This document provides an overview of sterilization principles and methods. It defines key terms like sterility, sterilization, and aseptic processing. It describes various sterilization methods including moist heat, dry heat, chemicals, and radiation. It outlines sterilization criteria used to evaluate effectiveness, including death/survival rates, D values, inactivation factors, death rate constants, Z values, Q values, and F values. Finally, it discusses sterilization validation and monitoring, covering the use of biological and chemical indicators.

1. Sterilization

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Outlines
Principles of Sterilization: Introduction
Methods of Sterilization: An introduction
Sterilization Criteria
Sterilization Validation and Monitoring

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I. Principles of Sterilization

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Introduction
◼ Sterility:
The total absence of viable microorganisms and it is an
absolute state
◼ Sterilization:
The inactivation or elimination of all viable
microorganisms and their spores, based on a probability
function. The sterilization process is usually the final
stage in the preparation of the product.
◼ Aseptic Processing:
Those operations performed between the sterilization of
an object or preparation and the final sealing of its
package. These operations are carried out in the
complete absence of microorganisms.
Definitions and Terminologies

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Introduction …(cont.)
◼ Disinfection:
A process which aims to reduce the number of harmful
(pathogenic) microorganisms in a particular situation. It is not
an absolute process i.e. will eradicate infective vegetative
organisms but not spores.
◼ Antiseptics:
Chemical substances applied to living tissues in humans or
animals in order to arrest or prevent the growth of
microorganisms by inhibiting their activity without necessarily
destroying them.
◼ Bactericide:
Any agent that destroys microorganisms

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Introduction …(cont.)
◼ Bacteriostat:
Any agent that arrests or retards the growth of microorganisms.
◼ Germicide:
Any agent that destroys microorganisms, but not necessarily
bacterial spores.
◼ Sterility Assurance Level (SAL):
A term related to the probability of finding a nonsterile unit
following a sterilization step. It usually is expressed in terms
of the negative power of 10 (i.e. 1 in 1 million = 10-6).
◼ Bioburden:
The number of viable microorganisms in or on an object or
population entering a sterilization step (usually expressed in
colony forming units per unit time).

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Introduction …(cont.)
The aim of sterilization process:
Is to destroy or eliminate microorganisms that are present on or in
an object or preparation, to make sure that this has been achieved
with an extremely high level of probability and to ensure that the
object or preparation is free from infection hazards.
The currently accepted performance target for
a sterilization process:
Is that it provide a probability of finding a nonsterile unit of less
than 1 in 1 million. That is, the process (including production,
storage, and shipment) will provide a Sterility Assurance Level
(SAL) equal to or less than 10-6.

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Contamination: General Facts
Some microbes (bacteria, molds, etc) multiply in the refrigerator,
others at temperatures as high as 60o.
Microbes vary in their oxygen requirements from strict anaerobes
that cannot tolerates oxygen to aerobes that demand it.
Slightly alkaline growth media will support the multiplication of
many organisms while others flourish in acidic environments.
Some microorganisms have the ability to use nitrogen and carbon
dioxide from the air and thus can actually multiply in distilled
water.
In general, however, most pathogenic bacteria have
rather selective cultural requirements, with optimum temperatures
of 30 to 37 and a pH of 7.0

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II. Methods of Sterilization

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Methods of Sterilization
Moist Heat Dry Heat Chemical (cold) Radiation Filtration
Saturated heat autoclaves
Superheated water autoclaves
Air oven steam autoclaves
Batch sterilizers
Continuous tunnel sterilizers
Ethylene oxide
Vaporized hydrogen peroxide
Hydrogen peroxide/steam
Other gases
Electromagnetic particulate Membranes

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III. Sterilization Criteria

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1. Death Rate or Survival Rate
Each sterilization method can be evaluated using experimentally
derived values (death and survival rates) representing the
general inactivating rates of the process.
Plotting the logarithm of surviving organisms
against time of exposure to the sterilization method.
In most instances, these data show a linear
relationship, typical first order kinetics, and suggest
that a constant proportion of contaminant
population is inactivated in any given time interval.
Based on such inactivation curves, it is possible to
drive values that represent the general inactivation
rates of the process. For example, based on such
data, it has become common to drive a decimal
reduction time or D value, which represents the
time under a stated set of sterilization exposure
conditions required to reduce a surviving microbial
population by a factor of 90%.

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1. Death Rate or Survival Rate… (cont.)
Death value (D): is the time required to reduce the population by 90% at specified
temperature. D value can be obtained from the death curve (i.e. the straight line
relationship between the log viable count of a bacterial population and time when the
population is exposed to a lethal temperature.
The graph shows three hypothetical
population of the same organism exposed
to the same killing agent (e.g. heat). Notice
that the populations A, B, and C die at the
same rate (all three lines are parallel)
although population C dies sooner than B
and B dies sooner than A because of the
initial sizes (ml of microorganism) are
smaller.

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1. Death Rate or Survival Rate…(cont.)
New Finding
It is sometimes known as Thermal death time was
found that complete killing of microorganisms is
approached by applications of 6 D values. By
extending the process to include 6 D values, most of
the remaining population is inactivated, reducing the
population of one organism surviving to one in 1
million.

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2. D Value and Inactivation Factor (IF)
From the D value for a particular combination of
organism/time/temperature an “inactivation factor”
(IF) can be calculated, e.g. if the D value of an
organism exposed to a temperature of 121 °C for 15
minutes was 2 minutes:
The IF would be 1015/2= 107.5

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3. Death Rate Constant (K)
- Microbiologist in various industries employee a value known as the death
constant (k) to compare susceptibilities of different microorganism to a
given control agent. The more rapidly a population can be sterilized, the
longer the death rate constant.
- The death rate constant is the same and independent of the initial
population size. It would only be different if were are comparing different
organisms. The death rate constant can be calculated using the following
formula:
  t
N
N
K /
)
/
(
log 0
10
=
Where:
T= Time in minutes of exposure to a killing agent
N0= Initial number of microorganisms
Nt = Final number of microorganisms after treatment

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3. Death Rate Constant (K)…(cont.)
Example
IF at time 0, the size of
population is 10,000,000
and after 3 minutes the
population is reduced to
10,000; the K value is
found to be 1.00 for
curve A. Calculate K
value for curve B and C?

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4. Z Value or Thermal Destruction Value
Z value relates the heat resistance of a microorganism
to changes in temperature. The Z value is the number
of degrees in temperature change required to produce
a 10-fold change in D value. Bacterial spores have a Z
value in the range 10-15 °C while most non sporing
organisms have Z values of 4-6 °C.

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4. Z Value or Thermal Destruction Value … (cont.)
Example:
If the D value for Bacillus stearothermophilus spores at
110 °C is 20 minutes and they have a Z value of 9 C,
this means that at 119 °C the D value would be 2.0
minutes and at 128 °C the value Z value would be
0.20 minutes.

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5. Q Value or Temperature Coefficient
Q value also gives a measure of the relative heat
resistance of different microorganisms and describes
the change in the death rate over a 10 °C change in
temperature. It does the same, like Z value, but is less
commonly used.

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6. F Values
- F value is a measure of the deadliness or lethality of the total
process of sterilization and equates heat treatment at any
particular temperature with the time in minutes at a designated
reference temperature that would be required to produce the
same lethality in an organism of stated Z value. The death rate
constant can be calculated using the following formula:
dt
F Z
Tc )
/
121
(
0 10 −
=
Where:
Tc= Load temperature at time dt
Z= 10 °C

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VI. Sterilization Validation
and Monitoring

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Indicators for Validation …(cont.)
A. Biological Indicators
- It involves incorporating a viable culture of a stated
species of microorganisms. The biological indicators
are usually used to check or monitor the efficacy of a
sterilization cycle.
- The efficacy of a biological indicator depends on:
1- Sensitivity
2- The viability of the organisms
3- The storage conditions before use and after the
incubation
4- The conditions of the culture after sterilization

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Indicators for Validation …(cont.)
A. Biological Indicators …(cont.)
Organism Sterilization Process
Bacillus subtilis Dry heat
Bacillus stearothermophilus Steam
Clostridium sporogenes Steam
Bacillus subtilis Ethylene oxide
Bacillus pumulis Ionizing radiation

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Indicators for Validation …(cont.)
B. Chemical Indicators
- They are used to indicate whether a particular
batch of product has been through a sterilization
process; they are not used to indicate whether a
specific process of sterilization was suitable or
successful.
- Disadvantage:
They undergo some physical and chemical changes
when exposed to the conditions of the sterilization
process.

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Indicators for Validation …(cont.)
B. Chemical Indicators … (cont.)
Indicator Main uses and specific feature
Browne’s tubes Color change at specified temperature for appropriate
length of time. Good for all sterilization processes.
They are not used as quantitative purposes.
Heat sensitive tape Used to steam sterilization. Tape that is heat sensitive,
which changes color after contact with steam
Chemical
degradation test
Check the degradation kinetic of a compound by U.V.
spectrophotometry. It is a useful technique for moist
heat sterilization
Indicator for
ethylene oxide
Change in color upon exposure to a given time and
concentration of ethylene oxide
Chemical dosimeters Monitor quantity of the radiation dose in radiation
sterilization.