1. STERILIZATION Definition:
The freeing of an article from all living organisms, including viruses, bacteria , spores & fungi: Pathogenic & non-pathogenic.

2. Uses Sterilization is used for :
Microbiology : Culture media, suspending fluids, reagents, containers & equipment
Hospital : medical & surgical instruments , surgical operations, I.V. infusions, hypodermic injections , diagnostic aspirations.

3. Methods Physical or chemical: Physical Methods: Heat Filtration
Irradiation
(2) Chemical Methods:
@ Strong disinfectants:
* Formaldehyde
* Ethylene oxide

4. Sterilization By Heat Moist Heat: @ Most effective and efficient.
@ Kills organisms by denaturing their enzymes &
proteins
@ Spores are killed by exposure to moist heat
at 121°C for minutes.
Dry Heat:
@ Kills organisms by oxidative destruction of
cell constituents.
@ Spores killed at 160°C for 1 hour

5. Factors influencing Sterilization by Heat:
Temperature & exposure time:
Higher temperature, shorter time and
vice versa.
2.No.of Vegetative cells + spores
@ Plenty of organisms, make sterilization less efficient.
@ So, clean before sterilizing

6. 4. Nature of material containing organisms
3. Nature of organism:
Vegetative cells & spores affect susceptibility of
organism to heat.
4. Nature of material containing organisms
@ Efficiency of sterilization is reduced by presence of organic matter that protect bacteria from lethal action of heat
@ Presence of a disinfectant enhances the kill.
@ Spores best killed in acidity or in alkalinity.

7. Holding Time: Thermal Death Point: Time required for killing organism.
(Not including heating-up time).
Thermal Death Point:
Lowest temperature to give complete killing in aqueous suspension within 10 min. at standard conditions.

8. Thermal Death Time:
Shortest time for complete killing at a stated temperature under standard conditions.
Decimal Reduction Time (D-value):
Time in minutes required to achieve a ten fold reduction in viability of a bacterial population at a given temperature under standard conditions.

9. Methods of Sterilization By Dry Heat:
Red Heat:
Article is held on flame until red hot (inoculating wires, forceps, spatulas)
2.Flaming:
Burning an article in spirit or gas flame (scalpels, needles)
Method will not produce sterilization.

10. 3. Hot-air oven: 4. Infra-red Radiation:
@ Oven has a thermostat + fan to circulate hot air
@ Works at 160˚C for 1 hour, to sterilize
glassware, metals , swabs, oils, powder
4. Infra-red Radiation:
@ Given by an electrically heated element
@ To sterilize glass syringes at 160°C & surgical instruments above 200°C in vacuum chamber in which N2 is passed during cooling time to avoid oxidation.

11. Methods of Sterilization by Moist Heat:
@ Employed at a temp. below 100°C, at 100˚C,
and above 100°C.
@ First two are disinfection methods, third is a
sterilization method.
Temperature Below 100°C:
@ This is the pasteurization process.
@ Used for milk, vaccines & utensils.

12. Milk: @ Pasteurized by 2 methods:-
Holder method: at 63°–66°C for 30 min.
Flash method: at 72°C for 20 seconds.
@ Both methods destroy only milk-borne organisms (Myco., Brucella, Salmonella)

13. Pasteurization is tested by :-
@ Phosphatase test : testing for presence of
phosphatase enzyme found in milk and
destroyed by pasteurization.
@ Methylene blue test : To indicate that
bacteria present in milk is destroyed.
Coxiella burneti is destroyed by flash method.

14. Vaccines:
Inactivated in the vaccine water bath, at 60°C for 1 hr
House articles:
Utensils, clothing, bedding, are disinfected at 70°– 80°C for several minutes.

15. Temperature at 100°C:
Boiling at 100°C:
@ Kills non-sporing bacteria within 5–10 min.
@ Used to disinfect blades, syringes,
@ Dry up articles on removing from boiler (sterilizer) to prevent contamination

16. Steaming at 100°C:
@ This is done by the steamer
(Koch Steamer)
@ It uses steam of boiling water at 100°C and at atmospheric pressure.

17. @ Steamer is used in two ways:-
Single exposure at 100°C for 90 minutes.
@ Time includes heating-up time.
@ Thermophilic & mesophilic
spores will survive this treatment.

18. 2. Tyndallization:
Exposure at 100°C for 20 – 45 minutes for 3 successive days.
@ Used for sterilizing sugars & gelatin
@ First steaming kills vegetative bacteria and spores germinating following day are killed by subsequent heating and so on.
@ Draw-back of tyndallization : spores not germinating in medium sterilized + thermophilic & anaerobic bacteria will escape killing.

19. Temperature above 100˚C:
@ Uses saturated steam - better than dry hot air because:-
Lethal action of moist heat is more
Quicker in :
heating up exposed particles
penetrating cotton stoppers, paper,
wrappers, surgical linen & hollow apparatus

20. How Does it Act?
@ Saturated steam when meets an
article, it condenses to a small
volume of water & liberates its
latent heat to article surface.
@ Avoid presence of air that
prevents steam penetration in article

21. What is the apparatus Used?
@ Autoclave : provides sterilization by dry saturated steam (steam at point of condensing to water)
@ Steam is under pressure higher than atmospheric.

22. Importance of Air Discharge:
@ Air is removed from autoclave:
a) Mixture of steam + air lower the
temperature.
b) Air hinders penetration of steam in
the load.
@ Air denser than steam, sinks down & makes a layer over load

23. @ Sealed bottles containing solutions
are autoclaved although air is
found in them – WHY?
Because water found in these solutions is heated up to steam temp. and so performing same work as moist heat.

24. Types of Autoclaves: Simple Autoclave:
@ Pressure-cooker type, simple, not jacketed, & used in laboratory.
@ Consists of a cylinder for water to be heated, articles placed on a tray
@ Has got a discharge valve, + pressure & temperature gauges.

25. @ Autoclave is opened at right time
because:
# If opened still under pressure, an
explosion occurs
# If opened below atmospheric pressure, evaporation of aqueous materials occurs.

26. Simple Autoclave Deficiencies :
Lacks control of air discharge
(nothing to show that discharge is complete)
2. Lacks means of drying load
after sterilization.
@ Suitable for load wrapped in paper to prevent contamination.

27. 2. Steam-Jacketed Autoclave:
@ Has got an automatic air & condensate discharge.
@ Load is dried up by steam circulating in jacket, & vacuum in autoclave.

28. 3. High Pre-Vacuum Sterilizers:
@ Have electric pumps creating a vacuum area in chamber.
@ First a vacuum is drawn, steam is admitted to chamber, load is heated very rapidly.
@ Temp. used 135˚C for 3 min. at 30 lb pressure & load dried by exhaustion of chamber

29. Advantages of high pre-vacuum sterilizers:
Operation time is shortened.
Damage to heat sensitive materials (sugars) is avoided.

30. Autoclave Control and Indicators:
Automatic process control,
* Advantages are:-
@ Saving time of an operator.
@ Safeguard against errors due to negligence.

31. 2. Recording Thermometer,
@ Draws graphic records of temperature changes inside chamber discharge channel.
@ Helps to avoid errors in timing the holding period.

32. 3. Thermocouple load temp.
measurement:
@ Thermocouple is inserted deeply inside load, its wire is carried to a potentiometer which reads temperature inside the load .

33. 4. Chemical indicators:
Browne’s Tubes, containing a red fluid that turns green on heating:
* at 115˚C for 25 minutes (Type 1)
* at 115˚C for 15 minutes (Type 2)
* at 160˚ C for 60 minutes (Type 3)
@ Tubes are stored below 20˚C to avoid change of color .
@ Satisfactory for routine work .

34. b) Bowies-Dick tape,
@ Used to test efficiency of high pre-vacuum sterilizers & high-pressure autoclaves
@ An adhesive tape is applied around load in the shape of (X).
@ After sterilization, tape changes color all over .

35. 5. Spore indicators,
@ Bacillus stearothermophilus spores are destroyed at 121˚C for 12 minutes.
@Spores placed within load and cultured after sterilizing

36. Disadvantages of Chemical and Spore Indicators:
@ Will not give a perfect efficiency of sterilization:
( heating may be inadequate in a site away from of indicator)
* To solve problem, autoclave is correctly operated & controlled by a thermometer & not by pressure gauge alone

37. Sterilization by Radiation
Ultra-violet Radiation:
@ U.V. rays induce thymine diamers in cell DNA, to destroy bacterial cell .
@ Produced by mercury vapor lamps, used to sterilize plastics

38. 2. Ionizing Radiation:
@ High-speed electrons, X-ray, gamma rays, using cobalt 60
@ Produces free DNA radicals that destroy bacteria .
Indicator for Radiation:
Micrococcus radiodurans that resists
radiation (has efficient DNA repair)

39. Sterilization by Filtration
@ Used to sterilize toxins, serum, antibiotics
@ Uses a filter of pore less than 0.75 µm for bacteria, & much smaller for viruses.
@ Some bacterial filters allow viruses, and Mycoplasma to pass through.

40. Test of Filter: @ Retain Serratia marcescens from broth culture.
Types of Filters:
Asbestos pad (Seitz) filter:
@ Consists of an upper cylinder and a lower funnel with an asbestos pad in between.

41. Uses of asbestos filter pads :
Rapid clarification of fluids.
Clarifying viscous fluids.
General sterilization.
4. Removing pyrogenes.
5. Removing small organisms.
6. Sterilizing serum.
Disadvantage:
Asbestos pads absorb some of filtrate.

42. 2. Sintered glass filters ;
Made up of ground glass, fused to make glass granules adhere together .
3. Cellulose membrane filters:
@ Less absorptive, has got a greater rate of filtration than Seitz filter.
@ Used to separate viruses.
* 2 types of membrane filters:
a) Cellulose nitrate( Gradocol membrane) .
b) Cellulose acetate: commonly used now.

43. @ Membrane is 120 µm thick, placed in two layers.
@ Bacteria are trapped in upper layer : better than Seitz filter
Micro-filters:
@ To filter small fluid volumes
@ May be membrane, or asbestos
@ May be syringe or centrifuge filter.