Evaluation of sterilization methods is carried out to ensure that the product produced by the design process is of the best quality. Evaluating sterilization methods provides a high degree of assurance that indicates whether a specific process will consistently produce a product that meets its predetermined specifications and quality assurance. So this action proves that any procedure, process, equipments, material activity, or system actually leads to the expected result and produces quality product.
Process of Microbial Destruction:
Destruction of microbes is carried out by several methods
such as heat, chemical, and radiation sterilization. Upon exposure to such
treatment, microorganisms die according to the logarithmic relationship between
the concentration or population of the living cells and the time exposure or
radiation dose. The relationship between microbial population and time may be
linear or non-linear. The D value or time required or dose required for one log
reduction in microbial population is calculated from the plots (Fig).
Fig: D Value curve
D value:
It is the rate of killing of microorganisms. It is used to
determine the time of contact for any sterilization process. It determines the
time required to reduce the microbial population by one decimal point i.e. it
is the time required for 90% reduction in the microbial population. Hence, the
time or dose it takes to reduce a thousand microbial cells to a hundred cells
is the D value.
Importance of D value validation:
• It is specific for each microorganism in an environment
subjected to a specific sterilizing agent or condition.
• The knowledge of the D value at different temperatures in
heat sterilization is necessary for the calculation of the Z value.
• The D value is used in the calculation of biological
factor F.
• Extrapolation of the D value predicts a number of log
reductions of the microbial population.
Factors affecting D value:
• The type of microorganism used as a biological indicator.
• The formulation component and characteristics.
• The surface on which the microorganism is exposed.
• The temperature, gas concentration, and dose of radiation.
D value is determined by:
• Survival curve method: The survival curve method is
based on plotting the log number of the surviving organism versus independent
variables such as time, gas concentration, or radiation dose.
• Fraction negative method: In this method, samples
containing similar spore populations are treated in an identical environment
and the number of samples still showing microbial growth after treatment and
incubation are determined. Data obtained by the survival curve method are
plotted semi-logarithmically.
Data points are connected by least square analysis.
Log N = a + bt
Where N is the number of the surviving organisms, t is time,
a is γ intercept and b is the slope of the line as determined by linear
regression.
The d value is the reciprocal of linear slope D = 1/ b.
Z value:
This value is used in the validation of the heat
sterilization process. The Z value is the reciprocal of the slope resulting
from the plot of the logarithm of the D value versus the temperature at which
the D value was obtained. The Z value may be defined as the temperature
required for one log reduction in the D value. The accepted standard (Z value)
for steam sterilization of Bacillus stearothermophilus spores and dried heat
sterilization for Bacillus subtilis are 10°C and 22°C respectively. These plots
are important because one can determine the D value of the indicator
microorganism at any temperature of interest. The magnitude of the slope
indicates the relative degree of lethality as temperature is increased or
decreased (Fig).
Fig: Z-value determination
F value:
It is the time in minutes at a specific temperature needed
to kill a population of cells or spores. The F value measures equivalent time,
not clock time that a monitored article is exposed to the desired temperature
e.g. 121.1oC. Mathematically, the F value is expressed by the rate
of motility per minute in function of temperature for a given pressure.
F value is calculated from the following equation:
F = ∆t ∑10(T−To)/Z
Where ∆t is the time interval for the measurement of product
temperature t, T is reference temperature To is 121.1oC for steam
sterilization.
Example: Food heated at 121.1°C for 2 minutes will give a value of F = 2. To get the same F-value of 2 using 111.1°C, the food needs to heat for 20 minutes.