sterilization and disinfection

This is a series of lectures on microbiology useful for undergraduate medical and paramedical students

1. STERILIZATION STERILIZATION
AND DISINFECTIONAND DISINFECTION
Dr. Ashish JawarkarDr. Ashish Jawarkar
M.D.M.D.
Parul Sevashram HospitalParul Sevashram Hospital

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

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

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

5. DISINFECTION:DISINFECTION:

Reducing the number of pathogenic
microorganismsto the point where
they no longer cause diseases.

6. SepsisSepsis: : Comes from Greek for decay or Comes from Greek for decay or
putrid. Indicates bacterial contamination.putrid. Indicates bacterial contamination.
AsepsisAsepsis: Absence of significant : Absence of significant
contamination.contamination.
Aseptic techniquesAseptic techniques are used to prevent are used to prevent
contamination of surgical instruments, contamination of surgical instruments,
medical personnel, and the patient during medical personnel, and the patient during
surgery. surgery.
Aseptic techniques are also used to Aseptic techniques are also used to
prevent bacterial contamination in food prevent bacterial contamination in food
industry.industry.

7. Bacteriostatic Agent: Bacteriostatic Agent:
An agent that An agent that inhibitsinhibits the growth of the growth of
bacteria, but does not necessarily kill bacteria, but does not necessarily kill
them. them.

8. BactericideBactericide: :
An agent that kills bacteria. Most do not kill An agent that kills bacteria. Most do not kill
Endospores.Endospores.
SporicideSporicide::
An agent that kills spores.An agent that kills spores.

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

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

11. Chemical AgentChemical Agent
Gas Liquids
SterilizationDisinfectionAnimate Inanimate
ChemotherapyAntisepticsSterilizationDisinfection

12. Mechanical Removal Mechanical Removal
MethodsMethods
Filtration
Air Liquids
Disinfection Sterilization

13. Physical Methods of SterilisationPhysical Methods of Sterilisation
Sterilisation By Dry Heat:Sterilisation By Dry Heat:
Hot Air Oven
•
Kills by oxidation effects
•The oven utilizes dry heat to
sterilize articles
• Operated between 50
o
C to
250/300
o
C.
•A holding period of 160
o
C for 1 hr
is desirable.
• There is a thermostat controlling
the temperature.
•Double walled insulation keeps the
heat in and conserves energy,

14. Uses:Uses:
To sterilise Forceps, Scissors, Scalpels, Swabs.To sterilise Forceps, Scissors, Scalpels, Swabs.
Pharmaceuticals products like Liquid paraffin, Pharmaceuticals products like Liquid paraffin,
dusting powder, fats and grease.dusting powder, fats and grease.

15. FLAMINGFLAMING
Inoculation loop Inoculation loop
or Wire, the tip of or Wire, the tip of
Forceps and Forceps and
spatulas are held spatulas are held
in a bunsen flame in a bunsen flame
till they are red till they are red
hothot..

16. Flaming Flaming

17. INCINERATIONINCINERATION
This is an excellent method of This is an excellent method of
destroying materials such as destroying materials such as
contaminated cloth, animal contaminated cloth, animal
carcasses and pathological carcasses and pathological
materials.materials.

18. IncinerationIncineration

19. Dry heat - HOT AIR OVENDry heat - HOT AIR OVEN
Holding temp & time: 160Holding temp & time: 160
00
C for 1 hrC for 1 hr
Used for glassware, forceps, swabs, water Used for glassware, forceps, swabs, water
impermeable oils, waxes & powdersimpermeable oils, waxes & powders
Before placing in hot air oven Before placing in hot air oven
Dry glassware completelyDry glassware completely
Plug test tubes with cotton woolPlug test tubes with cotton wool
Wrap glassware in Kraft papersWrap glassware in Kraft papers
Don’t over load the ovenDon’t over load the oven
Allow free circulation of air between the material Allow free circulation of air between the material

20. Dry heat - HOT AIR OVENDry heat - HOT AIR OVEN
Sterilization controls: to check whether the Sterilization controls: to check whether the
equipment is working properlyequipment is working properly
Chemical controls: Browne’s tubesChemical controls: Browne’s tubes
Color change from red to greenColor change from red to green
ThermocouplesThermocouples
Biological controls: paper strips containing10Biological controls: paper strips containing10
66
spores spores
of Clostridium tetaniof Clostridium tetani
Place strips in oven along with other material for the Place strips in oven along with other material for the
sterilization sterilization
Later culture the strips in thioglycollate broth or RCM at 37Later culture the strips in thioglycollate broth or RCM at 37
00
C C
for 5 daysfor 5 days
Growth in medium indicates failure of sterilizationGrowth in medium indicates failure of sterilization

21. Dry heat – Hot air ovenDry heat – Hot air oven

22. MOIST HEAT STERILIZATIONMOIST HEAT STERILIZATION
Kills microorganisms byKills microorganisms by coagulatingcoagulating their their
proteins. proteins.

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

24. PasteurisationPasteurisation
Process of killing of pathogens in the milk but Process of killing of pathogens in the milk but
does not sterilize it .does not sterilize it .
Milk is heated at 63Milk is heated at 63
oo
C for 30 mins.C for 30 mins.
(HOLDER METHOD)(HOLDER METHOD)
At 72At 72
oo
C for 15-20 Sec. Rapid cooling to 13C for 15-20 Sec. Rapid cooling to 13
oo
CC
(FLASH PROCESS)(FLASH PROCESS)

25. Water BathWater Bath

26. HOT WATER BATHHOT WATER BATH
To inactivate non sporing bacteria for the To inactivate non sporing bacteria for the
preparation of vaccines - preparation of vaccines - Special vaccineSpecial vaccine
bathbath at 60 at 60
oo
CC

for one hour is usedfor one hour is used
Serum or body fluids containing Serum or body fluids containing
coagulable proteins can be sterilized by coagulable proteins can be sterilized by
heating for 1 hr at 56heating for 1 hr at 56
oo
C in a water bath for C in a water bath for
several successive days.several successive days.

27. Inspissator

28. INSPISSATORINSPISSATOR
Sterilizes by heating at 80-85Sterilizes by heating at 80-85
oo
C for half an C for half an
hour for 3 successive dayshour for 3 successive days
Used to sterilize media such as Used to sterilize media such as
Lowenstein-Jensen & Loefller’s serumLowenstein-Jensen & Loefller’s serum

29. TEMPERATURE AT 100TEMPERATURE AT 100
OO
CC
Boiling:Boiling:
Kills vegetative forms of bacterial Kills vegetative forms of bacterial
pathogens. pathogens.
 Hepatitis virusHepatitis virus: Can survive up to 30 : Can survive up to 30
minutes of boiling.minutes of boiling.
EndosporesEndospores: Can survive up to 20 hours or : Can survive up to 20 hours or
more of boilingmore of boiling

30. Steam Sterilizer

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

32. For media containing sugar and gelatin For media containing sugar and gelatin
exposure of 100exposure of 100
oo
C for 20 min for 3 C for 20 min for 3
successive days is usedsuccessive days is used
The process is termed as The process is termed as
Tyndallisation /Intermittent SterilizationTyndallisation /Intermittent Sterilization

33. STEAM UNDER PRESSURE - STEAM UNDER PRESSURE - AUTOCLAVEAUTOCLAVE
Works on the principle of Works on the principle of Steam under Steam under
pressurepressure
Invented by Charles Chamberland in 1879. Invented by Charles Chamberland in 1879.

34. AUTOCLAVE

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

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

37. 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

38. ALCOHOLSALCOHOLS
Ethanol /Isopropyl alcohol are frequently Ethanol /Isopropyl alcohol are frequently
usedused
No action on sporesNo action on spores
Concentration recommended 60-90% in Concentration recommended 60-90% in
waterwater
UsesUses
Disinfection of clinical thermometer.Disinfection of clinical thermometer.
Disinfection of the skin – Venupuncture Disinfection of the skin – Venupuncture

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

40. USESUSES
FORMALDEHYDEFORMALDEHYDE
To preserve anatomical specimens To preserve anatomical specimens
Destroying Anthrax spores in hair and Destroying Anthrax spores in hair and
woolwool
10% Formalin+0.5% Sodium tetra borate 10% Formalin+0.5% Sodium tetra borate
is used to sterilise metal instrumentsis used to sterilise metal instruments

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

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

43. 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

44. ACRIDINE DYESACRIDINE DYES
Acridine dyes in use are orange in colourAcridine dyes in use are orange in colour
Effective against Gram positive than Gram Effective against Gram positive than Gram
negativenegative
Important dyes are Proflavine, Important dyes are Proflavine,
Acriflavine,Euflavine Acriflavine,Euflavine

45. HALOGENSHALOGENS
IodineIodine in aqueous and alcoholic solution in aqueous and alcoholic solution
has been used widely as a skin has been used widely as a skin
disinfectantdisinfectant
Actively bactericidal with moderate against Actively bactericidal with moderate against
spores spores
ChlorineChlorine and its compounds have been and its compounds have been
used as disinfectants in water supplies & used as disinfectants in water supplies &
swimming poolsswimming pools

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

47. USESUSES
Various combinations are used in the Various 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 in treatment Aqueous solutions are used in treatment
of woundsof wounds

48. GASESGASES
Ethylene OxideEthylene Oxide
Colourless ,Highly penetrating gas Colourless ,Highly penetrating gas
with a sweet ethereal smell.with a sweet ethereal smell.
Effective against all types of Effective against all types of
microorganisms including viruses microorganisms including viruses
and sporesand spores

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

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

51. 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

52. SURFACE ACTIVE AGENTSSURFACE ACTIVE AGENTS AND AND
METALLIC SALTSMETALLIC SALTS
Substances which reduce the surface Substances which reduce the surface
tension – tension –
Surface active agentsSurface active agents

53. Cations are widely used in the form of Cations are widely used in the form of
quaternary ammonium compounds.quaternary ammonium compounds.
Markedly bactericidal, active against Gram Markedly bactericidal, active against Gram
positive organisms.positive organisms.
No action on spores, tubercle bacilli, No action on spores, tubercle bacilli,
virusesviruses

54. METALLIC SALTSMETALLIC SALTS
The salts of silver, copper and mercury are The salts of silver, copper and mercury are
used as disinfectants.used as disinfectants.
Act by coagulating proteinsAct by coagulating proteins
Marked bacteriostatic, weak bactericidal Marked bacteriostatic, weak bactericidal
and limited fungicidal activityand limited fungicidal activity

55. Sterilisation by filtrationSterilisation by filtration
Filtration helps to remove bacteria from heat Filtration helps to remove bacteria from heat
labile liquids such as sera and solutions of labile liquids such as sera and solutions of
sugar, Antibioticssugar, Antibiotics..

The following filters are usedThe following filters are used
Candle filtersCandle filters
Asbestos filtersAsbestos filters
Sintered glass filterSintered glass filter
Membrane filtersMembrane filters

56. CANDLE FILTER

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

58. SEITZ FILTER
ASBESTOS DISCS

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

60. SINTERED GLASS FILTER

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

62. MEMBRANE FILTER

63. MEMBRANE FILTERSMEMBRANE FILTERS
Made of cellulose esters or other polymersMade of cellulose esters or other polymers
Uses Uses
Water purification & analysisWater purification & analysis
Sterilization & sterility testing Sterilization & sterility testing
Preparation of solutions for parenteral usePreparation of solutions for parenteral use

64. RADIATIONRADIATION
Two types of radiations are usedTwo types of radiations are used
NON –IONISINGNON –IONISING
IONISINGIONISING

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

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

67. ULTRASONIC AND SONIC VIBRATIONULTRASONIC AND SONIC VIBRATION
Bactericidal Bactericidal
Microorganisms vary in their sensitivity, Microorganisms vary in their sensitivity,
hence no practical value in sterilisation hence no practical value in sterilisation
and disinfectionand disinfection

68. THANK YOUTHANK YOU