is hastened, (after the heating cycle)
during the cooling period, by admitting filtered
N2 to the chamber.
21

22. FILTRATIONFILTRATION
 In order to sterilize solutions which is heat sensitive,
filtration is an excellent way to reduce the microbial
population.
 The filters simply remove the microbes instead of
killing them.
 Depth filters
 Consists of fibrous or granular materials that have been
bonded into a thick layer filled with twisting channels of
small diameter.
 The solution is passed through the filter which is sucked
through this layer under vacuum and microbial cells are
removed. The material used mostly is unglazed porcelain,
asbestos or other similar materials
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23. FILTRATIONFILTRATION
 Membrane filters
 Are also used and have replaced depth filters in recent
times.
 These filters are made up of cellulose acetate, cellulose
nitrate, polycarbonate, polyvinylidene fluoride, and other
synthetic materials.
 These filters vary in size with pore sizes mostly of 0.2 to
0.5 µm in diameter and used to remove most vegetative
cells, but not viruses, from solutions ranging in volume
from 1ml to many litres.
 These filters are mostly used to sterilize
pharmaceuticals, ophthalmic solutions, culture media,
oils, antibiotics and other heat sensitive solutions.
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24. FILTRATION
 The other way this method is used is in the
laminar flow biological safety cabinets where the
air is sterilized by filtration.
 These cabinets contain high-efficiency particulate
air (HEPA) filters, which remove 99.97% of
0.3µm particles.
 The safety cabinets are most useful as the
culturing of any organisms requires
contamination free air to reduce the growth of
other undesired organisms or for the preparation
of media, examining tissue cultures etc
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25.  Phenol and Phenolics
 Halogens
 Alcohols
 Heavy Metals
 Quaternary Ammonium Compounds
 Aldehydes
 Gaseous
 Peroxygens
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26. STERILIZATION : CHEMICAL
METHODS
 Useful for heat sensitive materials e.g. plastics and lensed
instruments endoscopes).
 The sterilising agent must be stable upon storage, odorless or with a
pleasant odor, soluble in water and lipids for penetration into
microorganisms, and have a low surface tension so that it can enter
cracks in surfaces.
 Peracetic Acid liquid sterilization:
 Peracetic acid was found to be sporicidal at low concentrations.
 It was also found to be water soluble, and left no residue after
rinsing. It was also shown to have no harmful health or
environmental effects.
 It disrupts bonds in proteins and enzymes and may also interfere
with cell membrane transportation through the rupture of cell walls
and may oxidize essential enzymes and impair vital biochemical
pathways.
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27. CHEMICAL STERILISATION
 Hydrogen Peroxide Sterilization:
 This method disperses a hydrogen peroxide solution in a
vacuum chamber, creating a plasma cloud.
 This agent sterilizes by oxidizing key cellular components,
which inactivates the microorganisms.
 The plasma cloud exists only while the energy source is turned
on. When the energy source is turned off, water vapor and
oxygen are formed, resulting in no toxic residues and harmful
emissions.
 The temperature of this sterilization method is maintained in
the 40-50°C range,
 well-suited for use with heat-sensitive and moisture-sensitive
medical devices.
 The instruments are wrapped prior to sterilization, and can
either be stored or used immediately. 27

28. GASEOUS STERILIZATION
 Ethylene Oxide Sterilizer:
 Ethylene oxide gas readily penetrates packing materials, even
plastic wraps and is both microbicidal and sporicidal and kills
by combining with cell proteins.
 Ethylene oxide alkylates DNA molecules and thereby
inactivates microorganisms.
 Ethylene oxide may cause explosion if used pure so it is mixed
with an inert gas e.g. Neon, Freon at a ratio of 10:90
 It requires high humidity and is used at relative humidity 50-
60% ; Temperature : 55-60°C and exposure period 4-6 hours.
 An ethylene oxide sterilizer consists of a chamber of 100-300-
Litre capacity and surrounded by a water jacket.
 Air is removed from sterilizer by evacuation, humidification
and preheated vaporized ethylene oxide is passed.
 After treatment, the gases are evacuated either directly to the
outside atmosphere or through a special exhaust system.
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29. GASEOUS STERILIZATION
 Activated alkaline Glutaraldehyde 2%:
 Immerse item in solution for about 20 minutes if
organism is TB. In case of spores, the immersion
period is extended to 2-3 hours.
 Batapropiolacetone (BPL)
 is occasionally used as a sterilizing gas in the liquid
form to sterilize vaccines and sera.
 Recently vapour-phase hydrogen peroxide
has been used to decontaminate biological safety
cabinets.
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30. ENUMERATION OF BACTERIA- MOST PROBABLE
NUMBER (MPN ) TECHNIQUE AND MEMBRANE
FILTER TECHNIQUE
30

31. Most Probable Number & Membrane Filter
methods
 These are methods used to enumerate the numbers of
bacteria in water samples.
 The Most Probable Number method is used to check
potability (if water is safe enough to be drinking
water) of water.
 The MPN method looks for the presence of potential
pathogenic bacteria that may be in the water due to
fecal contamination of the water supply.
 Water supplies are generally derived from ground
sources and have to be checked for safety levels of
bacterial contamination
31

32.  MPN method enumerates the enteric bacteria
called coliforms, specifically fecal coliforms (E.
coli)
 Coliforms are Gram negative bacilli that have
the ability to ferment lactose with the
production of acid and gas.
 Fecal coliforms are those coliforms that are
normally found in the feces of warm blooded
animals (including humans)
 MPN method thus enumerates the fecal
coliforms in water samples.
 E. coli is thus used as an indicator organism.
32

33.  MPN test includes 3 levels of testing:
 Presumptive,
 Confirmed,
 Completed.
 The presumptive test looks for presence of fecal
coliforms in the water sample by inoculating
lactose broths with the water sample.
 Those tubes that show presence of acid and gas
are scored + and those with no acid/gas as -.
 Three sets of lactose broths are inoculated with
varying dilutions of the sample:
 First set of 3 or 5 tubes inoculated with 10ml of
sample;
 Second set of tubes inoculated with 1ml of sample;
 Third set of tubes inoculated with 0.1ml of sample. 33

34. 34

35.  The combination of positives in the 3 sets is used
to figure out the MPN /100ml of water using the
table provided.
 The tubes that show positive in the
presumptive test should be confirmed to
contain E.coli.
 This done in the confirmed test using the
selective/differential medium EMB (that
uniquely highlights E.coli growth on it).
 The completed test is done only where legal
issues are involved wherein the bacterial culture
is then identified by a full complement of test
35

36. 36

37. 37

38. MPN
Advantages Disadvantages
 Relatively simple and
sensitive
 Can count a specific
type in the presence of
others
 Can use large sample
volumes
 Time consuming and
labor intensive
 Requires large
volumes of glassware
 Doesn’t give the “real”
value
 Doesn’t give isolated
colonies
38

39. MEMBRANE FILTER TECHNIQUE
39

40. MEMBRANE FILTER TECHNIQUE
 Samples are filtered through a membrane designed
to retain bacteria
 Filter is antiseptically transferred from the filtration
apparatus and placed on either a pad saturated with
media or agar plate.
 Plates are inverted and incubated at 35°C for 22-24
hours.
 The media is heated to near boiling
 All bacteria that produce a red colony with a metallic
(golden) sheen, are considered to be members of the
coliform group. However, some non-coliform bacteria
(ie. Proteus mirabilis) can produce sheen colonies.
 The MF test requires confirmation with LTB and
BGLBB. Only colonies that ferment lactose (found in
BGLBB) can be confirmed as coliforms.
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41. 41

42. PROCEDURE
1. Collect the sample and make
any necessary dilutions.
2. Select the appropriate
nutrient or culture medium.
Dispense the broth into a
sterile Petri dish, evenly
saturating the absorbent pad.
3. Flame the forceps, and
remove the membrane from
the sterile package.
4. Place the membrane filter
into the funnel assembly.
5. Flame the pouring lip of the
sample container and pour
the sample into the funnel.
6. Turn on the vacuum and
allow the sample to draw
completely through the filter.
42

43. 7. Rinse funnel with sterile buffered
water. Turn on vacuum and allow
the liquid to draw completely
through the filter.
8. Flame the forceps and remove the
membrane filter from the funnel.
9. Place the membrane filter into the
prepared Petri dish.
10. Incubate at the proper temperature
and for the appropriate time period.
11. Count the colonies under 10 - 15 X
magnification.
12. Confirm the colonies and report the
results.
43

44.  Counting range is 20-80 colonies per membrane
 Dehydrated media must be tightly closed and
stored properly
 Prepared media must be kept in the refrigerator
unless the method allows for a different storage
temperature
 Standard Units = CFU/100 mL (Colony Forming
Units)
44

45. ADVANTAGES
 Permits testing of large sample volumes.
 Reduces preparation time as compared to many
traditional methods.
 Allows isolation and enumeration of discrete colonies
of bacteria.
 Provides presence or absence information within 24
hours.
 Effective and acceptable technique. Used to monitor
drinking water in government laboratories.
 Useful for bacterial monitoring in the pharmaceutical,
cosmetics, electronics, and food and beverage
industries.
 Allows for removal of bacteriostatic or cidal agents
that would not be removed in Pour Plate, Spread
Plate, or MPN techniques.
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