1. Sterlization & Disinfection
Dr Rehma Dar
Assistant professor Pathology, KEMU

2. Learning Objectives What is Sterlization? What is Disinfection?
Chemical and physical methods of disinfection and sterilization
Principles and application of each method
Common disinfectants and antiseptics used in healthcare setting
Quality control of sterlization techniques

3. Historical background
The scientific use of disinfection and sterilization methods originated more than 100 years ago
Ignatz Semmelweis( ) and Joseph Lister( ) - important pioneers for the promotion of infection control

4. More than 100 years ago, Semmelweis demostrated that routine handwashing can prevent the spread of disease
He worked in a hospital in Vienna when maternity patients were dying an alarming rate
He recognized that medical students worked on cadavers during an anatomy class and afterwords they went to the maternity ward.
Students did not wash their hands between touching the dead and the living!!!
After administrating handwashing before examining the maternity patients the mortality rate decreased

6. In 1867, Lister introduced British surgery to hand washing and the use of phenol as antimicrobial agent for surgical wound dressings
• His principles were gradually and adopted in Britain and later in US
• This was the beginning of infection control

7. Sterilization
destruction of ALL forms of life, including the bacterial spores, viruses, prions
no degrees of sterilization: an all-or-nothing process
Physical or Chemical methods

8. Disinfection
A process that eliminates a defined scope of microrganisms, except most spores, viruses and prions
Purpose- prevent transmission of certain microorganisms with objects, hands or skin and prevent spreading the infection
Physical or Chemical methods
Most disinfectants are chemical agents applied to inanimate objects !

9. Commonly used terms
Aseptic techniques - prevent microbial contamination of materials or wounds
Antisepsis - disinfection of living tissues(e.g., in a wound)
Antiseptics are applied (do not kill spores) to reduce or eliminate the number of bacteria from the skin and mucus membranes
Sanitizer- an agent, usually a detergent, that reduces the numbers of bacteria to a safe level

10. Factors that influence the degree of killing
Types of organisms
Number of organisms
Concentration of disinfecting agent
Presence of organic material (e.g., serum, blood)
Nature (composition) of surface to be disinfected
Contact time
Temperature pH
Biofilms
Compatibility of disinfectants and sterilants

11. Rate of killing of microorganisms
N α 1/CT where N = No of survivors C = Concentration of agent T= Time of application of agent *CT referred to as dose

12. Types of organisms
Organisms vary in their ability to withstand chemical and physical treatment e.g.,
Spores – have coats rich in proteins, lipids and carbohydrates
Mycobacteria – cell walls are rich in lipids
Biofilms - microorganisms living together in communities
Prions – the most resistant known organisms to the action of heat, chemicals, and radiation !!!

13. Different types of organisms and their resistance to killing agents

14. Number of organisms
The total number of organisms determine the exposure time of killing agent
Composed of organisms with varying degrees of susceptibility to killing agents
the death curve is logarithmic!!!
higher numbers of organisms require longer exposure

15. The effect of exposure time versus number of organisms

16. Concentration of disinfecting agent
Proper concentration of disinfecting agents ensure the activation of target organisms, e.g.,
Povidone-iodine should be diluted with water before use because there is not enough free iodine to kill microorganisms in concentrated solution

17. Presence of organic material
such as blood, mucus, pus affects killing activity by inactivating the disinfecting agent, e.g,
by coating the surface to be treated, prevents full contact between object and agent (Glutaraldehyde)
easily inactivate bleach (Sodium hypochlorite)
For optimal killing activity, instruments and surfaces should be cleansed of excess organic material before disinfection !!!

18. Nature (composition) of surface to be disinfected
Some medical instrumentsare manufactured of biomaterials that exclude the use of certain disinfection and sterlization methods because of possible damage, e.g.,
endoscopic instruments cannot be sterilized by the heat in an autoclave

19. Contact time
The amount of time a disinfectant or sterilant is in contact with the object is critical! e.g.,
The spores of bacteria and fungi need a much longer time
Determine whether it is disinfecting or sterilizing the object

20. Temperature Disinfectants are generally used at room temp. (20-25 ̊C)
Activity is increased by an increased temp.
Decreased by a drop in temp

21. pH
The pH of the material to be desinfected or sterilized can have an effect on the activity of disinfecting or sterilizing agent

22. Biofilms Communities of microorganisms
can be on a surface of either inanimate or animate objects, e.g.,
catheters (criticial place!), pipes that carry water and dionizing columsused to make processed water
make disinfection more difficult
Concentration of the disinfectant and the contact time need to be increased

23. Compatibility of disinfectants
A common mistake is to belive that two disinfectants are better than one !
some of them may inactivate other, e.g.,
the bleach and quaternary ammonium compound together negate the activity of both

24. Classes of Medical materials
Medical materials are categorized into
Criticial materials
Semicriticial materials
Noncriticial materials

25. Criticial materials
Invade sterile tissues or enter the vascular system
most likely to produce infection if contaminated and therefore require sterilization

26. Semicriticial materials
have contact with mucos membranes
require high-level disinfection agents

27. Non-criticial materials
have contact with intact skin
require intermediate- level to low- level disinfection

28. Types of disinfectants
High-level disinfectants
activity against bacterial spores
Intermediate-level disinfectants
tuberculocidal activity but not sporocidal
Low-level disinfectants
a wide range of activity against microorganisms but no sporocidal or tuberculocidal activity

29. Classification of Disinfectants
Physical methods
Chemical methods

30. Physical methods Heat moist heat dry heat pasteurization boiling
Filtration
Radiaton

31. Heat The most common method used for elimination of microorganisms !
reliable effects
ease of use
economical

32. Heat Moist heat (Boiling/Autoclaving) Dry heat Pasteurisation
Mechanism: denaturation of proteins, membrane damage and cleavage of DNA.

33. Moist heat (Autoclave)
(heat under steam pressure)
destroy ALL microorganisms including spores
steam under 15lb/inch2 of pressure, at 121 ̊C temp., minutes of exposure in autoclaves or
2 atm./ 134 ̊C / 3 min.
destroy the prions at higher temperatures, ̊C for at least 1hour under 2 atm
Application: method of choice for heat-stable objects

34. Autoclaves

35. Dry heat (Hot air oven)
requires much longer exposure times and higher temperatures than moist heat
2 hours at 180 ̊̊C
Application: Heat stable substances that are not penetrated by moist heat e.g glassware or surgical instruments

36. Dry heat oven

37. Boiling & Pasteurisation
Boiling and pasteurisation achieve disinfection but not sterilisation (do not eliminates spores)!!!
Boiling
kills most microorganisms in 10 min. at 10 ̊C
Pasteurisation
62 ̊C for 30 minutes
Flash pasteurisation 72 ̊C for 15 seconds
Application: Milk borne pathogens Mycobacterium bovis, Salmonells, Streptococcus, Listeria, Brucella

38. Filtration
The membrane filters composed of plastic polymers or cellulose esters containing pores of certain size
Mechanism: Liquid is pulled (vacum) or pushed (pressure) through the filter matrix, organisms larger than the size of the pores are retained and small particles are retained by electrostatic attraction of particles to filters

39. Commonly used : Nitrocellulose with pore size of 0.22 μm
Application: Sterlization of solutions witth heat sensitive components.

41. Filtration of Air filters remove microorganisms larger than 0.3 μm
the high efficiency particulate air (HEPA) filters
Application: In laboratory hoods and in rooms of immunocompromised patients

42. Radiation
Two forms:
Ionizing
Non-ionizing

43. Ionizing radiation
gamma rays, X rays or electron beams
short wavelenght and high energy
Mechanism: formation of free radicals- damage DNA
Application: sterilizationof disposable supplies (syringes,sututres, bandages, catheters and gloves) and heat-sensitive pharmaceuticals

44. Non-ionizing
Ultraviolet rays (UV) : nm
Long wavelenght and low energy
poor penetrabilty
limited use
Mechanism: Thymine dimers – inhibiting DNA replication

45. Application: Disinfect smooth surfaces with UV lamps and to reduce airborn pathogens (surgical theaters)

46. Chemical methods
Physical methods are used mainly to achieve sterilization
whereas
Chemical agents are used mainly as disinfectants
But
some chemical agents may be used to sterilize (chemosterilizers)

47. Chemical agents Alcohols Aldehydes Halogens Phenols Surfactants
Heavy metals
Dyes
Gases (ethylene oxide, oxidants)

48. Mechanism of action
Reaction with components of the cytoplasmic membrane (sufactant compounds, alcohols)
Denaturation of cellular proteins (alcohols, phenols, aldehydes, oxidants)
Reaction with the thiol (-SH) groups of enzymes (heavy metals)
Damage of RNA and DNA (aldehydes, oxidants, dyes)
The agent can exert one or a combination of actions on microorganisms

49. Classes of chemical agents according to action
Disruption of Cell membranes
Alcohol, Detergents
Modification of Proteins
Halogens, Heavy metals, H2O2, Aldehydes. Ethylene oxide, Acids & alkalis
Modification of Nucleic acids
Dyes

50. Alcohols Ethanol 70%, isopropanol 70%, propanol 60%
Mechanism: Inactivate microorganisms by disorganize lipids in membrane and denaturing proteins but not sporocidal!
The most effective concentrations are between 60%-90% (water is needed in chemical reactions)
Application: Ethanol – clean skin before venipuncture and surgical disinfection of hands

51. Detergents/ Surfactants
also known as surface-active agents surfactants e.g quaternary ammonium compounds
Mechanism: disrupt cell membrane by interacting lipid within it.
moderate bactericidal effect (good against Gram(+) but not sporecidal, tuberculocidal
Application: disinfectionof noncriticial surfaces (bench tops, floors) and skin antisepsis

52. Phenols
Mechanism: not only damage membranes but also denaturate proteins
broad-spectrum, but not sporocidal or virucidal
Application: disinfection of hospital and household environment

53. Halogens Chlorine, Iodine, and their derivatives
Mechanism: oxidizing agent that crosslinks sulfhydryl groups in enzymes to form inactive disulfide
Chlorine acts after dissolution of chloride ions with water and also used in the form of hypochlorite (household bleach)
Application: disinfection of water and swimming pool

54. Iodine
Mechanism: inactivates sulfhydryl containing enzymes and binds to tyrosine in proteins
Application: skin antiseptic
Tincture Iodine (2% iodine solution and potassium iodide in ethanol) used to prepare skin prior to blood culture.
Iodophors complex of iodine with detergent used to prepare skin prior to surgery.

55. Heavy metals Mercury and silver have greatest antibacterial activity.
Mechanism: Inactivate enzymes by binding to the sulfhydryl groups of enzymes
Application: Silver nitrate(1% eyedrop solution) used in the prevention of eyes infections
Silver sulfadiazine used to prevent infection of burn wounds

56. Hydrogen peroxide
oxidizing agent – effectiveness limited by organism’s ability to produce catalase.
Bubbles produced on wounds as a result of O2 produced by tissue catalase on H2O2
Mechanism: inactivates sulfhydryl containing enzymes
Application: antiseptic to clean wounds and disinfect contact lenses.

57. Aldehydes
Formaldehyde - the most imporatant & glutaraldehyde (disinfectant and sterilizer!!!)
Mechanism: denaturate proteins and nucleic acids
Application: disinfection of surfaces and objects (plastic and rubber items)
Glutaraldehyde ( Cidex) – the sterilizer of choice for heat-sensitive medical equipment e.g endoscopes

58. Gases Ethylene oxide - the most commonly used for sterilization
Mechanism: alkylates proteins and nucleic acids
Application: widely in hospitals for heat sensitive materials like surgical instruments and

59. Dyes acridine dyes, brilliant green, crystal violet
Mechanism interact with bacterial nucleic acids
Application: used topically as antiseptics to treat e.g. mild burns

60. Quality Control of Sterilization
To ensure that potentially infectious agents are destroyed by adequate sterilization regimes
Three levels:
Physical
Chemical
Biological

61. Physical: includes measuring device control temperature, time and pressure
Chemical: substances that undergo a colour change or have melting points within the sterilizing range e.g
Browne's tubes, Bowie Dick tape
give an immediate indication of successful sterilization

62. Biological
Bacillus stearothermophilus spores
survives steam heat at 121 ̊C for 5 min. and is killed at 121 ̊C in 13 min.
validate the adequacy of steam sterilization
Bacillus subtilis spores
validate the adequacy of dry heat sterilization

63. Chemical indicators
Browne's tubes are glass tubes that contain heat sensitive dyes. These change colour after sufficient time at the desired temperature.
Before heat exposure, the contents of the tube appear red.
As heating progresses, the colour changes to green indicating adequate sterlization.

64. Bowie Dick tape is applied to articles being autoclaved
Bowie Dick tape is applied to articles being autoclaved. Before heat exposure, the tape is uniformly buff in colour.
After adequate heating, the tape develops dark brown stripes.
The pack on the left has been properly sterilised; that on the right has not.

65. Sterilization methods
Physical methods
Moist heat in autoclaves
Dry-heat in ovens
Gamma irradiaton
Filtration
Chemical agents
Ethylene oxide
Glutaraldehye (high concentration)
Disinfection methods
Physical methods
Boiling and pasteurisation
Ultraviolet radiation
Chemical agents
Alcohols
Aldehydes
Halogens
Phenols
Surfactants
Heavy metals
Dyes
Oxidants

66. Thank you