CSSD, STERILIZATION

1. Sterilization
methods.

2. Hot-Air
Oven
 Hot- air oven is the most widely used methods for Sterilization, which
works on the principle of Dry Heat Sterilization.
 Temperature: holding temperature at 160 C for 2 hours for effective
sterilization.
 Construction:
- Consists of an insulated double walled cabinet heated by electricity or
heating coils situated at bottom and is constructed to withstand a high
temperature.
- Walls are made of Stainless steel or Aluminum to prevent heat
conduction.
- There is a Motor and Fan fitted either at the bottom or back to circulate
hot air inside the chamber

3. - This hot air increases the
temperature inside the chamber
thus sterilizing it.
- A thermostat regulates the
temperature the temperature
at the desired level and a
thermometer is fitted for
recording the temperature.
- The shelves within the hot air
oven are perforated to allow
proper air circulation.

4. Materials sterilized :
• Glassware like glass syringes, petri dishes, flasks, pipettes and
test tubes.
• Surgical instruments like scalpels, forceps. Etc.
• Chemical such as liquid paraffin, fats, Glycerol, oil, and glove
powder, etc,.

5. Precautions:
 Overloading of hot air oven should be avoided.
 The material should be arranged in a manner so that free
circulation of air is maintained.
 Material to be sterilized should be dried completely.
 Cotton plugs should be used to close the mouths of test tubes,
flasks, etc,.
 Paper wrapping of the items should be done.
 Any inflammable material like rubber (except silicon rubber) should
not be kept inside the oven.
 The oven must be allowed to cool for two hours before opening the
doors, since the glassware may crack by sudden cooling.

6. Sterilization control:
 The effective sterilization done by hot air oven can be monitored by,
1. Physical method : Temperature monitoring .
2. Chemical method : Browne’s tube (green spot) is used. A green colour
is produced after 60 min at 160 C or 115 min at 150 C, suggesting
complete sterilization.
3. Biological method : Heat-resistant spores of a non-toxigenic strain of
Clostridium tetani or Bacillus subtilis are used to indicate efficiency of
dry heat sterilization.

7. Browne’s tube

8. Autoclave
 Principle : Steam under produces moist heat of very high temperature.
stem has higher penetrating capacity than dry heat.
- Steam lies in the latent heat liberated when it condenses on it
increases its water content with ultimate hydrolysis and break down of
the proteins and enzymes of microbes.
- High temperature combined with high moisture is one of the most
effective sterilization agent.
- Acts by coagulation and denaturation of bacterial cell wall.

9.  Temperature : 121 C for 15 min at 15 lbs.
 Construction : It is made of double walled metallic vessel made of thick
stainless steel or copper.
- one end of which has an opening meant for keeping the materials in to the
vessels for sterilization.
- the lid or door is provided with pressure gauze to measure the steam
pressue.
- At the top there will be a safety valve to permit the escape of steam from
chamber if it gets too high – to prevent explosion of the sterilizing chamber.
- there are valves to hold the steam and stem outlet.
- there is an exhaust valve below to let steam escape from bottom.

11. Precautions :
 The autoclave should not be overloaded , the material should be arranged
in such that every part of the material should be exposed to the steam.
 Air inside the chamber should completely evacuated by opening the outlet
to escape sir along with steam and then valve is closed.
 Sterilization time must be counted not from autoclave before switching on
but the time after achieving the required pressure.
 Ensure there is sufficient water in an autoclave before switching on. The
lid should be closed tightly.
 At the end of sterilization period, allow the steam pressure drop to zero
and only then open the lid.
 During sterilization the recommended steam pressure should not be
exceeded on any account.

12. Uses of Autoclave
It is particularly used for media containing water that
cannot be sterilized by dry heat.
• Surgical instruments
• Culture media
• Autoclavable plastic containers
• Plastic tubes and pipette tips.
• Solutions and water
• Bio hazardous waste
• Glassware.

13. Types of Autoclaves.
 Gravity displacement type autoclave : it is the most
common type used in laboratories. They are available in
various sizes.
1. Vertical type. (small volume capacity)
2.Horizontal type (large volume capacity).
 Positive pressure displacement type autoclave.
 Negative pressure(vacuum) displacement.

14. Vertical autoclave.

15. Horizontal autoclave.

16. Sterilization control:
 Biological method : spores of Geobacillus sterothermophilus
incarnated in a strip- are the best indicators, because they
are resistant to steaming.
the spores are killed in 12 min at 121 C.
 Chemical method : chemical indicators, autoclave tapes and
thermocouples are used.

17. Sterilization by Filtration.
 Is an effective method of sterilization to remove microorganism
from liquids and air.
 Filtration is used to prepare liquid that cannot withstand heat
like serum, vaccines ,sugar solutions, drugs, enzymes etc.
important in purification of water.
 Types of Filters : 1. Depth Filters.
2. Membrane filters.

18. Difference b/w Depth filter and Membrane filter.

19. Depth filters.
 Depth Filters consists of Fibrous or granular materials that have
been bonded into a thick layer filled with twisting channels of
small diameter.
 The solution containing microorganism is sucked through this layer
under vacuum, and microbial cells are removed by physical
screening or entrapment and also by adsorption to the surface of
the filter material.

20.  Advantage of depth filters. – they can retain a large mass of particles before
becoming clogged, flow rate of fluid is high, low cost.
 Disadvantage : Some of the particles still come out of the filtrate so not suitable to
filter the solutions containing Bacteria.
 Types of Depth Filters. 1. Candle filters
2. Asbestos filters
3. Sintered glass filters.
 Candle Filters : Manufactured in different pore sizes.prepared by mixing with
asdectos and organic matter like plaster of paris (POP) in different proportion
depending upon the pore size.
 It is of two types.
1. Diatomatious earth material(Berkefeld filters)
2. Unglazed porcelain material (Chamberland filters)

21. Berkefeld and chamberland filters.

22.  Seitz Filter/ Sterimat filters : Consists of
a compressed filter sheet of asbestos filter. The
fiber are 2-6 mm thick.
- It contains washed asbestos fibers, cotton
liner and other filter material.
- It is very soft and can be easily damaged.
-The filter disc are always fitted to filter
assembly for filtration purposes.
- The filter disc is fitted to a funnel like
structure through which the liquid to be filtered is
sucked through vacuum or negative pressure.

23.  Sintered glass filter : prepared by
heat-fusing finely powdered glass particles
of graded size.
 It is sealed into body of funnel and
thoroughly washed with sulphuric acid which
will oxidizes any organic matter if present,
and followed by washing with distilled
water.
 These may be classified into Extra Course,
Course, Medium. Fine & Ultrafine.

24. Membrane Filters
 Replaced by depth filters.
 Menbrane filters are made up of Cellulose
acetate , Cellulose nitrate and other synthetic
materials .
 These filters are porous membranes, over 0.1 mm
thick, and Average Pore Diameter(APF) of
0.22um which removes most of the bacteria.
 Are routinely used in water purification and
analysis, sterilization and sterility testing.

25. Mode of operation of membrane filters.
 The apparatus consist of a funnel
with a membrane filter attached
to its base.
 It is then fitted on to a vacuum
flask.
 The liquid to be filtered is poured
into the funnel.
 The vacuum are negative pressure
is applied to force the liquid
through the membrane.

26. Thank you