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Steam autoclaves generally follow the principles shown in Figure 1. Water is heated and
the steam pressure is built up in the jacket. Once sufficient pressure is obtained, the
steam is allowed to enter the chamber, thereby driving the air out of the drain port. Once
all the air has been replaced by steam, the drain valve
closes and steam pressure builds up in the chamber. The resultant pressure causes the
temperature in the chamber to rise to the required point, usually 121 or 135 °C.
Some type of autoclaves have a vacuum function that pumps out the air before the steam
is allow to enter. This method is much faster and more efficient.
Cycles Stages
Generally an autoclave cycle consists of 3 stages viz.
1)heating,
2)sterilizing,
3)de-pressure.
Vertical autoclaves may have a “water fill” stage before the heating stage. Autoclaves
fitted with vacuum facility may have vacuum stages before the heating stage and also as
part of the depressurizing stage.
Load Types
Autoclaves are used to sterilize several different types of loads:
_ Solid – metal, glass, plastic
_ Porous – linen, gowns, paper, gauze, complex instruments, hollow tubes
_ Liquid – water, saline, media
_ Laboratory waste – Petri dishes, sample bottles, syringes
2)Z value
3)F value
Autoclaving is the most effective and most efficient means of sterilization. All autoclaves must
go through the GMP process of autoclave validation during which, the various programs are
verified as conforming to the requirements detailed in the User Requirement Specification
(URS). They operate on a time/temperature relationship. These two variables are extremely
important. Higher temperatures ensure more rapid killing. Some standard temperature/pressures
employed are 115ºC/10 p.s.i., 121ºC/15 p.s.i., and 132ºC/27 p.s.i. Longer times are needed for
larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for
sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic
media, liquids, and many other things. When proper conditions and time are employed, no living
organisms will survive a trip through an autoclave.
It is not unusual to find people thinking 121° C is the temperature for sterilisation. The Fo-value
can be determined as per the following
Fo = 10 (T – 121.1)/10
Where T = temperature (° C) and Fo = equivalent sterilization time (minutes)
So given a Bioburden of 1215 CFU, with a D-value of 1.6 min/log at 121.1°C and a required
SAL of 10-6.
Then: Log (1215) = 3.08
Loge reduction = 3.08 log + 6 log = 9.08 log.
Ideal Cycle at 121.1°C (250°F) = (9.08 log)(1.6 min/log) = 14.53 minutes.
Positioning of the thermocouples (t/c's) during autoclave validation or indeed in any GMP
temperature mapping exercise is all about appreciating what is adding or subtracting heat from
the room or cabinet being qualified.
In the case of temperature mapping during autoclave validation, heat is added in the form of
pressurized wet steam, anything that can affect the distribution of the incoming steam, can affect
uniformity of temperature. Conversely anything that can take heat away from the chamber can
affect temperature uniformity.
Lets me say at this stage if you want to be pedantic and put t/c’s down the drain, the mapping
exercise will probable fail. However you are there to verify that product will be sterilized, and
product is never placed down the drain. Only the designated product containment area has to be
verified.
If this is new installation, then get hold of the Factory Acceptance Test (FAT). In the FAT the
chamber is subjected to detailed temperature transfer studies.
Even distribution of the in coming steam can be verified by placing a thermocouple sensor (t/c) in
each of the eight corners in the autoclave and one in the cabinet centre. (9 t/c’s)
Cooling due to heat loss will be maximum the further away you are from the steam inlet and the
closer you are to metal that will conduct heat out of the chamber. That is usually, the door, or doors
if double sided. The drain is also a heat sink that conducts heat out of the chamber. One t/c should
be placed as close to the drain as product would be, when the autoclave is in normal use and another
placed alongside the cabinet product temperature probe. This gives us an additional 2 t/c’s,
bringing the total for a standard sized autoclave to 11 t/c’s.
This is normally considered sufficient for 1.5 to 2.5 m3 autoclaves. Any bigger and I would
concentrate on heat loses i.e. add t/c’s to the top and bottom of the doors and or end wall.
It is most important to understand that it is impossible for autoclave validation to be successfully
executed while using none validated steam.
Your steam must be validated for – superheat – dryness – none condensable gases.
Another GMP essential is to carry out pre and post mapping, calibration of your
thermocouples. These should be calibrated against test standard instruments whose calibration is
traceable to national standards, and for which you have valid current calibration certification.
Lethality Calculation
Even if it is not possible to attain absolute sterility, it is possible to sterilize food products for
extended storage, so that they are safe for human consumption. This degree of sterility is referred
to as practical or commercial sterility. The F-Value is introduced as a standard on which to base
the sterilization of food products. The F-Value is defined as the number of minutes which it takes
to reduce the initial spore count of a certain microorganism to a desired safety level at a defined
lethal reference temperature.
To determine the time period required for a certain sterilization process the following data is
needed:
-The characteristic decimal reduction value of the endospores in the environment of the product
-Initial spore count per weight or volume unit (grams or ml) multiplied by the weight or volume
of the product quantity in the container
The technology to determine the required heat treatment beforehand in order to achieve
commercial sterility is available for not only heat sterilized food but for pasteurized products as
well. The parameters of the heat process can be tailored using the determined treatment.
An important role in this case is the heat resistance of those organisms, which may be destroyed
between 60C and 100C. As in the case of sterilization, there is a symbol indicating the
pasteurization value necessary for the sterility of the processed product which is the P-Value.
P-Value
Even if it is not possible to attain absolute sterility, it is possible to sterilize food products for
extended storage so that they are safe for human consumption. This degree of sterility is referred
to as practical or commerical sterility. The P-Value is introduced as a standard on which to base
the pasteurization of food products. The P-value is defined as the number of minutes which it
takes to reduce the initial spore count of a certain microorganism to a desired safety level at a
defined lethal reference temperature.
To determine the time period required for a certain pasteurization process the following data is
needed:
-The characteristic decimal reduction value of the endospores in the environment of the product
-Initial spore count per weight or volume unit (grams or ml) multiplied by the weight or volume
of the product quantity in the container
-Desired maximum probable survival of spores in the thermal processed product. An important
role in this case is the heat resistance of those organisms, which may be destroyed between 60C
and 100C.
1. Autoclave B.
steriothermophillus
spores
B. subtilis, 5230
spores
B. coagulance spores
Clostridium
sporogenes spores
B. subtilis, 5230
spores
Micrococcus
radiodurans
vegetative cells
In a mixture of air and steam, the presence of air will cause the temperature to be lower than
expected. The total pressure of a mixture of gases is made up of the sum of the partial pressures
of the components in the mixture.
Example
Consider a steam/air mixture made up of ¾ steam and ¼ air by volume. The total pressure is 4
bar. Therefore the steam only has an effective pressure of 3 bar as opposed to its apparent
pressure of 4 bar. The mixture would only have a temperature of 134°C rather than the expected
saturation temperature of 144°C. This could render autoclaving ineffective where a minimum
temperature is essential in order to kill bacteria. It is therefore of paramount importance during
the autoclave validation task to validate that all air has been removed from the chamber
None Condensable Gasses
The Non-Condensable Gas Test demonstrates that the attainment of sterilization conditions in all
parts of a steriliser load (particularly for porous load items) is not impaired by the presence of non-
condensable gases.
The measurement of non-condensable gases is made by cooling a steam sample with an efficient
condenser, using water siphoned from a tank at 200ml per minute. Minimum requirements are:
one metre head and water temperature below 28 degrees centigrade. Pressurised water is not
required. When the sampled steam is condensed any non-condensable gases present are released
and separated from the cooled condensate into sight glass columns.
B. Heat-Distribution Studies
The trips where the wires are soldered should not make contact with
the autoclave interior walls or any metal surface.
The principle is the location of the cool spot and the effect of the
load size and/or configuration on the cool spot location.
The difference in temperature between the coolest spot and the
mean chamber temperature should be not greater than •2.5°C .
c)Heat-Penetration Studies
1. A minimum F value
2. A design F value
3. A sterilization process time
To prove that the cool spot location has not changed or,
d. Equipment Qualification
Sterilization of medical device – validation and routine control of sterilization by moist heat :
Europiun standard EN 554 (1994)
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