STERILIZATION AND DISINFECTION

Training Objective
 At the end of this module, participants will be able to:
– Define Decontamination, Cleaning, and Disinfectant:

– Define and Differentiate Sterilization and Disinfection

– Understand and discusses methods of Sterilization

– Identify different types of disinfectants

 Definition of Terms
• Sterilization: A physical or chemical process that completely
destroys or removes all microbial life, including bacterial
spores.

• Disinfection: it is a process which reduces the number of

viable microorganisms to an acceptable level (no longer cause
disease) but may not inactivate some viruses and bacterial
spores.

• Disinfectant: Products used to kill microorganisms on

inanimate objects or surfaces.
 Definition of Terms Cont’d…
 Cleaning - It is a process which removes visible
contamination but does not necessarily destroy micro
organisms. It is necessary prerequisite for effective
disinfection or sterilization.

 Decontamination - process that removes pathogenic

microorganisms from an object to make it safe to handle.
 Introduction
Sterilization
To achieve and maintain sterilization of any instrument,
three definite stages are to be completed
 Pre sterilization cleaning: Manual or mechanical removal
of the organic matters (blood, saliva, feces, debris, etc)
which provide protective barrier for microorganisms and
prevents its destruction.
 Sterilization process: method and efficiency used for /
while sterilization
 Aseptic storage: maintenance of sterility during
transportation and storage is of utmost importance.
 Factors which influence the effectiveness
of Sterilization
 Number of Microbes: The more microbes present, the more
time it takes to eliminate population.
 Type of Microbes: Endospores are very difficult to destroy.
Vegetative
pathogens vary widely in susceptibility to different methods
of microbial control.
 Environmental influences: Presence of organic
material (blood, feces, saliva, pH etc.) tends
to inhibit antimicrobials.
 Time of Exposure: Chemicals & radiation are
more effective at longer times. In heat treatments,
longer exposure compensates for lower temperatures.
 Methods of Sterilization
I. Physical methods
1) Heat (Dry , Moist)
2) Filtration
3) Radiation (ionizing and non-ionizing)

II. Chemical methods (Ethylene Oxide &

Glutaraldehyde)
 1) Heat Sterilization
 Most common and one of the most effective methods of
sterilization.
 Factors influencing sterilization by heat are:-
i. Nature of the heat (Dry, Moist)
ii. Temperature & time
iii. Number of organisms present
iv. Presence or absence of bacterial spore
v. Type of material from which organism is to be
eradicated
 Heat sterilization ….Cont’d
• There are two types of Heat Sterilization
– Dry heat sterilization
– Moist Heat

Dry heat sterilization

 killing is due to :
 Dehydration and oxidation of organisms
 Protein denaturation and toxic effects of
elevated levels of electrolytes
 Dry heat sterilization
Kinds of Dry Heat sterilization
i) Hot air oven : is used to sterilize items, which do not get
damaged by high temperature, such as laboratory glass,
flasks, instruments with sharp cutting edges, etc.

Holding temperature & time of exposure are crucial

The spores of non-toxigenic strain of Bacillus subtilis

and Clostridium tetani are used as a microbiological

test (control) of dry heat sterilization.
 Dry heat sterilization…..Cont’d
ii) Flaming (Red Heat) : It is used to sterilize metallic
objects by holding them in a flame like bunsen burner
till they are red hot.
►Example : inoculating wires, needles, forceps etc.
III) Incineration:- is an excellent method of destroying
materials
such as contaminated cloth, animal carcasses,
pathological materials and etc which are no more
needed.
 B) Moist heat sterilization
 Principle: 1st exposure kills all vegetative bacteria &
spores, since they are in a favorable medium, will
germinate and be killed on subsequent occasions.

 The procedure was historically used for media

preparation .

 Causes denaturation and coagulation of proteins for

killing.
B) Moist heat sterilization Cont’d…
Kinds of moist Heat sterilization
1.Pasteurization: Heating at temperatures sufficient to inactivate harmful
organism in milk and other liquids. Does not achieve sterilization.
 The temperature employed is either 630C for 30mins (Holder
method) or720C for 15-20 seconds (Flash method) followed by
cooling.
 Is used for heat sensitive liquids, milk and pharmaceutical products.
 B) Moist heat sterilization Cont’d ….
2. Boiling: Heating to 100oC or more.
 Kills vegetative forms of the pathogens within
10 minutes or less.
 Endospores and some viruses are not
destroyed this quickly; may not achieve
sterilization as such.
B) Moist heat sterilization….Cont’d
3. Tyndallisation or intermittent sterilization:
named after John Tyndall.
 Exposure of 1000C for 20 min for 3 successive days.
 The process involves boiling for a period (typically 20
minutes) at atmospheric pressure (100oC), cooling and
incubating for a day and repeating for a 3 successive
days and finally boiling again.
 B) Moist heat sterilization….Cont’d
4. Autoclaving (Steam under pressure)
 Autoclave is a tough double walled chamber
in which air is replaced by pure saturated
steam under pressure which was invented by
Charles Chamberland in1879.

 Autoclaving is the most effective means of

sterilization and more effective than dry heat
in general, because of its:
 High penetrating capacity and large amount
of heat to a surface with which it comes in
contact.
B) Moist heat sterilization….Cont’d
 Denaturation and coagulation ability of
microbial proteins.

 To achieve sterility, a holding time of 15

minutes at 121 °C or 3 minutes at 134 °C at 15
psi (pressure in pound per square inch or 100
kPa- kilo pascal) above atmospheric pressure
is required.
B) Moist heat sterilization….Cont’d
 Indicators use to verify moist heat Sterilization
 Physical Indicator: an alloy designed to melt only
after being subjected to relevant holding time.
 Chemical indicator- Strips or tapes that change color
once the correct sterilization conditions have been
met.
 Biological indicator- Spores of Bacillus
stearothermophilus are used as the test organisms
as it is toughest organism for an autoclave to destroy.
• Its spores require an exposure of 15 mins at
121oc to be destroyed.
 2. FILTRATION
Generally removes most bacteria but viruses and some
small bacteria like Spirochetes , Chlamydia & Mycoplasmas
may pass through.

Used mainly for heat sensitive materials like vaccines,

sera, enzymes, antibiotics, and some culture media.
 2. FILTRATION Cont’d…
 Commonly used filter size that exclude smallest
bacterial cells is 0.22 um diameter.

 Currently, 0.01 um pore size filters are available,

that retain viruses and some large proteins.

 known example is the membrane filter made from

cellulose acetate.
 3. RADIATION

 Radiation can be used to kill or inactivate microorganisms.

 Two types radiation used for sterilization
A) Ionizing radiation: e.g., X-rays, gamma rays, and high
speed electrons .
B) Non-ionizing radiation:, e.g. ultraviolet light, and infrared
light.
 3. RADIATION Cont’d…
A) Ionizing Radiation
 X-rays, gamma rays and cosmic rays are highly lethal to DNA
and other vital constituents due to high penetration power.
 There is no appreciable increase in temperature, thus referred
to as cold sterilization.
 Has greater energy than U.V. light, therefore more effective.
 Used mainly in industrial facilities for sterilization of
disposable items like plastics, syringes, gloves, specimens
containers, Petri Dishes, swabs, catheters, etc.
 3. RADIATION Cont’d…
B) Non-ionizing radiation
 Two types of non-ionizing radiations used for sterilization:-
1) Ultraviolet
 Destroys microbial DNA.
 However, has limited sterilizing power because of poor
penetration into most materials.

2) Infrared
 Infrared is effective, however, it has no penetrating ability.
 It is most commonly used to purify air
 Sterilization by Chemical Methods
Effective against all types of microorganisms including viruses
and spores, but needs lengthy time for effectiveness.
A) Ethylene oxide (gaseous) sterilization
 Colorless, highly penetrating gas with a sweet ethereal
smell.
 Useful for sterilizing heat and moisture sensitive materials
 inactivate microorganisms.
 Highly flammable & in concentration greater than 3%,
highly explosive.
 II) Sterilization by Chemical Methods Cont’d….
 It requires high relative humidity (50-60%) and temperature
of 55-60°C with exposure period of 3-5 hours.
 Its disadvantages include lengthy cycle time, cost &
potential hazards to staff.

B) Glutaraldehyde ( 2%) sterilization

 Immerse item in solution for about 20 minutes if organism
is TB.
 In case of spores, the immersion period is extended to 4-6
hours.
 Disinfection
Process that eliminates / reduces defined
pathogens, but not necessarily spores
Categorized into 3 levels:
 High level,
 Intermediate level
 Low level
 Disinfection Cont’d…
Levels of (types) of disinfection
High-level disinfection: can be expected to destroy all
microorganisms, with the exception of large numbers of
bacterial spores.
Intermediate disinfection: inactivates Mycobacterium
tuberculosis, vegetative bacteria, most viruses, and most
fungi; does not necessarily kill bacterial spores.
 Low-level disinfection: can kill most bacteria, some
viruses, and some fungi; cannot be relied on to kill resistant
microorganisms such as tubercle bacilli or bacterial spores.
 Disinfection Cont’d…
 Level of Resistance of Microorganisms to disinfection

Mechanism of disinfecting or killing

Spores
Sterilization
bacterial, fungal
In increasing order of resistance

Mycobacteria, TB bacilli High Level Disinfection

Hydrophilic viruses Intermediate Disinfection

Vegetative fungi & bacteria Low Disinfection

Lipophilic viruses
 Disinfection…….Cont’d
Factors influencing effectiveness of Disinfectants:
 Temperature Concentration of the disinfectant

 Time
 Range or span of action
 Nature of the organism
 Level of cleanliness
 Disinfection Cont’d…
How chemical disinfectants work
The mechanism of action of most of the chemicals
are nonspecific and complex but most of them
effect microorganisms by one of the following
mechanisms.
1.Cell membrane injury.
2.Coagulation and Denaturation of proteins.
3.Interactions with functional groups of proteins.
 Types of Disinfectants
1. Phenol and Phenolics:
 Phenol (carbolic acid) was first used by Lister as a
disinfectant.
 Rarely used today because it is a skin irritant and has
strong odor.
 Phenolics are chemical derivatives of phenol
 Cresols (Lysol): Derived from coal tar.
 Biphenols: Effective against gram-positive staphylococci
and streptococci.

Advantages: Stable, persist for long times after applied, and

remain active in the presence of organic compounds.
 Types of Disinfectants Cont’d…
2. Halogens: are effective disinfectants.
A. Iodine:
 Iodine tincture (alcohol solution) was one of first
antiseptics used.

B. Chlorine:
– Used to disinfect drinking water, pools, and
sewage.
 Types of Disinfectants Cont’d…
3. Alcohols:
 Kill bacteria, fungi, but not endospores or naked viruses.
 Act by denaturing proteins and disrupting cell
membranes.
 Used to mechanically wipe microbes off skin before
injections or blood drawing.
 Not good for open wounds, because cause proteins to
coagulate.
 Ethanol (drinking alcohol): Optimum concentration is 70%.
 Isopropanol (Rubbing alcohol): Better disinfectant than
ethanol. Also cheaper and less volatile.
 Types of Disinfectant Cont’d…
4. Quaternary Ammonium Compounds (Quats):
 Cationic (positively charge) detergents.
 Effective against gram positive bacteria, less effective against
gram-negative bacteria.
5. Aldehydes:
 Include some of the most effective antimicrobials.
 Inactivate proteins by forming covalent crosslinks with several
functional groups
 Types of Disinfectants…….Cont’d
4. Quaternary Ammonium Compounds (Quats):
 Cationic (positively charge) detergents.
 Effective against gram positive bacteria, less effective against
gram-negative bacteria.
5. Aldehydes:
 Include some of the most effective antimicrobials.
 Inactivate proteins by forming covalent crosslinks with several
functional groups.
– Formaldehyde:
– Glutaraldehyde:
 Types of Disinfectants…….Cont’d
6. Gaseous Disinfectant/Sterilizers:
 Chemicals that sterilize in a chamber similar to an
autoclave.
 Denature proteins, by replacing functional groups with
alkyl groups.
Eg: Ethylene Oxide:
 Kills all microbes and endospores, but requires exposure of
4 to 18 hours.
 Types of Disinfectants…….Cont’d
7. Oxidizing Agents:
Oxidize cellular components of treated microbes.
 Disrupt membranes and proteins.

A. Ozone:
Used along with chlorine to disinfect water.
Helps neutralize unpleasant tastes and odors.
More effective killing agent than chlorine, but less stable and more
expensive.

B. Hydrogen Peroxide:
Effective in disinfection of inanimate objects,
It can be vaporize and used for decontamination.
 Summary
To achieve and maintain sterilization of any
instrument, three definite stages should be
completed (Pre sterilization cleaning, Sterilization
process and aseptic storage).

• Methods of Sterilization is classified as Physical

methods and Chemical methods.
Any comment, suggestion or Questions?