Sterilization is a process by which an article, surface or medium is free of all microorganism either in the vegetative or spore state.

1. 1
AUTOCLAVE VALIDATIONAUTOCLAVE VALIDATION

2. CONTENT
 Sterilization
 Principle Of Autoclave
 Definitions
 Types Of Autoclave
 Sterilization Cycle
 Load Types
 Basic Validation Approach
 Conclusion
 References
2

3. Sterilization:
Sterilization is a process by which an article, surface or medium is free
of all microorganism either in the vegetative or spore state.
Methods:
1. Physical method:
(a) dry heat sterilization: eg. incineration, red heat.
(b) moist heat sterilization: Eg. Pasteurization, tyndalization,
autoclave
(c) radiation/cold sterilization: eg. Uv ray, x ray
(d) filtration method: eg. Asbestos, membrane filter etc.
2. Chemical metod:
(a) gaseous sterilization: eg. Formaldehyde etc
(b) by using disinfectant: eg. Cresol, phenol.

4. Preparation of items for Autoclaving
In preparing items for autoclaving, containers should be unsealed and
articles should be wrapped in materials that allow steam penetration.
Large packages of dressings and large flasks of media require extra
time for heat to penetrate them.
Wrapping objects in aluminum foil is not recommended because it
may interfere with steam penetration.

5. Importance:
Moist heat in the form of pressurized steam is regarded as
the most dependable method for the destruction of all
forms of life, including bacterial spores. This method is
incorporated into a device called the autoclave.

6. Need of autoclaving:
Reliable sterilization with moist heat requires
temperatures above that of boiling water. These high
temperatures are most commonly achieved by steam
under pressure in an autoclave. Autoclaving is the
preferred method of sterilization, unless the material to be
sterilized can be damaged by heat or moisture.

7. Effectiveness of Autoclave
Sterilization in an autoclave is most effective when the
organisms are either contacted by the steam directly or are
contained in a small volume of aqueous (primarily water)
liquid. Under these conditions, steam at a pressure about
15 psi; attaining temperature (121oC) will kill all organisms
and their endospores in about 15 minutes.

8. Principle of Autoclaving
A basic principle of chemistry is that when the pressure of a gas
increases, the temperature of the gas increase proportionally.
For example, when free flowing steam at a temperature of 100o
C is
placed under a pressure of 1 atmosphere above sea level pressure –
that is, about 15 pounds of pressure per square inch (Psi) --- the
temperature rises to 121o
C. Increasing the pressure to 20 psi raises the
temperature to 126o
C. The relationship between temperature and
pressure is shown in table.
 In this way steam is a gas, increasing its pressure in a closed system
increases its temperature.
As the water molecules in steam become more energized, their
penetration increases substantially.
It is important to note that the sterilizing agent is the moist heat, not
the pressure.

9. Relationship between temperature & pressure:
Table The Relationship Between the
Pressure and Temperature of Steam
at Sea Level
Pressure (psi in excess of
atmospheric pressure)
Temperature (oC)
0 psi 100
5 psi 110
10 psi 116
15 psi 121
20 psi 126
30 psi 135

10. Working of Autoclave
Most autoclaves contain a sterilizing chamber into which articles are
place and a steam jacket where steam is maintained.
As steam flows from the steam jacket into the sterilizing chamber,
cool air is forced out and a special valve increases the pressure to 15
pounds/square inch above normal atmospheric pressure.
The temperature rises to 121.5o
C, and the superheated water molecules
rapidly conduct heat into microorganisms.
The time for destruction of the most resistant bacterial spore is now
reduced to about 15 minutes.
For denser objects, up to 30 minutes of exposure may be required.
Mechanisum: denaturation of critical proteins & nucleic acid within the
cell.

11. PRINCIPLE
11
HORIZONTAL STEAM AUTOCLAVE

12. Uses of Autoclave:
Sterilize culture media, instruments, dressings, intravenous
equipment, solutions, syringes, transfusion equipment, and numerous
other items that can withstand high temperatures and pressures.
The laboratory technician uses it to sterilize bacteriological media and
destroy pathogenic cultures.
The autoclave is equally valuable for glassware and metalware,

13. Limitations and Disadvantages of Autoclave
The autoclave also has certain limitations.
For example, some plasticware melts in the high heat, and sharp
instruments often become dull.
many chemicals breakdown during the sterilization process and oily
substances cannot be treated because they do not mix with water.
Heat requires extra time to reach the center of solid materials, such as
caned meats, because such materials do not develop the efficient
heat-distributing convection currents that occur in liquids.
Heating large containers also requires extra time

14. Different time requirements for sterilizing liquids in
various container sizes
Table The effect of Container Size on Autoclve
Sterilization Times for Liquid Solutions
Container Size Liquid Volume Sterilization Time
(min)
Test Tube:
18×150 mm
10 ml 15
Erlenmeyer Flask:
125 ml
95 ml 15
Erlenmeyer Flask:
2000 ml
1500 ml 30
Fermentation Bottle:
9000 ml
6750 ml 70

15. Definations:
 Sterility assurance level: The probability of a single viable microorganism
being present on a sterilized product is one in one million after completion of
sterilization process.
 Bioburden: The number/type of viable microorganism contaminating a
product.
 D-value: Time in minutes at any defined temperature to destroy 90% viable
microorganism is called D value.
 it indicate the efficiency of sterilization process.
 Z-value: is a no. of degrees of temp. change required to produce a 10 fold
change in D value.
 it relates the heat resistance of a microorganism to change in temp.
 F-value: The number of minutes to kill a specified number of microorganisms
with a specified z-value at a specific temperature.
 used to calculate the probable no. of survivors remaining in a load as,
where, D = D value at 121°C of organism.
N = initial population number/ unit volume .˳
N = final population number/ unit volume.
15

16. 16

17. Autoclave types
 There are several types of autoclave that we can find on the market.
1.Positive Pressure Displacement :
This type is the improvement of downward displacement type.
The steam is created within a second in separate unit (steam generator).
The steam is released to the sterilization chamber to start the sterilization
progress.
2. Negative Pressure Displacement :
most recommended types of autoclave .
very accurate & achieve high sterility assurance level.
disappointing fact is that too expensive .
3. Downward Displacement :
also called as the gravity displacement autoclave.
heats water that becomes steam.
steam then forces the air in the chamber to go through the drain hole.
drain hole is closed once the temperature is sufficient for the serialization process
to begin.

18. 4. Triple Vacuum Displacement :
similar to the negative pressure displacement type.
unit is named "triple vacuum" because there are three process of
removing the air and the steam's pulse .
5. Type "N" or type "B“
Type "N" does not use vacuum to remove air from the chamber.
Contrary, type "B" uses vacuum pump in operation.

19. Sterilization Cycle
Steam enters chamber & conditions load.
Air displaced through chamber drain.
Steam processes load at selected time& temp.
Steam is removed from chamber & pressure released.
19

20. Sterilization Cycle
A steam sterilization cycle consists of three basic phases:
 Heating (come-up) phase - Steam enters the sterilizer jacket and air
is removed from the sterilizer chamber, either by gravity displacement or
mechanically (pre vacuum).
Sterilization (exposure) phase - Load is exposed to steam at a set
temperature (measured and controlled by a temperature sensor in the drain
line) for a set time.
Cool-down (drying) phase - Sterilizer chamber is exhausted to
atmospheric pressure followed by circulating air through the chamber or by
drawing a deep vacuum. Jacket heat is maintained during the drying phase.
20

21. Sterilizer Design
The validation of a steam sterilization cycle is dependent on
the equipment chosen. The sterilizer & its support systems
must be designed & constructed to deliver the effective cycles
repeatedly & consistently.
Qualification of the autoclave consist of proper design,
installation according to design & operational testing to ensure
design criteria & specification are met.
All steam sterilization cycles are based on direct contact with
saturated steam. saturated steam is water vapour in equilibrium
with liquid water.
Saturated steam can exist only along the phase boundry i.e.
relation between its temperature & pressure is fixed.
21

22. 22

23. Characteristics of modern steam sterilizer
Pressure vessel
Steam jacket & insulation
Safety door
Thermostatic steam trap
Temperature control system
Cycle timer
Microbial retentive vent filter
Chamber pressure indicator
23

24. Load types:
Autoclaves are used to sterilize several
different types of loads:
 Solid – metal, glass, plastic
 Porous – gowns, paper ,gauze,
complex instruments, hollow tubes
 Liquid – water, saline, media
 Laboratory waste – Petri dishes, sample bottles, syringes
24

25. VALIDATION
WHO validation definition
 The documented act of proving that any procedure, process,
equipment, material, activity, or system actually leads to the
expected results.
 A system must be qualified to operate in a validated process
 Qualify a system and/or equipment
 Validate a process
 Qualification versus validation, e.g. you
qualify an autoclave, whereas you validate
a sterilization process.
25

26. BASIC VALIDATION APPROACH
26

27. INSTALLATION QUALIFICATION
Installation tests should include checking:
• That the electrical supply is suitable for the autoclave.
• That the temperature and humidity are sufficiently well
controlled.
• That there is no interference to or from other equipment.
• The accuracy of indicators has been certified to national
standards.
• The temperature and pressure of the chamber during a
sterilization cycle are within specifications.
27

28. INSTALLATION QUALIFICATION
The leakage into the chamber during a vacuum cycle does not
exceed the specified maximum.
 That there is no leakage of steam, water or effluent at any
point during the sterilization cycle.
That both water quality and steam quality comply with the
sterilizer specification
 The chamber is capable of withstanding its specified
maximum pressure. This test may be a legal requirement and
should be performed by a suitably certified organization.
28

29. Mechanical equipment specification (Chamber, valves, traps,
stainers, filters, regulators, vacuum pump, heat exchanger,
condenser, etc.)
Control & instrumentation specifications (programmable logic
controller, operator interface, printer/recorder, control valves,
transducers, pressure & temperature transmitters, resistance
temperature devices, switches, level sensors, interlocks,
photocells.
Site specifications/utilities(power, grounding, surge protector,
uninterruptible power supply, breakers, water, clean steam,
plant steam, drain, isolation valves)
29

30. Approval documentation (e.g. pressure vessel, electrical) ,bill
of materials, vendor specification sheets, purchase orders,
preventive maintenance program, piping installation
verification, operating & maintenance manuals.
Control system documentation (system configuration/block
diagram, flow sheets, display layouts, general process limits,
data monitoring, software inventory, electronic application
code listing, system security.
30

31. OPERATIONAL QUALIFICATION
The OQ process is intended to demonstrate that the components of the
autoclave operate properly & that the autoclave is deemed ready for
performance or load testing.
An OQ may include the following checks:
 Operational tests(operator/supervisory/maintenance modes, doors,
abort & emergency stop, programmable parameter, menu navigation,
power-up & shut down, procedure select/start control switch &
interlock tests.
 Leak/air removal/steam penetration/vacuum hold test
 Jacket mapping
 Saturated steam check
 Empty chamber tests
31

32. Empty chamber heat distribution studies
Intent of this study to demonstrate the temperature uniformity
& stability of the sterilizing medium throughout sterilizer.
Temperature uniformity may be influenced by type, size,
design & installation of sterilizer.
In production size sterilizer, 15-20 thermocouples are used &
distributed geometrically throughout sterilizer.
Thermocouple should be placed in the exhaust drain which is
adjacent to the sensor that control vessel temperature.
Temperature deviation should not greater than +2.5 degree
celsius of the mean chamber temperature.
32

33. Thermocouple locations
33

34. Container Mapping
Intent of this study to determine coolest point within liquid filled
containers.
A sufficient no of thermocouples should be positioned in the upper, middle
& lower portions of the containers.
The profile point having the lowest temperature is designated as the cold
spot.
The no. of thermocouples positioned within the container will be dependent
upon the container volume.
34

35. PERFORMANCE QUALIFICATION
Loaded chamber steam penetration tests:
 intent of this study is to determine the coolest point within a
specified load.
 Load cool points are dependent on the type of objects that
compose the loads i.e. liquid filled containers, process
equipment.etc.
The penetration thermocouples are positioned within liquid
filled containers at the cool point previously determined by the
container mapping studies.
35

36. Loaded chamber microbiological challenge
studies
 The microorganisms most frequently used to challenge moist heat
sterilization cycles are Bacillus stearothermophilus & Clostridium
sporogenes.
 These studies are typically conducted concurrently with the heat
penetration studies.
 After the sterilization cycle is complete, the spore strips are recovered &
subjected to microbiological test procedures.
 Strips are aseptically immersed in a suitable growth medium ( soyabean
digest medium is typical) & incubated for up to 7 days.
 Incubation temperature far B. stearothermophilus is 50-55 degree celsius.
36

37. Conclusion:
If the temperature in an autoclave does not reach 121°c, not all
spore will destroyed in the typical cycle time of 20min.
Validation is therefore required to ensure that the autoclave is
operating correctly and that complete sterilization is being achieved.
To ensure that qualification and validation are performed correctly,
the test should only be carried out by suitably qualified & experienced
personnel & organization.

38. References
1) Textbook of microbiological methods, by Collins & Lyne’s,7th
edition, K.M. Varghese Company, Mumbai.
2) Indian Pharmacopoeia 1996 vol , Govt. of India , appendix-
12,monograph a-140.
3) Pharmaceutical process validation, Robert A. Nash, Alfred
H.Wachter. Marcel, 3rd
edition,volume-129,Marcel Dekker, page
no 176-190.
4) Validation of pharmaceutical processes, sterile products, II
edition, by Frederick J.Carleton, James P. Agalloco, Marcel
Dekker. Page no.413-449.
5) The theory & practice of industrial pharmacy, by Leon Lachnan.
Herbert A. Lieberman, Joseph L. Kanig. 3rd
edition,page no.625.
6) Pharmaceutical Quality Assurance, M.A. Potdar, Nirali
Prakashan, Pune.
38

39. Web references
www.pharmtech.com
www.validation-online.net
www.pda.org
www.askaboutvalidation.com
www.intercal.co.za
www.fda.com
www.google.com
www.pharmaweb.net
www.sterilizers.com
39