STERILIZATION

Define the term sterilization?

• The complete removal of all forms of microorganisms (both vegetative and spore forms) from
a surface or an object by physical or chemical methods
• Mainly due to: oxidation of cell component, denaturation (proteins, nucleic acids, RNA) and
loss of membrane permeability.
• There is no single sterilization process that is suitable for all type of product and there is
diversity in sterilization
The Association for the Advancement of Medical Instrumentation (AAMI) defines sterilization
as: A process designed to remove or destroy all viable forms of microbial life, including
bacterial spores, to achieve an acceptable sterility assurance level of (SAL) of 10^-6 is
appropriate for items intended to come into contact with compromised tissue, which has lost
the integrity of natural body barriers. This would include sterile body cavities, tissues and
vascular system
• Sterility assurance level (SAL) of 10^-6 means: that there is less than or equal to one chance
in a million that a particular item is contaminated or unsterile following a sterilization process.
Uses of sterilization
1- sterilization for surgical procedures and medicines :gloves, surgical instruments, syringes,
drugs and other supplies etc
2-sterilization in microbiological works : preparation of culture media, reagents and equipment

The major sterilizing agents commonly used in healthcare facilities today are
a) saturated steam (autoclave), b) ethylene oxide gas, c) hydrogen peroxide gas plasma, and d)
liquid chemicals. Dry heat is also used, although less commonly. And a new sterilizing agent,
ozone, has recently become available for use
Factors affecting sterilization effectiveness: Sterilization requires contact, time,
temperature and with steam sterilization requires high pressure
• The effectiveness depends on 4 factors: Order of Resistance
1- The type of microorganism present: (according
to order of resistance)
2- The number of microorganisms present
3-The amount and type of organic material
that protects the microorganisms.
4- The number of cracks and crevices on
an instrument that might harbor microorganisms.
• What’s Been Done Wrong in sterilization ?
• We fixate on killing the most resistant microbe we can imagine. This results in over-treatment
of many items especially product, media & elastomeric materials.
• This creates numerous problems including; degradation of product, loss of flexibility, loss of
integrity, increased particles, change in essential properties, changes in color, etc.
 Physical method
 Sunlight:
• The microbicidal activity of sunlight is mainly due to the presence of ultraviolet rays in it. it is
responsible for spontaneous sterilization in natural conditions.
• In tropical countries, the sunlight is more effective in killing germs, due to combination of
ultraviolet rays and heat but it is not sporicidal
• Sun light kills bacteria in suspended in. it provides natural method of disinfection of water
bodies such as tanks and lakes.
 Heat :
• Heat sterilization is the most widely used and reliable method of sterilization, involving
destruction of enzymes and other essential cell constituents.
• This method of sterilization can be applied only to the thermostable products
A- Dry heat sterilization: No water (170℃ for 1 hour or 160 ℃ for 2 hours)
• Advantages of dry heat: it has good penetrability and non-corrosive nature
• Uses: for sterilizing glass-wares and metal surgical instruments and for sterilizing non-aqueous
thermo-stable liquids (e.g. Oils, glycerin) and thermostable powders and it applicable for
sterilizing glass bottles which are to be filled aseptically.
• Mechanism: Dry heat kill the organisms using the destructive oxidation method. This helps
destroy large contaminating bio-molecules such as proteins. The temperature is maintained for
almost an hour to kill the most difficult of the resistant spores
• Examples of Dry heat sterilization are:
1. Red heat 2. Flaming 3. Incineration 4. Hot air oven 5- Infra red ray's tunnel
 expose items to 160 °C for 1 hour.

As ointments and powder

B- Moist heat sterilization (steam sterilization)
Classified into : 1- Temperature <100 ℃ 2-Temperature of 100 ℃ 3-Temperature >100 ℃
1) Temperature >100 ℃ ( Autoclave)
• It is one of the best methods of sterilization in hospitals.
• Moist heat sterilization is done with the help of an instrument called an autoclave.
• An autoclave works under the same principle as the pressure cooker where water boils at
increased atmospheric pressure ,because of increased pressure the boiling point of water is
>100 °C. (the principle of producing steam under pressure).
The water is boiled in an autoclave at 121-134℃ at a pressure of 15psi(temperature at 121℃for
15 minutes or 134 ℃ for 10 minutes)
 Mechanism of action of autoclave: Moist heat destroys microorganisms by the irreversible
coagulation and denaturation of enzymes and structural proteins
• The autoclave is a tough double walled chamber in which air is replaced by pure saturated steam
under pressure.
• Air in the chamber is evacuated and filled with saturated steam. The chamber is closed tightly, the
steam keeps on filling into it and the pressure gradually increases.
• The items to be sterilized 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
(the amount of heat released when liquid droplets formed at a fixed temperature)which adds to
already raised temperature of steam so that all the microorganisms and spores are killed.
 Advantages of Autoclaves: 1-Temp > 100 ℃ therefore spores killed. 2-Condensation of steam
generates extra heat and allows the steam to penetrate into porous materials.
Note: Autoclavable items must be used for invasive procedures in operating room , dental
equipment , and for thermostable aqueous injections in final sealed containers (ampoules or vials)
and eye drops, surgical dressing and microbiological media
• Autoclave not suitable for : oils and powders
Autoclave
Monitoring of autoclaves
1. physical method: use thermocouple to measure accurately the temperature.
2. chemical method : use heat sensitive chemical that changes color at the right temperature and
exposure time e.g. a) autoclave tape ( commonly used) b) browne’s tube.
3. biological method : spore test, a kit containing a spore of Bacillus stearothermophilusis added
during the sterilization process ,then cultured later on to ensure that spores have been killed.
2-

Pasteurized milk has

no phosphatase
enzyme and (-ve with
Methylene blue)
the process of increasing the viscosity of a fluid, or even of causing it to solidify
3-
Survivor curves
• When populations of microorganisms are exposed to a killing process (heated or treated with
antimicrobial chemicals), they are generally killed in an exponential fashion (die at a constant
rate), independent of the initial number of organisms
• This can be represented graphically by plotting log survivors against exposure time ( Three
types of curves are obtained, all having a linear portion)
• (A) : Continuous linearity
• (B) : Initial shoulder, is due to increase in number of
cells through germination or activation of spores
(if present)
• (C): Biphasic, irregular tailing is due to presence of
resistant mutants Figure. Typical survivor curves for bacterial spores
exposed to moist heat or gamma radiation.
Expressions of resistance to sterilization
Thermal death point ( lowest temperature required to kill all microorganisms in suspension
within certain time (usually 10 min).
Thermal death time (TDT) (extinction time): shortest time required to kill all microorganisms
in suspension at certain temperature.
Decimal reduction time (D value): The time taken at a fixed temperature, or a radiation dose
required to achieve a 90% reduction in viable cells (i.e. a 1 log cycle reduction in survivors).
It could be used as a measure for the degree of resistance of an organism at particular
temperature or a radiation dose.
• In other words, for each minute the treatment is applied, 90% of the remaining population is
killed. If the death curve is plotted logarithmically, the death rate is constant.
For example, suppose a population of 1 million microbes has been treated for 1 minute, and
90 % of the population has died. we are now left with 100,000 microbes. if the population is
treated for another minute, 90% of those microbes die, and we are left with 10,000 survivors.
• Z value: the increase in temperature needed to reduce the d value of an organism by 90 % (i.e.
1 log cycle reduction). it assess the influence of temperature changes on thermal resistance.
THANKS