This document provides information on various sterilization methods. It begins with an introduction to sterilization and its history. It then defines key terms and describes various physical and chemical sterilization methods such as heat, filtration, radiation, and chemicals. Specific techniques like autoclaving, pasteurization, and tyndallization are explained. Advantages, uses, and controls for different methods are also summarized.

2. CONTENT
●Introduction
●History of sterilization
●Definition
▪Types of sterilizaton −
●Physical method
●Chemical method
●Advantages
●Disadvantages
●Applications

3. INTRODUCTION
 Microorganisms cause contamination,infection and
decay, it becomes necessary to remove or destroy
them from materials or from areas.
 This is the object of Sterilization.
 Methods to remove or kill microorganism are
known as Sterilization.
 The time requied for sterilization is inversely
proportional to the temperature of exposure and
can be expressed as Thermal death time.

4. HISTORY OF STERILIZATION
Denis Papin (1679)
A French physicist, Denis Papin invented a pressure
cooker that would trap boiling water,convert it into
steam,& was found to cleanse objects by cooking
them.

5. Charles Chamberland (1879)
A French Microbiologist, created a new version of
pressure cooker, called the AUTOCLAVE to be used
in medical microbiology.

6. LOUIS PASTEUR (1860)
2 major contributions to the art of sterilization came in
the 1860’s when-
 Louis pasteur wrote extinsively on how germ
cause disease & the technique of pasteurization.
 Joseph Lister developed a technique that used
Carbolic acid as a spray to disinfect instruement.

7. DEFINITION
STERILIZATION
Medium is made free of all microorganism either in the
vegetative or spore form.
DISINFECTION
-Destruction or reduce the number of pathogens,
-Capable of producing infections but not spores.
ANTISEPTICS
-Chemical disinfectants,
-Which can safely be applied to living tissues,
-Prevent infection by inhibiting the growth of bacteria.
GERMICIDES
-Substances that can kill bacteria.

8. TYPES OF STERILIZAION
PHYSICAL METHOD
 Heat
 Radiation
 Filtration
 Sunlight
 Ozone
CHEMICAL METHOD – sporocidal agent---
Ethylene oxide, Formaldehyde ,Gutaraldehyde
,Hydrogen peroxide,peracitic acid , O-phtalic acid,
plasma sterilization .

9. HEAT STERILIZATION-
 Most Reliable
 Commonly employed method.
Factors influencing sterilization by heat are-
 Nature of heat
 Temperature & Time
 No. of microbes present
 Characterstics of the organism
 Type of material.
DRY HEAT
TYPE OF
HEAT
MOIST
HEAT

10. DRY HEAT
 Kills the microorganism
 By denaturation of bacterial protein,
 Oxidative damage &
 By toxic effect of elevated levels of electrolytes.
DRY HEAT
Flaming Incineration Hot air oven
Red heat

11. RED HEAT
 Inoculating wires or loops
 Tips of forceps and
 Needles are held in flame of a Bunsen burner till
they become red hot.

12. FLAMING
 Glass slides, scalpels and mouths of culture tubes
are passed through Bunsen flame without allowing them
to become red hot.
Incineration
 Excellent method for destroying biomedical waste.
 A instrument named incinerator used for this
purpose.

13.  Materials are reduced to ashes by burning.
 Plastics such as PVC and polythene can be dealt with
similarly.
Guidelines on “Design and construction of biomedical
waste incinerator” recommend various design feature of
an incinerator as well as the air pollution control device

14. HOT AIR OVEN
 Most widely used
 Oven is electrically heated and is fitted with fan to
ensure adequate and even distribution of HOT AIR
in the chamber.

15. PRINCIPLE OF HOT AIR OVEN-
 The heat is absorbed by the surface of the item to be
sterilized, which then penetrates to the centre, untill the
entire items reaches the desired temperature.
Temperature & Time-
-150°C for 2 hours 30 min.
-160°C for 2 hours.
-170°C for 1 hour.
-180°C for 30 min.

16. USES-
Sterilization of- (At 160°C for 2 hrs)
 Glasswares like- Glass syringes, petridishes ,flasks,
pipettes & test tubes.
 Surgical instruments- forceps, scissors, scalps etc.
 Parmaceuticals products-liquid paraffin, fats & grease etc.
 For oils, glycerols & dusting powder
(British Pharmacopoeia recommends a holding time of 1 hr at
150°C)

17. PRECAUTIONS-
 It should not be overloaded.
 Material should be arranged
 Allows free circulation of air.
 Glassware should be dry fitted with cotton plugs.
 Petridishes & pipettes should be wrapped in craft paper.
 Rubber material should not be kept inside the oven.
 Allowed to cool for 2 hrs before opening the door, since
the glasswares may crack by sudden cooling.

18. STERILIZATION CONTROL-
 The spores of Bacillus subtilis subsp. niger
(NCTC10075 or ATCC 9372) Are kept inside the oven.
These spores should be destroyed if the sterilization is
proper.
 Thermocouples may also be used.
 Browne’s tube with red colour is available, after
proper sterilization a green colour is produced.(after 1
hr. at 160°C).

19. MOIST HEAT – KILLS THE MICROORGANISM BY
DENATURATION AND COAGULATION OF PROTEINS.
MOIST
HEAT
Temperature
below 100°C
Temperature at
100°C
Temperature
above 100°C

20. TEMPERATURE BELOW 100°C
A. Pasteurization of milk.
B. Inspissation.
C. Vaccine bath.
D. Low temperature steam
formaldehyde (LTSF) sterilization.

21. A. PASTEURIZATION OF MILK
 Holder method- 63°C for 30 min.
 Flash method- 72°C for 20 min.
Followed by cooling quickly to 13°C.
 All nonsporing pathogens such as mycobacteria,
brucella and salmonellae are killed except Coxiella
burnetii is relatively heat-resistant and may survive in
holder method.

23. B. INSPISSATION
 Instrument used –Inspissator.
 Serum or egg medium- lowenstein jensen’s and
loeffler’s serum slope are render by heating at 80-85°C
temp. for half an hour daily on three consecutive days.

24. C. VACCINE BATH
 Bacterial vaccines are heat inactivated in special
vaccine bath at 60°C for 1 hour.
 Serum and body fluid containing coagulable
proteins can be sterilized by heating for 1 hour at
56°C in a water bath or several successive days.

25. D. LTSF - LOW TEMPERATURE STEAM FORMALDEHYDE
STERILIZATION
 Steam at atmospheric pressure 75°C
 Formaldehyde vapour is used.
 Bacillus stearothermophilus has been used as biological
control to test the efficacy of LTSF sterilizers.

26. TEMPERATURE AT 100°C
1.Boiling
2. Steaming
3. Tyndallization

27. 1.BOILING
 Boiling of the items in water at 100°C for 15 minutes may kill most
of vegetative forms but not the spores.
 Not suitable for sterilzation of surgical instruments.
 When better methods are not available, boiling may be used for
glass syringes and rubber stoppers.
2. Steaming
 Koch’s or Arnold’s steamer is used.
 Useful for those media which are decomposed
At high temperature of autoclave.
 Articles are kept on a perforated tray exposed to steam 100°C at
atmospheric pressure for 90 min.

28. 3. TYNDALLIZATION
 An exposure to 100°C for 20 minutes on
three successive days is used.
 Also called Fractional sterilization.
 John Tyndall discovered tyndallization.
 May fail to kill spores of cetain anaerobes and thermophiles
Principle-
-1st exposure kills all the vegetative bacteria,
-And in the intervals between the heatings the remaining spores
germinate into vegetative forms,
-which are killed on subsequent heating.
Uses
To sterilize gelatin and egg,serum or sugar containing
media.

29. TEMPERATURE ABOVE 100°C
Autoclave
 Physical method of sterilization under moist heat.
 Killed bacteria , viruses and even spores present in
the material put inside of the vessel using steam
under pressure.
 Similar to pressure cooker.
Principle- water boils when its vapour pressure
equals that of the surrounding atmosphere.
So when the atmospheric pressure is raised , the
boiling temperature is also raised.

31. COMPONENTS OF AUTOCLAVE
1.A pressure chamber-
 large cylinder (vertical or horizontal) in which the
materials to be sterilized are placed.
 made up of stainless steel and placed in a
supporting case.
 A steam jacket (water compartment)
2.A lid-
 fastened by screw clamps and rendered air tight by
an asbestos water.
 It bears-discharge tap for air and steam discharge.
 A pressure gauge.
3.An electrical heater-
Heats the water to produce steam.

32. PROCEDURE -
 Cylinder filled with sufficient water.
 Material to be sterilized is placed inside the
pressure chamber.
 The lid is closed ,electrical heater is put on.
 The safety valve is adjusted to required pressure.
 After the water boils the steam and air mixture is
allowed to escape through the discharge tap till all
the air has been displaced.
 Steam pressure rises inside and when it reaches
the desired set level, the safety valve opens and
excess steam escape out.

33.  Holding period is counted from this point of time ,
which is about 15 min in most cases.
 After the holding period- Electrical heater is
stopped and the allowed to cool till the pressure
gauge indicates that the pressure inside is equal to
the atmospheric pressure.
 Discharged tap opens slowly and air is allowed to
enter the autoclave.
 The lid is now opened and the sterilized materials
are removed.
Sterilization conditions-
-121°C for 15 min at pressure of 15 pound(psi).
-126C for 10 min at pressure of 20 psi.
-133C for 3 min at pressure of 30 psi.

34. USES OF AUTOCLAVE-
 Sterilize culture media, rubber material, gowns, dressing,
gloves etc.
 Useful for materials which cannot withstand the higher
temperature of hot air oven.
Precautions-
 It should not be used for waterproof material(oil and
grease)
 Do not overfill the chamber.
 Material should not touch the sides or top of the
chamber.
 Polyethylene trays should not be used.

35. TYPE OF AUTOCLAVE
 Gravity displacement type-
 Most common used in laboratories.
●Vertical type &
●Horizontal type.
 Positive pressure displacement type
 Negetive pressure displacement type

36. STERILIZATION CONTROL
 Biological indicator-
Spore of Geobacillus stearothermophilus (formely called
Bacillus stearothermophilus)-
-Are the best indicator because,
-They are resistant to steaming
-Their spores are killed in 12 min. at 121C.
 Chemical indicator- Autoclave tape
-Browne’s tube

37. TABLE- Indicators used for monitering the sterilization process of
autoclave (steam sterilizer), ethylene oxide sterilizer and plasma
sterilizers .
1.Physical or mechanical indicators:- distal displays of the serilizer
equipment such as Temperature,time and pressure, etc.
2.Chemical indicators:- To moniter satisfactory process of
sterilization,uses chemical sensitive materials which undergo a color
change if sterilization parameter is achieved
TYPE-I ●Process indicator
●External pack indicator
TYPE-II ●Called Bowei-dick test ,used only for steam sterilizers
●Done before the first load
●To check air removal, air leaks & steam penetration
●To check autoclave functioning well.
TYPE-III Single parameter indicator, obsolete now.

38. TYPE-IV ●Internal pack indicator,used inside each pack.
●Designed to measure any two of the critical varibles:- time,
steam quality & temp.
TYPE –V ●Also an internal pack indicator
●measure all three variable: time,steam quality & temp.
TYPE-VI ● Emulating indicator
● it is cycle specific.
3. Biological indicators-
-Bacterial spores used to check effectiveness
-Highly resistant spores
1) Geobacillus stearothermophilus for autoclave
2) Bacillus atrophaeus for ethylene oxide sterilizer & dry heat

39. FILTRATION
 Excellent way to remove the microbial population in
solution
 For Heat labile materials like vaccine, antibiotics
,toxins , serum and sugar solution as well as for
purification of air.
Types of filtration-
1.Depth filter 2.Membrane filter

40. DEPTH FILTER
 Porous filter,
 Retain particles throughout the depth of the filter
 It composed of random mats of metallic ,polymeric
 These filters relay on the density and thickness of the
filter to trap particles rather than pore size.
TYPE- Candle filters ,Asbestos filters, Sintered glass
filters.

41. ADVANTAGE-
-it can retain a large mass of particles
-Flow rate of fluid is high
-Low cost
DISADVANTAGE-
-Some of the particles still come out in the filter
-Not suitable for filtration of solution containing
bacteria

42. TYPES -
1.Candle filters-
-Manufactured in Different grade of porosity
-Used for purification of water for industrial & drinking
purpose
2 Type- Unglazed ceramic (chamber land filter)
-Diatomaceous earth filter (berkfeld filters)

43. 2.SINTERED GLASS FILTERS
 Prepared by heat fusing finely powdered glass
particles of graded sizes.
 They have low absorptive property and can be
cleaned easily, but are brittle and expensive.

44. 3. MEMBRANE FILTERS
 Made up of cellulose acetate ,cellulose nitrate,
polycarbonate, polyvinylidene fluoride,or other
synthetic materials.
 Porous,Most widely used for bacterial filtration.
 Retain all the particles on the surface that are larger
than their pore size.
Pore size –
 diameter of 0.22µm- Removes bacteria.
 Filters of 0.45µm- Retain coliform bacteria
 Filters of 0.8µm- Remove airborne microbes.

45. FILTRATION OF LIQUID
Done for following purposes:-
 To sterilized sera,sugar and antibiotics solutions
 Separation of toxins and bacteriophages from bacteria
 To obtain bacteria free filterate of clinical samples for
virus isolation.
 Purification of water.

46. FILTRATION OF AIR –
DELIVER BACTERIA FREE AIR-
HEPA filters- high efficiency particulate air filter
It removes 99.97% of articles that have a size of 0.3µm

47.  ULPA filter- ultra low particulate air
It can remove from the air atleast 99.99% of dust,
pollen,mould,bacteria and any air borne particles with
a size of 0.12µm
STERILIZATION CONTROL -
 By Brevundimonas diminuta and serratia
marcescens.

48. RADIATION
 Radiation kills germs that can cause disease and
neutralizes other harmful organisms.
TYPES-
1. IONIZING RADIATION-
 It includes x-rays, gamma rays, cosmic rays.
 Mechanism- it cross breakage of DNA without
temperature rise(cold sterilization).

49. Uses-
 gamma radiation used for sterilization of
disposable plastic supplies,
such as-disposable rubber, infusion sets and
catheters.
 Bone and tissue graft and irradiation of pores.

50. ADVANTAGES
 High penetration power
 Rapidity of action
 Temperature is not raised
Sterilization control- Efficacy of ionising radiation is
tested by using Bacillus pumilus.

51. 2.NON IONIZING RADIATION-
 It includes infra red and UV radiations
 They are quiet lethal but do not penetrates
glass,dirt films,water, hence there uses is restricted.
 UV rays –
250-300nm wavelength for 30min use for disinfection
of clean surfaces in OT, laminar flow ,as well as
water treatment.

52. SUNLIGHT
 Direct sunlight is a natural method of sterilization of
water in tank ,rivers ,and lakes.
 Direct sunlight has an active germicidal effect due to its
content of ultraviolet and heat rays.
 Bacteria present in natural water sources are rapidly
distroyed by exposer to sunlight.

53. OZONE STERILIZATION
 Ozone has extremely great oxidative power and
ready to react to germs, viruses and a host of
microbes.
 Useful for disinfect the water and food
 It said to be 50% more powerful and act 3000 times
faster than chlorine at 100 times the strength.
“ Ozone(O3)kills microbes in high voltage
electricity, when healthy bacterial cell exposed to
ozone oxidative burst puncturing of the cell
,causing cell death”.

54. REFERENCES
1.Essential of microbiology by Apurba Sastry
2. C. P. Baveja
3. Ananthanarayan and Paniker’s

55. THANK YOU