Microbiology of Sterilization

Heat sterilization is a function of probability dependent upon the number of challenge

microorganisms, the heat resistance of these microorganisms, and the amount of heat exposure.
Use of the F, D, and z-values allows sterilization engineers the ability to compare the effectiveness
of various sterilization cycles using a mathematical model. These terms are used extensively in
both the pharmaceutical and food industries and are generally well accepted by regulatory agencies
as demonstration of sterilization effectiveness. The three factors that play the most important roles
in the sterilization process are time, temperature, and resistance:
Time
The amount of time at the process temperature is the key element of the sterilization process. If the
process temperature is not constant throughout the cycle, the lethal rate accumulated over the cycle
can still be calculated.
Temperature
Consideration should be given to the temperature of the material being sterilized throughout the
entire process. Mathematical calculations are available to include those portions of the cycle which
are not at the optimal temperature but, due to their magnitude, impart some measurable lethality to
the microbial population.
Resistance
Some microorganisms have limited resistance to sterilization, but others, such as bacterial spores,
are highly resistant. This is the underlying reason why bacterial spores are usually selected as the
microbiological challenge (bioindicator) for sterilization processes.
D-Value (Decimal Reduction)
D-value is defined as the time in minutes required for a one-log, or 90%, reduction of a
specific microbial population under specified lethal conditions. For steam sterilization it is
determined at a constant temperature. There are two primary mathematical methods used for the
determination of D-value: the Survivor Curve Method and the Fraction Negative Method.
The letter D indicates that the D value is also referred to as the Decimal Reduction Time, the
word decimal being defined as a mathematical value founded on the number 10.
z-Value (Temperature Coefficient)
Although D-value determinations are carried out at a single temperature under isothermal
conditions, it has been proven experimentally that the resistance of a microorganism may change
with alterations in temperature. This change in the rate of microbial inactivation with a change
in the temperature is known as the z-value (2,3). The z-value is defined as the number of
degrees of temperature change necessary to change the D-value by a factor of 10. The z-value
allows integration of the lethal effects of heat as the temperature changes during the heating and
cooling phases of a sterilization cycle.

F-Value (Lethal Rate or Lethality Factor)

The F-value is a measurement of sterilization effectiveness. F (T, z) is defined as the equivalent
time at temperature T delivered to a container or unit of product for the purpose of
sterilization, calculated using a specific value of z. The F-value is used, along with the number of
challenge microorganisms, the type of suspending medium, the z value, and the D-value, to
evaluate the microbiological effectiveness of the sterilization cycle. When the values used are a
temperature of 121.1C and a z-value of 10 C, the process lethality can be defined as the F0-value.
The Lethal Rate can be calculated quite easily by the following formula, or can simply be
referenced from numerous publications

L=log-1(T0-Tb) /z
L=10 (T0-Tb) /z

where:

T0 = Temperature within the item being heated

Tb = Reference temperature
z = z-value of the challenge organism
F Physical : A term used to describe the delivered lethality calculated based on the physical
parameters of the cycle.
F0-value
The term F0 is defined as the number of equivalent minutes of steam sterilization at a temperature
of 121.1C delivered to a container or unit of product calculated using a z value of 10C.
Therefore, wherever a value is stated in terms of F0, it is referring to the equivalent time at
precisely 121.1C. If, for example, a cycle has a stated F0 value of 8, the sterilization effectiveness
of that cycle is equivalent to exactly 8 minutes at precisely 121.1C regardless of the process
temperature and time actually achieved in the cycle.
The lethal rate (L) can be used to determine the amount of lethality delivered in the process using
the following formula (based on the assumption that the lethal effect obtained at different
temperatures is additive):
x

F0= Lt
t 1

where:
t = 1 is the first time increment with an F0 value, and
x is the last time increment with an F0 value
and assumes Tb = 121.1C and z = 10C
F Biological :

A term used to describe the delivered lethality measured in terms of actual kill of
microorganisms on or in a BI challenge system. The F Biological - value is calculatedas
DT x LR, where DT is the D-value of the BI system at the reference temperature (T) and LR
is actual logarithmic reduction (log N0 log NF) of the BI population achieved during the
cycle.

D-Value
Most of the moist heat resistant microbiological spores known are found in two genera:
Clostridium, which is usually a strict anaerobe, and Bacillus, an aerobe that may be facultative
anaerobic. The most commonly used species are Bacillus subtilis, Geobacillus stearothermophilus,
Bacillus coagulans (ATCC51232), Clostridium sporogenes, and Clostridium
thermosaccharolyticum.

Factors Affecting Sterility

Sterility is a probability function and the factors that affect it can be expressed by the following
simple equation:
PNSU = N0 - DR
PNSU Probability of Non-Sterile Unit
N0 the pre-sterilization microbial population level
DR the decimal reduction that occurs during a sterilization process
The equation shows that sterility depends upon control of the pre-sterilization microbial load
(bioburden), as well as the extent of bioburden inactivation that occurs during the sterilization
process.
Proving experimentally that microbial contamination has not occurred cannot be accomplished
with any degree of certainty.

Calculation of Process Lethality

x

F 0 = Lt
t 1

t = 1 is the first time increment with an F0 value, and

x is the last time increment with an F0 value
NF or PNSU or SAL
log NF = F(T,z)/DT + log N0

Rearranging for F

F(T,z) = (log N0 log NF ) x DT

N0 = initial microbial level (BI or bioburden)
NF = PNSU or SAL post-sterilization microbial level
DT = BI or bioburden resistance value
F(T,z) = Lethality (calculated or recorded)
Also.
log N0 = log NF + F(T,z)/DT