This document defines various terms related to disinfection and sterilization such as sterilization, disinfection, antiseptics, sanitizers, asepsis, and provides classifications of different disinfection methods and agents used for achieving sterilization and disinfection in hospitals. It also discusses factors affecting the efficacy of sterilants/disinfectants and properties of an ideal disinfectant.

1. Disinfection
By Dr. Rakesh Prasad Sah
Associate Professor, Microbiology

2. Definition
• Sterilization : process of 100 % killing of m.os. either pathogenic
or non-pathogenic including spores from any article, surface or
objects.
• Process that destroys all (100%) pathogenic microorganisms but may
or may not destroy bacterial spores is called Disinfection.
• Agents  Disinfectants.

3. Definitions
• Antiseptics  type of disinfectant  safe to apply on body surface
(skin & mucosa)  destruction of organisms  present on the body
surfaces  type of disinfection is termed as Asepsis.
• Sanitizer: an agent reduces but may not eliminate m.os. To safe
level.
• Decontamination  process  removes/destroys all pathogenic
m.os. From an object to make it safe to handle.

4. Definitions
• Asepsis : refers to a technique that aids in preventing the onset of
infection in uninfected tissue.
• Cidal : Killing of m.os.
• Static : inhibit the growth of m.os.
• Germicide : agents which kills pathogenic m.os. (germs). includes
both antiseptics and disinfectants.

5. Factors affecting Efficacy of Sterilant/Disinfectant
• Organism load
• Nature of organisms
• Concentration
• Contact time
• Temperature
• Stability
• Local pH
• Relative humidity
• Organic matter
• Pus, serum, blood & stool can interfere with antimicrobial
activity of disinfectant. (hypochlorites), Overcome by
• Overcome by mechanical cleaning of instrument or
surface/floor before it is subjected for disinfection or
sterilization.
• Increase exposure time or concn. Of agent.
• Biofilm
• Prevents entry of disinfectants which embedded
inside biofilm.

6. Property of an Ideal Disinfectant
• Broader microbicidal activity.
• Fast acting.
• Not affected by environmental factors such as organic matter.
• Nontoxic.
• Compatible with surfaces/materials to which it is used.
• Environmental friendly.

7. Classification of disinfection methods
Disinfectant Examples
1 Alcohols Ethyl alcohol, isopropyl alcohol
2 Aldehydes
Formaldehyde, glutaraldehyde, Ortho-
phthalaldehyde
3 Phenolic compounds
Cresol, lysol, chlorhexidine, chloroxylenol,
hexachlorophene
4 Biguanide Chlorhexidine gluconate
5 Halogens Chlorine, iodine, iodophors
6 Oxidising agents Hydrogen peroxide , Peracetic acid

8. Classification of disinfection methods
7 Heavy metal salts Mercuric chloride, copper salts
8 Surface active agents Quaternary ammonium compounds and
soaps
9 Dyes aniline dyes and acridine dyes
10 Gas sterilization- • Low temperature steam formaldehyde
• Ethylene oxide (ETO)
• Betapropiolactone (BPL)
• Plasma sterilization

9. Type of Disinfectants
High level Disinfectants
(HLD)
• Capable of killing
bacterial spores  used
in sufficient
concentration under
suitable conditions.
• Kill all microorganisms.
Intermediate level
Disinfectant (ILD)
• Kills all
microorganisms
but not spores.
Low-level Disinfectants
(LLD)
• Kills vegetative bacteria
and enveloped viruses.
• Variable action on non-
enveloped viruses and
fungi. No action on
Tubercle bacilli and
spores.

10. Level of sterilant/ disinfectants according to
their microbicidal action
Level of
disinfectant/
sterilant
Bacterial
spores
Tubercle
bacilli
Non-
enveloped
viruses
Fungi Vegetative
bacteria
Enveloped
viruses
Sterilant Yes Yes Yes Yes Yes Yes
Disinfectant
High level +/- Yes Yes Yes Yes Yes
Intermediate
level
No Yes Yes Yes Yes Yes
Low level No No +/- +/- Yes Yes

11. Agents used in the hospital for achieving
sterilization, disinfection and cleaning
Agents Physical methods Chemical methods
Sterilant
Agents of sterilization  Steam sterilizer
(autoclave)
 Dry heat sterilizer (hot air
oven)
 Filtration
 Radiation: Ionizing and
non-ionizing (infrared)
 Others: Incineration,
microwave
 Ethylene oxide sterilizer
 Plasma sterilizer

12. Agents Physical methods Chemical methods
Disinfectants
High-level
disinfectants
No physical methods in this
category
 Aldehydes- glutaraldehyde,
orthophthaldehyde, formaldehyde
 Peracetic acid
 Hydrogen peroxide
Intermediate–level
disinfectants
 Heat-based methods:
Pasteurization, boiling and
steaming
 Ultraviolet (non-ionizing)
radiation
 Alcohols–ethyl alcohol and
isopropyl alcohol
 Phenolics–phenol, cresol, lysol
 Halogens–iodine and chlorine
Low-level
disinfectants
No physical methods in this
category
 Quaternary ammonium compound
(QAC)
 Chlorhexidine

13. Agents Physical methods Chemical methods
Cleaning
Agents of cleaning Automated washers such as
ultrasonic washers, washer-
disinfector and automated
cart washers
 Enzymatic solution
 Detergent
 Soap (antimicrobial or plain
soap)

14. High Level Disinfectants (HLD)
Capable of killing all microorganisms
Bacterial spores
Sufficient concentration under suitable conditions
MOA –Combine with nucleic
acids, proteins  inactivate
them, probably by cross-linking
and alkylating the molecules.

15. Glutaraldehyde
• Used for semi-critical items (endoscope, cystoscope)
• Also used for aerial disinfection and cleaning (fogging
and cleaning of floor and surfaces of critical areas e.g.,
Bacillocid Extra) (aerial disinfection  process of disinfecting
suspended particles in the air for a period of time)
• Concentration - 2% or 2.4% (e.g. Cidex); available in
inactive form and once activated - remains active only
for 14 days.
• Duration- 20 minutes (10–14 hrs for spores)

16. • Advantages: Remains active in the presence of organic matter, has
excellent material compatibility.
• Disadvantages: Pungent odour, can produce eye irritation,
occupational asthma and contact dermatitis.

17. Ortho-phthalaldehyde (0.55%)
• Used for disinfection of semi-critical items, has many advantages over
glutaraldehyde—
 Does not require activation
 Better odor
 Less eye irritation
 Acts faster (5–10 min).
• OPA does not kill spores effectively and stains skin gray.

18. Formaldehyde
• Excellent HLD.
• Health-care uses are limited:
o Irritating fumes and pungent odor.
o Potential carcinogen, corrosive (metals) and
causes skin irritation and asthma (when
inhaled).

19. • Formaldehyde is largely restricted to non-patient
care area only; used for—
– Preservation of anatomical specimen and
– Stool specimen and as an embalming agent.
• Fumigation of closed areas (e.g. OT) is an obsolete
practice.
• Tablets and gaseous form of formaldehyde- used for
sterilization of surgical instruments – is unreliable.

20. Peracetic Acid
• Used in automated machines and for manual immersion(0.1–
0.2%; 5–15 min)
• Used to sterilize medical (e.g., endoscopes, arthroscopes),
surgical, and dental instruments.
• Used in combination with H2O2  for hemodialyzers.
• Disadvantages: Expensive, has material compatibility issues,
causes chemical irritation and eye damage.

21. Hydrogen Peroxide (H2O2)
• MOA- produces destructive hydroxyl free radicals 
that can attack various cell components.
• H2O2 can be degraded by catalase-producing
organisms to water and oxygen, which can be
overcome by using higher concentrations.

22. Uses
• Sporicidal only at >4–5%.
• 3% H2O2 – Environmental surface disinfection, fogging & for wound cleaning
• 3–6% H2O2 – Disinfect soft contact lens, tonometer biprisms, ventilators,
fabrics, and endoscopes, etc.
• 6–7.5% H2O2 - used as chemical sterilant in plasma sterilization.
• Vaporized H2O2 - Industrial sterilization of medical devices and for
decontamination of large and small area.

23. Advantages:
• It is rapid in action, nontoxic.
• Has detergent properties with good
cleaning ability.
• Active in the presence of organic
material.
Disadvantages:
• Expensive
• Material compatibility issue
(contraindicated for use on Cu, brass, Zn2+
etc).
• Can produce chemical irritation and
corneal damage.
• Should be properly stored in dark
containers.

24. Intermediate Level Disinfectants
• Ethyl alcohol and Isopropyl alcohol - most
popular alcohol used in hospitals.
• Mode of Action: Rapidly bactericidal to
most organisms except spores - act by
denaturation of proteins.

25. Uses
• Alcohol (60–80%)  ideal is 70% ethanol is
used for various purposes.
 Alcohol based handrub (ABHR), e.g.
Sterillium.
 Disinfecting smaller non-critical
instruments e.g. thermometer.
 Disinfection of small medical items/surfaces
e.g. vaccine vials.
 Disinfection of external surfaces of
equipment e.g. stethscope.
 Disinfection of non-critical surfaces e.g. lab
bench area.

26. Disadvantages
• Flammable and must be stored in a cool, well-ventilated area.
• Evaporate rapidly.
• May damage tonometer tips and lenses.
• Inactivated by organic matter.

27. Phenolics
• Phenol (carbolic acid) - first widely used
antiseptic and disinfectant (Joseph Lister )
• Phenol and its derivatives - produced by
distillation of coal tar.
• MOA: Act as protoplasmic poison, disrupt the
cell wall and cell proteins

28. Used as disinfectants
• Cresol & lysol - Disinfecting environmental surfaces
(e.g. bedside tables, bedrails, and laboratory surfaces)
and noncritical medical devices.
• Toxic to skin - not used as antiseptics.
• 5% phenol - Disinfection of sputum specimen
(mycobactericidal).
Used as antiseptics
• Chloroxylenol (active ingredient dettol) -Compatible
with skin and are widely used as antiseptics.

29. • Advantages: only ILD that retain activity in the presence of organic
materials.
• Disadvantages: can cause hyperbilirubinemia in infants

30. Halogens
• Iodine and chlorine have antimicrobial activity.
• Exist in free state and form salt with sodium and other metals.

31. Iodine
• MOA- acts by disruption of protein and nucleic
acid.
• Two preparations are available
 Tincture of iodine (2% iodine in potassium
iodide)
 Iodophor (e.g. povidone iodine) - iodine with
carrier (povidone) which helps in sustain
release.
o Non staining and free of skin toxicity
o Brands- Wescodyne and betadine

32. Used as antiseptics
surgical skin
preparation
hand scrub ointment for
wound
cleaning
mouth wash
Used as disinfectant for medical equipment.

33. Chlorine and Hypochlorite
• MOA –All preparations yield hypochlorous acid (HClO) -causes oxidation of
cellular materials and destruction of vegetative bacteria and fungi.

34. Chlorine and Hypochlorite
Preparations: Chlorine occurs as—
• Free chlorine
• Hypochlorite—it is available in two preparations.
 Liquid form (sodium hypochlorite or household bleach)
 Powder form (calcium hypochlorite or bleaching powder)
 Other forms: sodium dichloroisocyanurate (NaDCC) tablets and chlorine
dioxide

35. Uses (free chlorine)
• Disinfection of municipal water supplies
and swimming pool water.
• Also employed in the dairy and food
industries

36. Uses (sodium hypochlorite)
• Available at 5.25– 6.15% - equivalent to 50,000 ppm
of available chlorine.
• It should be used in appropriate dilutions (by adding
with water) for disinfection of various hospital
supplies.
• Contact time: 10-20 minutes.

37. S.N
o.
Volume %age of Sod.
hypochlorite
Dilution
1 Large blood spill 0.5% 1:10 dilution or 5000 ppm
2 Small blood spill 0.05% 1:100 dilution or 500 ppm
3 Pre-treatment of liquid
waste before disposal
1% 1:5 dilution or 10000 ppm
4 Laundry items 0.1% 1:50 dilution 1,000 ppm
5 Surface disinfectant 0.5% 1:10 dilution or 5000 ppm
6 C. Difficile >0.5% 5000 ppm

38. A
Advantages:
• Hypochlorites are broad
spectrum (sporicidal)
• Rapid in its action
• Non-flammable
• Low cost
• Widely available.
Disadvantages:
• Inactivated by organic matter
• Toxic to skin and mucosa, &
carcinogenic,
• Daily preparation (unstable,
evaporates on exposure to
sunlight)
• Corrosive, damages fabrics,
carpets

39. LOW-LEVEL DISINFECTANT
• Destroy vegetative bacteria and enveloped viruses,
• variable action on non-enveloped viruses and fungi,
• but no action on tubercle bacilli and spores.

40. Quaternary Ammonium Compound (QAC)
• Commonly used in ordinary environmental
sanitation of noncritical surfaces, such as
floors, furniture's, and walls.
• Disinfecting non-critical medical equipment
(e.g., blood pressure cuffs).
• QAC are also good cleaning agents as they
have surfactant like action

41. • MOA:
– Inactivation of energy producing enzymes,
– denaturation of essential cell proteins, and
– disruption of the cell membrane.
• QAC formulations: Benzyl ammonium chloride - most popular QAC.
• Does not act in the presence of hard water.
• Newer generation of QACs (e.g. didecyl dimethyl ammonium
bromide) -remain active in hard water and are better compatible.

42. Chlorhexidine Gluconate (CHG)
• MOA  acts by disruption of
cytoplasmic membrane.
• Uses: CHG is widely used in antiseptic
products, at various concentrations:
 Hand hygiene product:
 Hand rub (0.5%),
 Hand wash (4%)
 Mouthwash (0.1-0.2%)

43. Chlorhexidine Gluconate (CHG)
 Body wash solutions (used before
surgery)
 Skin disinfectant before surgery (2%)
 Antiseptics for wound cleaning 
commerically available as “Savlon” 
combination of CHG 0.3%, cetrimide
(quarternary ammonium compound)
and isopropyl alcohol.

44. • Advantages:
– Residual activity (prolonged action than alcohol hand rub) and
– is less irritant.
• Disadvantages:
– Slower in action.
– Activity is pH dependent and is greatly reduced in the presence of
organic matter.
– Produces dermatitis on prolonged use as handrub.

45. Spaulding’s Classification of Medical Devices
Risk category Definition Recommended
method
Medical equipment or surfaces
Critical device
(high risk)
Items that enter a
normally sterile site of
body including blood.
Sterilization •Surgical instruments
•Implants/prosthesis
•Rigid endoscopes
•Needles
•Scalpels
Semi-critical device
(intermediate risk)
Items in contact with
mucous membranes or
non intact skin but does
not penetrate them.
•High level
Disinfection (HLD)
•Sterilization is
preferred.
•Respiratory equipment,
•Non-invasive flexible endoscopes,
•Laryngoscope
•Endotracheal tubes
Non-critical
(low-risk)
Items in contact with
intact skin
•Low level
Disinfection (ILD or
LLD)
•Intermediate level
disinfection
•Stethscope
•Blood pressure cuffs
•Thermometers
•ECG machine
•Oximeters
•Bedpans

46. Disinfection of Operation Theatre
• Basic Principle
– Cleaning is more important than
sterilization and Disinfection?
– However
– Cleaning
• Removes contaminants, dust,
organic matter.
– Disinfection
• Reduces number of microbes.

47. Disinfection of Operation Theatre
Surface
disinfection
Cleaning with a cleansing agent
disinfection by using aldehyde-based disinfectant

48. Disinfection of Operation Theatre
First cleaning of the day (before cases begin)
In between cases (Cleaning 3 to 4 feet perimeter around the OT table)
Disinfection of OT is carried out
in following situations :
Terminal cleaning of OT after the last case
Detailed wash-down of the OT complex once a week
During renovation or construction of OT or nearby places.

49. Fogging (aerial disinfection ):
• Spraying of a disinfectant (e.g.
glutaraldehyde,H2O2 or QAC based
product) with the help of a fogger
machine.

50. Cleaning (Decontamination)
• Reduction in the pathogenic microbial population to a level at
which items are considered safe without protective attire.
• Achieved by
– manual cleaning with soap and detergents to eliminate debris or
organic matter from medical devices or surfaces.

51. Central Sterile Supply Department (CSSD)
• Performs sterilization of
– Medical devices
– Equipment and
– Consumables
• Processing area of CSSD - four unidirectional zones starting from an
unsterile area to a sterile area separated by physical barrier.

52. • Decontamination area – washing
by manual or automated washers
• Packaging area –items are
enclosed safely to allow
penetration of sterilant
• Sterilization area –steam sterilizer
or ETO or plasma sterilizer
• Sterile storage area

53. Methods To Test Efficacy Of
Sterilant/Disinfecant
• Rideal Walker test
• Chick Martin test
• Kelsey Sykes test
• In - Use test

54. SPORICIDAL AGENTS
1. Ethylene oxide
2. Formaldehyde
3. Glutaraldehyde
4. Hydrogen peroxide
5. O-Phthalic acid
6. Peracetic acid
7. Autoclave
8. Hot air oven
9. Plasma sterilization

55. Methods of sterilization/disinfection used in different clinical situations
Material Method of sterilization/disinfection
Clinical thermometer Isopropyl alcohol
Paraffin, glass syringe, flask, slide, oil, grease, fat,
glycerol
Hot air oven
OT, entryway, ward, lab fumigation ,
Preservation of anatomical specimen, woolen
blanket
Formaldehyde gas > UV > BPL
Cystoscope, bronchoscope Orthophthaldehyde> glutaraldehyde 2%
(cidex)
Heart lung machine, respirator, dental equipments Ethylene oxide
Vaccine, sera, antibiotic, sugar solution, antibiotic &
body fluids
Filtration
Sharp instruments Cresol
Milk Pasteurization

56. Methods of sterilization/disinfection used in different clinical situations
Germicide & their
concentrations
Level of
disinfectant
Bacteria &
enveloped
viruses
Fungi Un-
envelope
d viruses
M.
tuberculosis
Spore Inactivated
by organic
matter
Glutaraldehyde (2%) High/CS + + + + + -
Formaldehyde (3-8%) High/CS + + + + + -
H2O2 (3-25%) High/CS + + + + + +/-
Chlorine (100-1000 ppm of
free chlorine)
High + + + + +/- +
Isopropyl alcohol (60-95%) Intermediate + + +/- + - +/-
Phenol (0.4-5%) Intermediate + + +/- + - -
Iodophore (30-50ppm of
free iodine)
Intermediate + + + +/- - +
Quaternary ammonium
compounds (0.4-1.6%)
Low + +/- - - - +
Chlorhexidine gluconate
(2-4%
Low + +/- +/- +/- +/- +

57. Common chemical disinfectants used in the hospital and
their applications

58. Common chemical disinfectants used in the hospital and
their applications

59. Common chemical disinfectants used in the hospital
A. Sterillium (Alcohol hand rub); B. Cidex (2% Glutaraldehyde used for disinfection of endoscopes); C. Bacillocid
(Glutaraldehyde-based combination product used for OT surface cleaning and fogging); D. Microshield (4% Chlorhexidine
used as hand wash); E. Dettol (Chloroxylenol used as skin antiseptic); F. Betadine (Povidone Iodine used as skin
antiseptic); G. Sodium hypochlorite solution (used as laboratory disinfectant)