1. The document discusses various sterilization techniques including physical agents like heat, radiation and chemical agents. 2. Heat sterilization methods include dry heat using an oven or flame and moist heat using steam under pressure in an autoclave or at lower temperatures for pasteurization. 3. Radiation sterilization uses ionizing radiation like X-rays or gamma rays for cold sterilization of items that cannot withstand heat.

1. STERILIZATIONSTERILIZATION
TECHNIQUESTECHNIQUES
COURSE : B.SC. (MICRO)COURSE : B.SC. (MICRO)
SUBJECT : ELEMENTARY MICROBIOLOGYSUBJECT : ELEMENTARY MICROBIOLOGY
UNIT 4.2UNIT 4.2

2. INTRODUCTIONINTRODUCTION
 Microorganisms are the agents ofMicroorganisms are the agents of
contamination, infection, and decay.contamination, infection, and decay.
 Hence it becomes necessary toHence it becomes necessary to
remove them from materials andremove them from materials and
areas.areas.
 Early civilization practiced salting,Early civilization practiced salting,
smoking, pickling and exposure tosmoking, pickling and exposure to

3. In mid 1800s Lister developedIn mid 1800s Lister developed
Aseptic techniquesAseptic techniques to preventto prevent
contamination of surgical wounds.contamination of surgical wounds.
Prior to this development:Prior to this development:
• Nosocomial infections causedNosocomial infections caused
death in 10% of surgeries.death in 10% of surgeries.
• Up to 25% mothers delivering inUp to 25% mothers delivering in
hospitals died due to infection.hospitals died due to infection.

4. DEFINITIONDEFINITION
STERILIZATIONSTERILIZATION
The process of freeing an article fromThe process of freeing an article from
microorganisms including theirmicroorganisms including their
spores.spores.

5. AsepsisAsepsis: Absence of significant: Absence of significant
contamination.contamination.
 Aseptic techniques are used to preventAseptic techniques are used to prevent
contamination of surgical instruments,contamination of surgical instruments,
medical personnel, and the patientmedical personnel, and the patient
during surgery.during surgery.
 Aseptic techniques are also used toAseptic techniques are also used to
prevent bacterial contamination in foodprevent bacterial contamination in food
industry.industry.

6. Bacteriostatic Agent:Bacteriostatic Agent:
An agent thatAn agent that inhibitsinhibits the growth ofthe growth of
bacteria, but does not necessarily killbacteria, but does not necessarily kill
them.them.

7. Bactericide:Bactericide:
An agent that kills bacteria. Most do not killAn agent that kills bacteria. Most do not kill
Endospores.Endospores.
Sporicide:Sporicide:
An agent that kills spores.An agent that kills spores.

8. Methods of SterilisationMethods of Sterilisation
Microbial ControlMicrobial Control
MethodsMethods
Physical AgentsPhysical Agents Chemical AgentsChemical Agents
Mechanical RemovalMechanical Removal
MethodsMethods

9. Physical AgentsPhysical Agents
Heat Radiation
Dry Moist
Incineration
Dry Oven
Steam Under
Pressure
Boiling Water/Hot Water
Pasteurization
Sterilization
Ionizing Non Ionizing
X Ray, Cathode,
Gamma
Disinfection
UV
Sterilization Disinfection

10. 1.Physical agents1.Physical agents
I.I. SunlightSunlight
II.II. HeatHeat
III.III. RadiationRadiation

11. sunlightsunlight
 The bactericidal activity of sunlight is mainlyThe bactericidal activity of sunlight is mainly
due to the presence of ultra violet rays.due to the presence of ultra violet rays.
 It is responsible for spontaneous sterilizationIt is responsible for spontaneous sterilization
in natural conditions.in natural conditions.
 the sunlight is more effective in killing germsthe sunlight is more effective in killing germs
due to combination of ultraviolet rays anddue to combination of ultraviolet rays and
heat.heat.

13. HeatHeat
 Heat acts by oxidative effects as well asHeat acts by oxidative effects as well as
denaturation and coagulation of proteins.denaturation and coagulation of proteins.
 Heat is considered to be most trustfulHeat is considered to be most trustful
method of sterilization for which articles thatmethod of sterilization for which articles that
can withstand heat.can withstand heat.
 Those articles that cannot withstand highThose articles that cannot withstand high
temperatures can still be sterilized at lowertemperatures can still be sterilized at lower
temperature by prolonging the duration oftemperature by prolonging the duration of
exposure.exposure.

14. Dry heatDry heat
 Dry heat acts by protein denaturation,Dry heat acts by protein denaturation,
oxidative damage and toxic effects ofoxidative damage and toxic effects of
elevated levels of electrolytes.elevated levels of electrolytes.

15. Dry heatDry heat
Flaming:Flaming:
 Inoculation loopInoculation loop
or Wire, the tipor Wire, the tip
of Forceps andof Forceps and
spatulas arespatulas are
held in aheld in a
bunsen flame tillbunsen flame till
they are red hotthey are red hot..

16. Dry heatDry heat
INCINERATIONI:INCINERATIONI:
This is an excellent methodThis is an excellent method
of destroying materials suchof destroying materials such
as contaminatedas contaminated cloth,cloth,
animal carcassesanimal carcasses andand
pathologicalpathological materials.materials.

17. Moist heatMoist heat
 Moist heat acts by coagulation andMoist heat acts by coagulation and
denaturation of proteins.denaturation of proteins.

18. MOIST HEAT STERILISATION IS CARRIED OUTMOIST HEAT STERILISATION IS CARRIED OUT
WITH FOLLOWING METHODSWITH FOLLOWING METHODS
 Temp below 100Temp below 100oo
C:C: “Pasteurisation”“Pasteurisation”
 Temperature at 100Temperature at 100oo
C:C: Boiling.Boiling.
 Steam at atmospheric pressure:Steam at atmospheric pressure: Koch/Arnold’sKoch/Arnold’s
steamer.steamer.
 Steam under pressure:Steam under pressure: Autoclave.Autoclave.

19. Temp below 100Temp below 100oo
CC
Pasteurisation:Pasteurisation:
 originally employed by Louis Pasteur.originally employed by Louis Pasteur.
 Use in food and dairy industry.Use in food and dairy industry.
 Process of killing of pathogens in the milkProcess of killing of pathogens in the milk
but does not sterilize it .but does not sterilize it .
 There are two methods of pasteurization:- theThere are two methods of pasteurization:- the
holder method (heated at 63oC for 30holder method (heated at 63oC for 30
minutes) and flash method (heated at 72oCminutes) and flash method (heated at 72oC
for 15 seconds) followed by quickly coolingfor 15 seconds) followed by quickly cooling
to 13oC.to 13oC.

20. Vaccines(water bath):Vaccines(water bath):
 The contaminating bacteria in a vaccineThe contaminating bacteria in a vaccine
preparation can be inactivated by heating in apreparation can be inactivated by heating in a
water bath at 60oC for one hour.water bath at 60oC for one hour.
 Only vegetative bacteria are killed and sporesOnly vegetative bacteria are killed and spores
survive.survive.
Temp below 100Temp below 100oo
CC

21. Temp below 100Temp below 100oo
CC
Serum bath:Serum bath:
 The contaminating bacteria in a serumThe contaminating bacteria in a serum
preparation can be inactivated by heating in apreparation can be inactivated by heating in a
water bath at 56oC for one hour on severalwater bath at 56oC for one hour on several
successive days.successive days.
 Only vegetative bacteria are killed and sporesOnly vegetative bacteria are killed and spores
survive.survive.

22. TEMPERATURE AT 100TEMPERATURE AT 100OO
CC
Boiling:Boiling:
Kills vegetative forms of bacterialKills vegetative forms of bacterial
pathogens.pathogens.
 Hepatitis virus: Can survive up to 30Hepatitis virus: Can survive up to 30
minutes of boiling.minutes of boiling.
Endospores: Can survive up to 20 hoursEndospores: Can survive up to 20 hours
or more of boilingor more of boiling

23. STEAM AT ATMOSPHERIC PRESSURESTEAM AT ATMOSPHERIC PRESSURE
 Steam is generated using a steamerSteam is generated using a steamer
(Koch/ Arnold)(Koch/ Arnold)
 Consists of a Tin cabinetConsists of a Tin cabinet
 Has a conical lid to enable the drainageHas a conical lid to enable the drainage
of condensed steamof condensed steam
 Perforated tray above ensuresPerforated tray above ensures
materials are surrounded by steam.materials are surrounded by steam.
 For routine sterilization exposure of 90For routine sterilization exposure of 90
mins is usedmins is used

24.  For media containing sugar and gelatinFor media containing sugar and gelatin
exposure of 100exposure of 100oo
C for 20 min for 3C for 20 min for 3
successive days is usedsuccessive days is used

25. STEAM UNDER PRESSURE -STEAM UNDER PRESSURE - AUTOCLAVEAUTOCLAVE
 Works on the principle ofWorks on the principle of Steam underSteam under
pressurepressure
 Invented byInvented by Charles ChamberlandCharles Chamberland inin 18791879..

26. AUTOCLAVE

27.  Autoclave consists of a vertical or aAutoclave consists of a vertical or a
horizontal cylinder.horizontal cylinder.
 One end has an opening which isOne end has an opening which is
meant for keeping materials to bemeant for keeping materials to be
sterilised.sterilised.
 The lid is provided with a PressureThe lid is provided with a Pressure
gauge, to measure the pressuregauge, to measure the pressure
 A safety valve is present to permit theA safety valve is present to permit the
escape of steam from the chamberescape of steam from the chamber

28.  Articles to be sterilised are placed inArticles to be sterilised are placed in
the basket providedthe basket provided
 Sterilisation is carried out underSterilisation is carried out under
pressure at 121pressure at 121ºº for 15 mnts.for 15 mnts.

29. 1.Physical agents1.Physical agents
II.II. RadiationRadiation
 Two types of radiations are usedTwo types of radiations are used
a)a) Ionizing :-x ray , cathod , gammaIonizing :-x ray , cathod , gamma
- sterilization- sterilization
b)b) Non ionizing :-uvNon ionizing :-uv
-disinfection-disinfection

30. IONISING RADIATIONSIONISING RADIATIONS
 X- rays, gamma rays & cosmic rays.X- rays, gamma rays & cosmic rays.
 ​​High effective strong powerHigh effective strong power
 No appreciable increase in theNo appreciable increase in the
temperature –temperature – COLD STERILISATIONCOLD STERILISATION
 Sterilise plastics Syringes, catheters,Sterilise plastics Syringes, catheters,
grease fabrics metal foilsgrease fabrics metal foils

31. Non- Ionising radiationNon- Ionising radiation::
 Electromagnetic rays with longerElectromagnetic rays with longer
wavelengthwavelength
 Absorbed as heatAbsorbed as heat
 Can be considered as hot air sterilisationCan be considered as hot air sterilisation
 Used in rapid mass sterilisation ofUsed in rapid mass sterilisation of
prepacked Syringes and cathetersprepacked Syringes and catheters
Eg: UV raysEg: UV rays

32. ULTRASONIC AND SONIC VIBRATIONULTRASONIC AND SONIC VIBRATION
 BactericidalBactericidal
 Microorganisms vary in theirMicroorganisms vary in their
sensitivity, hence no practical value insensitivity, hence no practical value in
sterilisation.sterilisation.

33. Chemical AgentChemical Agent
Gas Liquids
Sterilization Disinfection Animate Inanimate
Chemotherapy Antiseptics Sterilization Disinfection

34. CHEMICAL AGENTSCHEMICAL AGENTS
Chemical agents act byChemical agents act by
 Protein coagulationProtein coagulation
 Disruption of the cell membraneDisruption of the cell membrane
 Removal of Sulphydryl groupsRemoval of Sulphydryl groups
 Substrate competitionSubstrate competition

35. ALCOHOLSALCOHOLS
 Ethanol /Isopropyl alcohol areEthanol /Isopropyl alcohol are
frequently usedfrequently used
 No action on sporesNo action on spores
 Concentration recommended 60-90% inConcentration recommended 60-90% in
waterwater
UsesUses
 Disinfection of clinical thermometer.Disinfection of clinical thermometer.
 Disinfection of the skin – VenupunctureDisinfection of the skin – Venupuncture

36. ALDEHYDESALDEHYDES
 Formaldehyde & Glutaraldehyde areFormaldehyde & Glutaraldehyde are
frequently usedfrequently used
 Formaldehyde is bactericidal,Formaldehyde is bactericidal,
sporicidal & has a lethal effect onsporicidal & has a lethal effect on
viruses.viruses.
 Glutaraldehyde is effective againstGlutaraldehyde is effective against
Tubercle bacilli, fungi and virusesTubercle bacilli, fungi and viruses

37. USESUSES
FORMALDEHYDEFORMALDEHYDE
 To preserve anatomical specimensTo preserve anatomical specimens
 Destroying Anthrax spores in hair andDestroying Anthrax spores in hair and
woolwool
 10% Formalin+0.5% Sodium tetra10% Formalin+0.5% Sodium tetra
borate is used to sterilise metalborate is used to sterilise metal
instrumentsinstruments

38. USESUSES
GLUTARALDEHYDEGLUTARALDEHYDE
 Used to treat corrugated rubberUsed to treat corrugated rubber
anesthetic tubes, Face masks, Plasticanesthetic tubes, Face masks, Plastic
endotracheal tubes, Metal instrumentsendotracheal tubes, Metal instruments
and polythene tubingand polythene tubing

39. DYESDYES
Two groups of dyes are usedTwo groups of dyes are used
Aniline dyesAniline dyes
Acridine dyesAcridine dyes

40. ANILINE DYESANILINE DYES
 Are Brilliant green, Malachite green &Are Brilliant green, Malachite green &
Crystal violetCrystal violet
 Active against Gram positive bacteriaActive against Gram positive bacteria
 No activity against tubercle bacilliNo activity against tubercle bacilli

41. ACRIDINE DYESACRIDINE DYES
 Acridine dyes in use are orange inAcridine dyes in use are orange in
colourcolour
 Effective against Gram positive thanEffective against Gram positive than
Gram negativeGram negative
 Important dyes are Proflavine,Important dyes are Proflavine,
Acriflavine,EuflavineAcriflavine,Euflavine

42. HALOGENSHALOGENS
 IodineIodine in aqueous and alcoholicin aqueous and alcoholic
solution has been used widely as asolution has been used widely as a
skin disinfectantskin disinfectant
 Actively bactericidal with moderateActively bactericidal with moderate
against sporesagainst spores
 ChlorineChlorine and its compounds have beenand its compounds have been
used as disinfectants in water suppliesused as disinfectants in water supplies
& swimming pools& swimming pools

43. PHENOLSPHENOLS
 Obtained by distillation of coal tarObtained by distillation of coal tar
 Phenols are powerful microbicidalPhenols are powerful microbicidal
substancessubstances
 Phenolic derivatives have been widelyPhenolic derivatives have been widely
used as disinfectants for variousused as disinfectants for various
purposes in hospitalspurposes in hospitals
 Eg: Lysol, cresolEg: Lysol, cresol

44. USESUSES
 Various combinations are used in theVarious combinations are used in the
control of pyogenic cocci in surgical &control of pyogenic cocci in surgical &
neonatal units in hospitals.neonatal units in hospitals.
 Aqueous solutions are used inAqueous solutions are used in
treatment of woundstreatment of wounds

45. GASESGASES
Ethylene OxideEthylene Oxide
Colourless ,Highly penetratingColourless ,Highly penetrating
gas with a sweet ethereal smell.gas with a sweet ethereal smell.
Effective against all types ofEffective against all types of
microorganisms includingmicroorganisms including
viruses and sporesviruses and spores

46. USESUSES
 Specially used for sterilising heart-Specially used for sterilising heart-
lunglung
machines,respirators,sutures,dentalmachines,respirators,sutures,dental
equipments, books and clothing.equipments, books and clothing.
 Also used to sterilise Glass, metal andAlso used to sterilise Glass, metal and
paper surfaces ,plastics, oil,somepaper surfaces ,plastics, oil,some
foods and tobacco.foods and tobacco.

47. FORMALDEHYDE GASFORMALDEHYDE GAS
 Widely employed for fumigation ofWidely employed for fumigation of
operation theatres and other roomsoperation theatres and other rooms

48. BETA PROPIOLACTONEBETA PROPIOLACTONE
 Used in fumigationUsed in fumigation
 For sterilisation 0.2% BPL is usedFor sterilisation 0.2% BPL is used
 Has a rapid biocidal activityHas a rapid biocidal activity
 Very effective against virusesVery effective against viruses

49. Mechanical RemovalMechanical Removal
MethodsMethods
Filtration
Air Liquids
Disinfection Sterilization

50. Sterilisation by filtrationSterilisation by filtration
Filtration helps to remove bacteria fromFiltration helps to remove bacteria from
heat labile liquids such as sera andheat labile liquids such as sera and
solutions of sugar, Antibioticssolutions of sugar, Antibiotics..
The following filters are usedThe following filters are used
Candle filtersCandle filters
Asbestos filtersAsbestos filters
Sintered glass filterSintered glass filter
Membrane filtersMembrane filters

51. CANDLE FILTER

52. CANDLE FILTERSCANDLE FILTERS
 Widely used for purification of waterWidely used for purification of water
Two typesTwo types
(a) Unglazed ceramic filter –(a) Unglazed ceramic filter –
Chamberland filterChamberland filter
(b) Diatomaceous earth filters –(b) Diatomaceous earth filters –
Berkefeld filterBerkefeld filter

53. ASBESTOS FILTERASBESTOS FILTER
 Disposable single use discsDisposable single use discs
 High adsorbing tendencyHigh adsorbing tendency
 CarcinogenicCarcinogenic
Eg: Seitz filterEg: Seitz filter

54. SINTERED GLASS FILTERSINTERED GLASS FILTER
 Prepared by heat fusing powderedPrepared by heat fusing powdered
glass particles of graded sizeglass particles of graded size
 Cleaned easily, brittle, expensive.Cleaned easily, brittle, expensive.

55. MEMBRANE FILTERSMEMBRANE FILTERS
 Made of cellulose esters or otherMade of cellulose esters or other
polymerspolymers
UsesUses
 Water purification & analysisWater purification & analysis
 Sterilization & sterility testingSterilization & sterility testing
 Preparation of solutions for parenteralPreparation of solutions for parenteral
useuse

57. Reference:Reference:
1.1. Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig,Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig,
Tata McGraw HillTata McGraw Hill
2. Brock Biology of Microorganisms (13th Edition) by Michael T.2. Brock Biology of Microorganisms (13th Edition) by Michael T.
Madigan, John M. Martinko, David Stahl.Madigan, John M. Martinko, David Stahl.
3. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R.3. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R.
FunkeFunke
4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne
M. Willey, Linda MM. Willey, Linda M

58. THANK YOUTHANK YOU