The document provides a comprehensive overview of autoclaves, including their function as steam sterilizers using high pressure and temperature to eliminate microorganisms. It discusses the mechanisms of steam sterilization, the importance of air removal, and the regulatory considerations involved in autoclave usage. Additionally, it outlines advantages and disadvantages of steam sterilization, as well as critical design aspects and microbiological assurance levels associated with the sterilization process.

1. Training on Autoclave
By
Dhaval Surti
@Pharmatalks_group

2. What is An Autoclave?
• An autoclave is like a large pressure cooker; it operates
by using steam under pressure as the sterilizing agent.
High pressures enable steam to reach high
temperatures, thus increasing its heat content and
killing power.
• Autoclave and Steam Sterilizer words are used for same
equipment interchangeably
• Charles Chamberland invented first real steam
sterilizer in 1880.
• First steam sterilizer looked like pressure cooker and
also worked in same way.

3. How steam kills microorganism?
• Autoclave operates using steam under high pressure as
sterilizing agent.
• High pressures enables steam to reach high
temperature, thus increasing heat content and killing
power.
• Most of heating power of steam comes from its latent
heat of vaporization. This is the amount of heat
required to make water hot.
• It takes 80 calories to make 1 litre of water to boil, but
540 calories to convert that boiling water to steam
• Therefore steam at 100 C has almost seven times more
heat than boiling water

4. How steam kills microorganism?
• Steam is able to penetrate objects with cooler
temperatures because once the steam contacts a
cooler surface, it immediately condenses to water,
producing a concomitant 1870 fold decrease in
steam volume.
• This creates a negative pressure at the point of
condensation and draws more steam to the area.
• Condensations continues to so long as the
temperature of the condensing surface is less than
that of steam; once temperatures equilibrate, a
saturated steam environment is formed.

5. How steam kills microorganism?
• Moist heat is thought to kill microorganisms by
causing coagulation of essential proteins.
• When heat is used as sterilizing agent, the
vibratory motion of every molecule of a
microorganism is increased to levels that
induce cleavage of intramolecular hydrogen
bonds between proteins.
• Death is therefore caused by an accumulation
of irreversible damage to all metabolic
functions of the organism

6. Role of Air in Steam Sterilization
• Air and steam do not mix readily.
• Air is heavier than steam.
• If items to be sterilized are wrapped too tightly
than entrapped air cannot escape forming cool
pockets inside packages preventing steam from
reaching sufficient temperature to kill all
microorganisms.
• Heat carrying capacity of air is much less than
steam
• Debris in drain strainer may form air layer which
preventing effective operation of steam sterilizer

7. Role of Air in Steam Sterilization

8. Why Air Must Be Removed?
• Air is enemy since the heat power is steam
• Air does not mix readily with steam and sinks
to the bottom of chamber
• Entrapped air pockets touches the surfaces,
impairing killing effect of steam

9. Why Air Must Be Removed?

10. Equilibration Time
• As per EN 285 2016, Equilibration time is
‘period which elapses between the attainment
of the sterilization temperature at the
reference measurement point and the
attainment of the sterilization temperature at
all points within the load’.
• Equilibration time 15 seconds for the
Autoclaves of size less than 800 L
• 30 seconds for the Autoclaves of size more
than 800 L

11. Usage of Autoclave
• Filling Assemblies includes filling needles and tubing
• Components for filling, filtration etc (vessels, containers,
glassware, sampling bottles)
• Garments
• Rubber stoppers, aluminium seals
• Filters, silicone tubing, gloves
• Documents and labels
• Components for environmental monitoring in aseptic areas
• Components for area sanitization like buckets, sponge mops,
wipes, sanitization bottles, etc
• Accessories like forceps, tools, clamps, bio-breathable papers
• And so on…

12. Regulatory Observations
• Inadequate control of autoclave sterilization
cycles
• Single door autoclaves instead of double door
• The porous load steriliser data did not meet
current equilibration requirements
• Leak rate and air removal testing was not
frequent enough
• Sterilizer load patterns were not adequately
defined in SOP

13. Advantages of Steam
Sterilization
• It is preferred way of destroying
microorganism
• It is non-toxic
• Kills spores
• Inexpensive
• Cycles are fast

14. Disadvantages of Steam
Sterilization
• Steam can be hazardous certain pieces of
equipment
• If steam is not pure than it can get into very
small lumens and block them
• Pitting or discoloration of metal when come in
contact with steam that wasn’t pure
• Not suitable for moisture sensitive materials

15. Importance of Steam Quality
• Saturated steam is required for steam
sterilization
• Super heated steam causes sterilization failure
due to lack of heat transfer to load contents
• Unsaturated steam contains water droplets,
lacks sufficient heat for steam sterilization
and causes wet packs in load
• Measures of steam quality are as follows:

16. Importance of Steam Quality
Item Description Limits
Non condensable
gases (NCG)
Air and other gases, which do not
condense under the conditions of
steam sterilization and prevent the
attainment of sterilization conditions
in any part of the load
≤ 3.5%
Superheat Steam whose temperature, at any
given pressure, is higher than that
indicated by the equilibration curve for
the vaporization of water
≤ 25°C
Dryness Value The dryness fraction is a measure of
the amount of moisture carried by the
steam being supplied and used for
sterilization
0.90-0.95
Contaminants Clean steam condensate tests as per
EP
As per EN 285

17. Leak Test
• (1.3 mBar/min = 0.0195 PSIA/min or 0.039”
Hg/min)
• Mark, Sterilizers are prone to leakage because of
the nature of the process – pressure/vacuum and
hot/cold, all of which stress the valves and sealing
systems. Add on to this the complexity and the
number of fittings/valves present combined with
environmental debris
• The EMA requirement to conduct leak rate tests
derives from the Orange Guide – Moist Heat 66 –
“There should be frequent leak tests on the
chamber when a vacuum phase is part of the
cycle”.

18. Typical Autoclave Cycle
• Steam sterilization is generally carried out at 121°C
(250°F) for 15 minutes or at 134°C for 3–4 minutes.
• Temperatures can be reduced to 115°C, and even as low
as 105°C, depending upon the bioburden, integrity, heat
resistance, and characteristics of the material being
sterilized. This approach is beng mainly used for liquid
products terminal sterilized
• Low-temperature steam processes (65°–80°C) have been
used (e.g., steam-formaldehyde); however, other
combinations could also be used.
• Load items must be clean, dirty load item may protect
bacterial protein resulting in steam non penetrating and
non-sterilization.

19. Steam Sterilizer Design Aspects
• Autoclave is composed of autoclave chamber along with
its auxiliary system and equipment
1. A Heating System
2. A Pressure System
3. A Vacuum System
4. A Sensor System
5. A Control Panel
6. A Computer or PLC System
7. An Instrumentation System
8. A Datalogging System
9. Safety Systems

20. Steam Sterilizer Design Aspects

21. Steam Sterilizer Design Aspects

22. Steam Sterilizer Design Aspects

23. Steam Sterilizer Design Aspects

25. Steam Sterilizer Design Aspects
• Critical Parameters are Steam Quality, Steam
Pressure, Temperature and Time
• Temperature sensors located in drain and in
the chamber. More than one sensors are placed
for overall monitoring of chamber temperature
and drain temperature
• Temperature control can be on drain sensor,
OR drain sensor as well as chamber sensor
• Automated cycles with data monitoring and
alarms

27. Steam Sterilizer Design Aspects

28. Steam Sterilizer Design Aspects

29. Steam Sterilizer Design Aspects

30. Mechanism of Sterilization
• Most of the heating power of steam comes
from its latent heat of vaporization. This is the
amount of heat required to convert boiling
water to steam.
• This amount of heat is large compared to that
required to make water hot. For example, it
takes 80 calories to make 1 liter of water boil,
but 540 calories to convert that boiling water
to steam. Therefore, steam at 100º C has
almost seven times more heat

31. Mechanism of Sterilization
• Steam is able to penetrate objects with cooler
temperatures because once the steam contacts a
cooler surface it immediately condenses to water,
producing a concomitant 1,870 fold decrease in
steam volume. This creates negative pressure at
the point of condensation and draws more steam
to the area. Condensations continues so long as the
temperature of the condensing surface is less than
that of steam. These properties ensure rapid
heating of surfaces, good penetration of dense
materials, and coagulation of proteins.

32. Why steam is so Effective?

33. Validation of Sterilization Cycle

34. Validation of Sterilization Cycle
Type Goods to be
sterilized
Typical Pre-
vacuum pulses
Air Removal
Hard Goods
(Non-porous
load)
SS Equipment,
containers, SS
piping,
Glassware
1 or more
(typically 3)
Easy air removal
and steam
penetration
Wrapped Goods
(Porous load)
Garments,
filters, rubber
stoppers, filters,
silicone tubing
3 or more More difficult air
removal/steam
penetration
Liquids Media, terminal
sterilized
products
None Air overpressure
process, heat
and cool without
vacuum

35. Validation of Sterilization Cycle

43. Load items must be clean, dirty load item
may protect bacterial protein resulting in
Load items must be clean, dirty load item
may protect bacterial protein resulting in
Load items must be clean, dirty load item
may protect bacterial protein resulting in
Load items must be clean, dirty load item
may protect bacterial protein resulting in
Load items must be clean, dirty load item
may protect bacterial protein resulting in

44. Microbiological Aspects

45. Microbiological Aspects
• Sterility Assurance Level
The probability of a single viable microorganism
being present on a sterilized unit is one in one
million after the item has undergone a
sterilization process; often called a six log
reduction
For potential products, desire a SAL of 106

46. Microbiological Aspects

47. Microbiological Aspects

48. Microbiological Aspects

49. Microbiological Aspects

50. Microbiological Aspects

51. Microbiological Aspects

52. Microbiological Aspects

53. Hope this is of some Help
•This is not all inclusive but
can help/direct to
understand more about
autoclave
•Thank You For Your Time
•DHAVAL SURTI
t.me/Pharmatalks