STERILIZATION BY

PHYSICAL
METHODS

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 Why we need Sterilization
• Microorganisms capable of causing infection are
constantly present in the external environment and on
the human body.
• Microorganisms are responsible for contamination and
infection.
• The aim of sterilisation is to remove or destroy them
from materials or from surfaces.

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 How can microorganisms
be killed?
1 Denaturation of proteins (e.g. wet heat, ethylene oxide)
2 Oxidation (e.g. dry heat, hydrogen peroxide)
3 Filtration
4 Interruption of DNA synthesis/repair (e.g. radiation)
5 Interference with protein synthesis (e.g. bleach)
6 Disruption of cell membranes (e.g. phenols)

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 Classification
There are two types of 2. Chemical sterilization includes:
sterilization: physical and  Alcohols
chemical.
 Aldehydes
1. Physical sterilization
 Phenolics
includes:
  Oxidizing agents
heat
  Quaternary ammonium compou
radiation nds
 filtration
 ethylene oxide gas
 Others

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 Definitions:
Sterilisation :
– It is a process by which an article, surface or medium is made
free of all microorganisms either in vegetative or spore form.
Disinfection :
– Destruction of all pathogens or organisms capable of
producing infections but not necessarily spores.
– All organisms may not be killed but the number is reduced to
a level that is no longer harmful to health.

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 Antiseptics :
Antiseptics :
– Chemical disinfectants which can safely applied to
living tissues and are used to prevent infection by
inhibiting the growth of microorganisms.
Asepsis :
– Technique by which the occurrence of infection into an
uninfected tissue is prevented.

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 Factors that influence efficacy of
disinfection/sterilization
1 Contact time
2 Physico-chemical environment (e.g. pH)
3 Presence of organic material
4 Temperature
5 Type of microorganism
6 Number of microorganisms
7 Material composition
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 Uses of sterilisation:
1. Sterilisation of materials, instruments
used in surgical and diagnostic
procedures.
2. Sterilisation of Media and reagents used
in the microbiology laboratory.
3. Food and drug manufacturing to ensure
safety from contaminating organisms.
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 Understanding the Terminology
• a suffix indicating that the antimicrobial agent will kill or destroy a certain group of
microorganism
• suffix “cide” – meaning to kill
• viricide – destroys virus
• fungicide – destroys fungi
• bactericide – destroys bacteria
• Suffix “static/stasis” – meaning to stand still
• a suffix indicating that the agent will prevent the growth or multiplication of the
type of organism but are not killed outright
• bacteriostatic - prevents the growth of bacteria
• fungi static – prevents the growth of fungi

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 Relative Resistance of Microbial
Forms
Highest resistance Moderate resistance Least resistance

bacterial endospore protozoan cyst most bacterial vegetative cells

(Bacillus & Clostridium) some fungal spores ordinary fungal spores & hypae
some naked virus enveloped virus
vegetative bacteria that Yeasts
have higher resistance Trophozoites
( M. tuberculosis, S.aureus,
Pseudomonas)

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 What to sterilize?
• It is mandatory to sterilize :
– all instruments that penetrate soft tissues and bone.
– Instruments that are not intended to penetrate the
tissues, but that may come into contact with oral
tissues.
• If the sterilization procedure may damage the
instruments, then, sterilization can be replaced by
Disinfection procedure
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 Ideal sterilization/disinfection process
• Highly efficacious
• Fast
• Good penetrability
• Compatible with all materials
• Non-toxic
• Effective despite presence of organic material
• Difficult to make significant mistakes in process
• Easily monitored
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 Control of Microbial Growth:
Rate of Microbial Death
When bacterial populations are heated or treated with an
antimicrobial chemicals, they usually die at a constant rate.

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 Figure 9.1 A plot of microbial death rate

Number of living microbes

90% die Constant percentage

of the extant population
1 min is killed each minute

90% die

1 min

Time (min)
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 Methods
1.Physical
methods
2.Chemical
methods
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 Physical methods:
• Physical methods:
1. Sunlight
2. Heat
a. Dry heat
b. Moist heat
3. Filtration
4. Radiation
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 Chemical methods
• Chemical methods:
1. Alcohols
2. Aldehydes
3. Phenols
4. Halogens
5. Oxidizing agents
6. Salts
7. Surface active agents
8. Dyes
9. Vapor phase disinfectants

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 Physical Methods

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 How to Sterilize
Materials Method
1 Inoculating wires and loops Red heat
2 Glass ware- syringes, petridishes, testtubes, flasks etc. Hot –air oven

3 Disposable syringes, and other disposable items Gamma radiation

4 Culture media Autoclaving

5 Culture media containing serum and egg Tyndallisation
6 Toxin , serum, sugar, and antibiotic solutions Filtration
7 Cystoscope and endoscope Glutaraldehyde

8 Infected soiled dressings Incineration

9 Skin Iodine, alcohol
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10 Milk Pasteurisation
 Physical Methods of Microbial Control
Dry Heat:
 Direct Flaming: Used to sterilize inoculating loops
and needles. Heat metal until it has a red glow.
 Incineration: Effective way to sterilize disposable items (paper
cups, dressings) and biological waste.
 Hot Air Sterilization: Place objects in an oven. Require 2 hours
at 170oC for sterilization. Dry heat is transfers heat less
effectively to a cool body, than moist heat.

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 Physical Methods of Microbial Control
• Heat-Related Methods
– Moist heat
• Pasteurization
– Used for milk, ice cream, yogurt, and fruit juices
– Not sterilization
» Heat-tolerant microbes survive
– Pasteurization of milk
» Batch method
» Flash pasteurization
» Ultrahigh-temperature pasteurization
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© 2012 Pearson Education Inc.
 Physical Methods of Microbial Control
Moist Heat (Continued):
 Pasteurization: Developed by Louis Pasteur to prevent the
spoilage of beverages. Used to reduce microbes
responsible for spoilage of beer, milk, wine, juices, etc.
 Classic Method of Pasteurization: Milk was exposed to 65 oC
for 30 minutes.
 High Temperature Short Time Pasteurization (HTST): Used
today. Milk is exposed to 72oC for 15 seconds.

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 Inspissation:
1. Inspissation:
 Heating at 80-85°C for half an hour daily on three
consecutive days
 Serum or egg media are sterilised
2. Vaccine bath:
 Heating at 60°C for an hour daily in vaccine bath
for several successive days.
 Serum or body fluids can be sterilised by heating
at 56°C for an hour daily for several successive
days.
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 Sun light:
• Sun light:
– Active germicidal effect
due to its content of
ultraviolet rays .
– Natural method of
sterilisation of water in
tanks, rivers and lakes.

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 Heat :
• Factors influencing:
• Nature of heat
• Temperature and duration
• Characteristic of organism and spores
• Type of material

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 Heat effectively kills Majority of
Microbes
Heat :
• Principle:
– Dry heat kills the organism by
• denaturation of the bacterial proteins,
• oxidative damage
• toxic effect of elevated levels of electrolytes.

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 Heat :
• Dry heat:
1.Red heat
2.Flaming
3.Incineration
4.Hot air oven
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 Dry-Heat Sterilization
• Involves heating at atmospheric pressure and
often use a fan to obtain uniform temperature by
circulation.
• Heat at 180º for half hour , 170º for 1 hr., or 160º
C for 2 hrs.
• Times are the periods during which object is
maintained at the respective temp.
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 Dry heat:
• Dry heat:
1. Red heat: Materials
are held in the flame of
a bunsen burner till
they become red hot.
» Inoculating wires or
loops
» Tips of forceps
» Needles
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 Dry heat:
• Dry heat:
2. Flaming: Materials are
passed through the flame
of a bunsen burner
without allowing them to
become red hot.
» Glass slides
» scalpels
» Mouths of culture
tubes
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 Incineration:
• Materials are reduced to
ashes by burning.
• Instrument used was
incinerator.
• Soiled dressings
• Animal carcasses
• Bedding
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• Pathological material
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 Dry-Heat Sterilization
Disadvantages
• Disadvantages:
– Less reliable than autoclaving
– Large temp difference may arise within device.
– sharp instruments get dulled
– Many materials do not tolerate dry heat

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 Hot air oven:
• Most widely used method
• Electrically heated and fitted with a fan to even
distribution of air in the chamber.
• Fitted with a thermostat that maintains the chamber
air at a chosen temperature.
• Temperature and time:
» 160 C for 2 hours.
» 170 C for 1 hour
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» 180 C for 30 minutes.
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 Uses of Hot Air Oven
– Sterilisation of
1.Glassware like glass syringes, petri
dishes, pipettes and test tubes.
2.Surgical instruments like scalpels,
scissors, forceps etc.
3.Chemicals like liquid paraffin, fats etc.

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 – Precautions :
1. Should not be overloaded
2. Arranged in a manner which allows free circulation of air
3. Material to be sterilized should be perfectly dry.
4. Test tubes, flasks etc. should be fitted with cotton plugs.
5. petridishes and pipetts should be wrapped in paper.
6. Rubber materials and inflammable materials should not be
kept inside.
7. The oven must be allowed to cool for two hours before
opening, since glass ware may crack by sudden cooling.

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THANK YOU