Sterilisation and disinfection methods lecture notes for Allied Health Sciences and Nursing Students. Various methods of sterilisation and disinfection used in health care settings in order to prevent hospital acquired infection.

1. Sterilisation and Disinfection for
allied health science students and
nurses
Dr. I. Kannan Ph.D
Associate Professor
Department of Microbiology
Tagore Medical College and Hospital
Chennai – 600127. INDIA
Lecture series for Allied Health Sciences students and Nurses

2. DEFINITION
STERILISATION
• The process of freeing an article, surface or medium
from microorganisms either in vegetative form or
spores.
STERILANT
The agent capable of sterilising objects.

3. DEFINITIONS
DISINFECTION
• Reducing the number of pathogenic microorganisms
to the point where they no longer cause diseases .
DISINFECTANTS
Agents (mostly chemical) used to disinfect inanimate
objects.

4. DEFINITION
Sepsis: Comes from Greek for decay or putrid.
Indicates bacterial contamination.
Asepsis: Absence of significant contamination.
• Aseptic techniques are used to prevent
contamination of surgical instruments, medical
personnel, and the patient during surgery.
• Aseptic techniques are also used to prevent bacterial
contamination in food industry.

5. DEFINITION
ANTISEPTICS
Chemicals applied to living tissue to prevent / inhibit
pathogen growth.

6. Terminal disinfection
• Application of disinfection measures after the patient
has been removed, e.g., by death, or has ceased to
be a source of infection.
• the objective is to prepare the rooms or areas for
subsequent occupancy of patients for them to be
treated without the risk of acquiring an infection.

7. Sterilisation
Physical
method
Chemical
method

8. PHYSICAL METHODS OF
STERILISATION

9. Physical Methods
1) Sunlight
2)Drying
3)HEAT
4)FILTRATION
5)RADIATION
6)Ultrasonic/sonic vibration

10. HEAT

11. There are methods:
• Dry heat
• Moist heat

12. •Dry heat sterilisation

13. Mode of action
• Denaturation of bacterial protein
• Oxidative damage
• Toxic effects of elevated levels of electrolytes

14. Red heat
• Inoculation loop or
Wire, the tip of
Forceps and
spatulas are held
in a bunsen flame
till they are red hot.

15. Flaming
• A variation on flaming is to dip the object in 70%
ethanol (or a higher concentration) and merely touch
the object briefly to the Bunsen burner flame, but not
hold it in the gas flame. The ethanol will ignite and
burn off in a few seconds.

16. Incineration
• The infective material is burnt into ashes in the
incinerator.
• This is an excellent method of destroying materials
such as contaminated cloth, animal carcasses and
pathological materials.

17. Hot air
• Dry heat utilizes hot air that is free from water vapour.
• Dry-heat destroys microorganisms by causing
coagulation of proteins.
• It is less penetrating and requires longer exposure
than moist heat

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

19. Hot air oven
Uses:
• To sterilise glasswares
• To sterilise Forceps, Scissors, Scalpels, Swabs.
• Pharmaceuticals products like Liquid paraffin, dusting powder,
fats and grease.

20. Sterilisation control
• Spores of non-toxigenic strain of Clostridium tetani
• Thermocouples
• Browne’s tube

21. •Moist heat sterilisation

22. Moist heat sterilization
• Kills microorganisms by denaturation and
coagulating their proteins.
• More penetrating power than dry heat.

23. Methods in moist heat sterilisation
• Temp below 100oC: “Pasteurisation”, Inspissator.
• Temperature at 100oC: Boiling.
• Steam at atmospheric pressure: Koch & Arnold’s
steamer.
• Steam under pressure: Autoclave.

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

25. Inspissation
• Sterilizes by heating at 80-85oC for half an hour for 3
successive days
• Used to sterilize media such as Lowenstein-Jensen &
Loefller’s serum

26. Inspissator

27. Hot water bath
• To inactivate non sporing bacteria for the preparation
of vaccines - Special vaccine bath at 60oC for one
hour is used
• Serum or body fluids containing coagulable proteins
can be sterilized by heating for 1 hr at 56oC in a water
bath for several successive days.

28. Water Bath

29. Temperature at 100oc
Boiling:
Kills vegetative forms of bacterial pathogens.
• Hepatitis virus: Can survive up to 30 minutes of
boiling.
Endospores: Can survive up to 20 hours or more of
boiling

30. Steam at atmospheric
pressure
• Steam is generated using a steamer (Koch/ Arnold)
• Consists of a Tin cabinet
• Has a conical lid to enable the drainage of condensed
steam
• Perforated tray above ensures materials are
surrounded by steam.
• For routine sterilization exposure of 90 mins is used

31. Steam Sterilizer

32. Tyndallisation /Intermittent
Sterilization
• For media containing sugar and gelatin exposure of
100oC for 20 min for 3 successive days is used
• The process is termed as Tyndallisation
/Intermittent Sterilization

33. Tyndallisation /Intermittent Sterilization
• The first exposure kills all the vegetative forms
• In the intervals between the heating the remaining
spores germinate into vegetative forms which are
killed on subsequent heating.

34. Steam under pressure - Autoclave
• Works on the principle of Steam
under pressure
• Invented by Charles Chamberland in 1879.

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

36. • Articles to be sterilised are placed in the basket
provided
• Sterilisation is carried out under pressure (15 lbs) at
121º C for 15 mnts.

37. An autoclave

38. Sterilisation control
• Thermocouple
• Bacterial spores- Bacillus stearothermophilus
• Autoclave tapes
• Chemical indicators-Browne’s tube contains
red solution which turns green at 121OC for
15 minutes

39. FILTRATION

40. Sterilisation by filtration
Filtration helps to remove bacteria from heat labile
liquids such as sera and solutions of sugar,
Antibiotics.
To obtain bacteria - free filtrates of clinical sample for
virus isolation.
Bacterial toxins and bacteriophages can be obtained by
passing the culture through filters
Filter disc - to concentrate bacteria from liquids e.g.
testing water sample for cholera vibrio and typhoid
bacilli.

41. Types of filters
The following filters are used
Candle filters
Asbestos filters
Sintered glass filter
Membrane filters

42. Candle filters
• Widely used for purification of water
Two types
(a) Unglazed ceramic filter – Chamberland filter and
Doulton filter
(b) Diatomaceous earth filters – Berkefeld filter and
Mandler filter

43. CANDLE FILTER

44. Asbestos filter
• Disposable single use discs
Disadvantage:
• High adsorbing tendency
• Carcinogenic
Eg: Seitz filter and Sterimat filter

45. SEITZ FILTER
ASBESTOS DISCS

46. Sintered glass filter
• Prepared by heat fusing powdered glass particles of
graded size
• Cleaned easily, brittle, expensive.

47. SINTERED GLASS FILTER

48. Membrane filters
• Made of cellulose esters or other polymers
• For sterilisation – 0.22µm pore size
• For water analysis – 0.45µm pore size
Uses
• Water purification & analysis
• Sterilization & sterility testing
• Preparation of solutions for parenteral use

49. MEMBRANE FILTER

50. HEPA filters
• High-efficiency particulate air (HEPA) filters are used
to filter the air flowing into aseptic environments (like
operation theatre).

51. RADIATION

52. Radiation
Two types of radiations are used
NON –IONISING
IONISING

53. Non- Ionising radiation:
• Infra red rays
–Absorbed as heat.
–Has low penetrating power
–Can be considered as hot air
sterilisation
–Used in rapid mass sterilisation of
prepacked Syringes and catheters

54. UV radiation
• Ultraviolet light irradiation is useful only for
sterilisation of surfaces and some transparent objects.
• Action: Denaturation of proteins and interferes in the
DNA replication.

55. UV radiation
• UV irradiation is routinely used to sterilize the interiors
of biological safety cabinets between uses and
operation theatre,
• But is ineffective in shaded areas, including areas
under dirt.
• It also damages some plastics, such as polystyrene
foam if exposed for prolonged periods of time.

56. IONISING RADIATIONS
• X- rays, gamma rays & cosmic rays.
• High penetrative power
• Action: lethal to DNA and other biomolecules.
• No appreciable increase in the temperature –
COLD STERILISATION
• Sterilise plastics Syringes, catheters, grease
fabrics, metal foils

57. Gamma rays
• Gamma rays are very penetrating and are commonly
used for sterilisation of disposable medical
equipment, such as syringes, needles, cannulas and
IV sets.

58. Ultrasonic and Sonic vibration
• High frequency sound – beyond the sensitivity of
human ear.
• They are known to disrupt the cells.
• Not used commonly used due to variable result.
• More effective to kill gram negative organism.

59. CHEMICAL METHODS OF
STERILISATION

60. Ideal disinfectant/antiseptic
• Have wide spectrum of activity
• Be active in presence of organic matter
• Effective in acid/alkali media
• Have speedy action
• Have high penetrating power
• Be stable
• Be compatible with other disinfectants

61. Ideal disinfectant/antiseptic
• Not corrode metals
• Not cause local irritation or sensitisation
• Should not interfere in healing process
• Not be toxic
• Cheap and should easily available

62. Factors that influence the potency of
disinfectant
• Concentration of substance
• Time of action
• pH of the media
• Temperature
• Nature of organism
• Presence of extraneous materials

63. Chemical methods
Chemical agents act by
• Protein coagulation
• Disruption of the cell membrane
• Removal of Sulphydryl groups
• Substrate competition

64. Alcohols
• Ethanol /Isopropyl alcohol are frequently
used
• Acts by denaturing bacterial proteins
• No action on spores except methyl alcohol
• Concentration recommended 70% in water
• Alcohol-water mixtures are more penetrating
than pure alcohols
Uses
• Disinfection of clinical thermometer.
• Disinfection of the skin – Venupuncture

65. Aldehydes
• Formaldehyde & Glutaraldehyde are frequently used
• Active against the amino group in the protein
molecule
• Formaldehyde is bactericidal, sporicidal & has a lethal
effect on viruses.
• Glutaraldehyde is effective against Tubercle bacilli,
fungi and viruses

66. Uses
FORMALDEHYDE
• To preserve anatomical specimens
• Used to prepare vaccines and toxoids
• Destroying Anthrax spores in hair and wool
• 10% Formalin+0.5% Sodium tetra borate is used to
sterilise metal instruments

67. Uses
GLUTARALDEHYDE
• Used to treat corrugated rubber anesthetic tubes,
Face masks, Plastic endotracheal tubes, Metal
instruments and polythene tubing

68. Oxidising agents
• Hydrogen peroxide (H2O2) is a typical
oxidising agent
• hydrogen peroxide is actually not a very
effective antiseptic or disinfectant
• This is because bacteria and body
tissues contain enzymes (catalase) that
inactivate hydrogen peroxide
• On the other hand, the oxygen released
upon inactivation can help oxygenate
deep wounds and thus kill strict-
anaerobe contaminants

69. Dyes
Two groups of dyes are used
Aniline dyes
Acridine dyes

70. Aniline dyes
• Are Brilliant green, Malachite green & Crystal violet
• Lethal effect due to their reaction with the acid groups
in the cell
• Active against Gram positive bacteria
• No activity against tubercle bacilli

71. Acridine dyes
• Acridine dyes in use are orange in colour
• Impair DNA and thus destroy the reproductive
capacity of the cell
• Effective against Gram positive than Gram
negative
• Important dyes are Proflavine,
Acriflavine,Euflavine

72. Halogens
• Iodine in aqueous and alcoholic solution has been
used widely as a skin disinfectant
• Actively bactericidal with moderate against spores
• Chlorine and its compounds have been used as
disinfectants in water supplies & swimming pools

73. Iodine
• Iodine is often employed as a tincture or as an
iodophor
• A tincture is an alcohol solution of a iodine.
• Iodine tinctures may be employed as antiseptics

74. Iodine
• Iodophors are organic compounds that slowly release
of iodine
• thus increasing penetration while simultaneously
steadily supplying iodine over long periods
• Betadine and Isodine are examples of iodophors

75. Chlorine
• Drinking water is commonly disinfected using
hypochlorite
• Hypochlorite may either be added directly (i.e., in the
form of bleach) or created within water by bubbling
chlorine gas through the water

76. Phenols
• Obtained by distillation of coal tar
• Phenols are powerful microbicidal substances
• Causes cell membrane damage.
• Phenolic derivatives have been widely used as
disinfectants for various purposes in hospitals
• Eg: Lysol, cresol, chlorhexidine

77. Uses
• First antiseptic discovered and used by Joseph Lister.
• Various combinations are used in the control of
pyogenic cocci in surgical & neonatal units in
hospitals.
• Aqueous solutions are used in treatment of wounds

78. Gases
Ethylene Oxide
–Colourless ,Highly penetrating
gas with a sweet ethereal smell.
–Effective against all types of
microorganisms including viruses
and spores
–Action due to alkylating amino,
carboxyl, hydroxyl and sylphydryl
groups in protein molecule

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

80. Formaldehyde gas
• Widely employed for fumigation of operation theatres,
wards, sick rooms and laboratories.
• Sterilisation of instruments and heat sensitive
catheters, clothing, bedding, furnitures , books etc.
• It is produced by adding 150 g of Potassium
permangnate to 280 ml of formalin in 1000 cu.ft of
room volume.

81. Beta propiolactone
• Used in fumigation
• For sterilisation 0.2% BPL is used
• Has a rapid biocidal activity
• Very effective against viruses

82. Surface active agents
Substances which reduce the surface tension – Surface
active agents

83. • Cations are widely used in the form of quaternary
ammonium compounds. Eg. Cetrimide.
• Markedly bactericidal, active against Gram positive
organisms.
• No action on spores, tubercle bacilli, viruses

84. Metallic salts
• The salts of silver, copper and mercury are used as
disinfectants.
• Act by coagulating proteins
• Marked bacteriostatic, weak bactericidal and limited
fungicidal activity

85. Metallic salts
• Silver nitrate has been used to treat
the eyes of newborns to kill any
Neisseria gonorrhea that may have
been acquired during passage down
the birth canal
• the treatment of ponds with copper
sulphate (which is blue in water
solution) as an anti-algal
• Selenium compounds are effective
antifungals

86. Checking of disinfectants
• Rideal-Walker Test
• Chick Martin Test
• Kelsey-Sykes Test
• In-Use Tests

87. Rideal-Walker Test
• Suspensions of same quantity of organisms are
subjected to different concentration of phenol and
disinfectant to be tested.
• The dilution of the disinfectant which sterilizes the
suspension is divided by corresponding phenol
concentration.
• This gives phenol coefficient.
• The phenol coefficient of 1.0 means that the
disinfectant is as effective as phenol.

88. Chick Martin test
• Modification of Rideal-Walker test.
• The disinfectant acts in presence of organic matter.

89. Kelsey-Sykes test
• This test gives a measure of the capacity of a
disinfectant to retain its activity when repeatedly used.
• It is also named as capacity test.

90. In Use test
• The liquid phase of the disinfectant solutions in actual
use is examined quantitatively for viable organisms.
• Then a ‘use’ dilution is determined.

91. STERILISATION OF HOSPITAL
INSTRUMENTS

92. Spaulding classification
• Critical – Objects which enter normally sterile tissue
or the vascular system and require sterilization
• Semi‐critical – Objects that contact mucous
membranes or non‐intact skin and require high‐level
disinfection, which kills all but high‐levels of bacterial
spores
• Non‐critical – Objects that contact intact skin but not
mucous membranes, and require low‐level
disinfection

93. Processing critical instruments
• Penetrate or enter normally sterile tissue or spaces,
including the vascular system. Example: Surgical
instruments (elevators, bone files, rongeurs, forceps,
etc.)
• Must be sterilized between uses or used as single use
disposable devices

94. Processing semi‐critical objects
• Contact mucous membranes and non‐intact skin.
Example: Mouth mirrors, cheek retractors,
handpieces
• Must be sterilized or immersed in high‐level
disinfectant

95. Non‐critical instruments and devices
• Contact intact skin. Example: BP cuffs,
electrocardiogram (EKG) leads, stethoscopes
• Disinfect using a low level disinfectant