This document discusses health care associated infections (HCAIs) and various methods for sterilization and disinfection. It begins by outlining some common types of HCAIs and their impacts, including increased morbidity, hospitalization time, costs, and antibiotic resistance. It then discusses the most frequent bacteria and viruses that cause HCAIs. The document goes on to define and compare sterilization, disinfection, antisepsis, and cleaning. It also outlines various physical and chemical agents used for sterilization and disinfection, such as heat, radiation, and chemical disinfectants like alcohols and aldehydes. Throughout, it provides details on the appropriate uses and effectiveness of different sterilization

1. Dr. Abhijeet Mane
Assistant Professor
Department of Microbiology
&
Coordinator, Infection Control Department, Bharati
Hospital
BVDUMC, Pune
Sterilisation & Disinfection
April 29, 20181

2. April 29, 20182
 Health Care Associated Infections (HCAIs) - a major
problem for patient safety.
 central line-associated bloodstream infections
 catheter-associated urinary tract infections
 ventilator-associated pneumonia
 surgical site infection
 What is impact of HCAIs?
 Morbidity
 Increase hospitalisation
 Increase cost
 Inconvenience to pt and family
 Increase antibiotics = increase drug resistance

3. Most common bacteria and viruses
causing HAIs
April 29, 20183
 Acinetobacter
baumannii
 Bacteroides fragilis
 Burkholderia cepacia
 Clostridium difficile
 Carbapenem-resistant
Enterobacteriaceae
 Enterococcus faecalis
 Escherichia coli
 Hepatitis A
 Hepatitis B
 Hepatitis C
 Human
Immunodeficiency Virus
 Influenza
 Klebsiella pneumoniae
 Methicillin-resistant
Staphylococcus aureus
 Psuedomonas
aeruginosa
 Stenotrophomonas
maltophilia
 Mycobacterium
tuberculosis
 Vancomyin-resistant
Staphylococcus aureus
 Vancomycin-resistant
Enterococci

4. What is sterilization?
 Definition:
 Process by which an article, surface or medium is
freed of all living micro-organisms either in
vegetative or spore state.
April 29, 20184

5. What is disinfection?
 Definition:
 The destruction or removal of all pathogenic
organisms capable of giving rise to infection.
April 29, 20185

6. Antisepsis
 The term is used to indicate the prevention of
infection, usually by inhibiting the growth of
bacteria in wounds or tissues.
 Achieved by antiseptics
 Chemicals or disinfectants which can be safely
applied on skin or mucous membrane to prevent
infection by inhibiting the growth of bacteria.
April 29, 20186

7. Bactericidal agents
 Bactericidal agents / germicides
 those who are able to kill bacteria.
 Bacteriostatic agents
 Only prevent multiplication of bacteria, but they
remain alive.
April 29, 20187

8. What is cleaning?
 Definition:
 Important preparatory step before sterilisation or
disinfection, by removing soil and other dirt.
 DECONTAMINATION
 The process of rendering an article or area free of
contaminants, including microbial, chemical,
radioactive and other hazards.
April 29, 20188

9. Agents for Sterilisation
 Physical agents
 Sunlight
 Drying
 Dry heat: flaming, incineration, hot air oven
 Moist heat: pasteurization, boiling, steam under
pressure
 Filtration: candles, asbestos pads, membranes
 Radiation
 Ultrasonic and sonic vibrations
April 29, 20189

10. Agents for sterilisation
 Chemicals
 Alcohol
 Ethyl, isopropyl, trichlorobutanol
 Aldehydes
 Formaldehyde,glutaraldehyde
 Dyes
 Halogens
April 29, 201810

11. April 29, 201811
 Phenols
 Surface active agents
 Metallic salts
 Gases:
 Ethylene oxide, formaldehyde (g), beta propiolactone

12. PA : sunlight
 Action primarily due to UV rays
 however, effects vary due to places
 Eg: in tropical country, the germicidal effect is better
than 4 seasoned countries.
 Bacteria in water are readily destroyed by
sunlight.
April 29, 201812

13. PA : drying
 Moisture is essential for growth of bacteria.
 Drying in air has deleterious effect on many
bacteria.
 However, spores are unaffected. Therefore, it is
not really unreliable.
April 29, 201813

14. PA : heat
 Most reliable method of sterilization and should
be the method of choice.
 The factors influencing sterilization by heat:
 Nature of heat-dry or moist
 Temperature and time
 Number of microorganisms present
 Characteristics of organisms –species, strain,
sporing capacity
 Type of material from which organism have to be
eliminated.
April 29, 201814

15. Killing effect of dry heat
 Killing effect is due to protein denaturation,
oxidative damage and toxic effect of elevated
level of electrolytes.
April 29, 201815

16. Killing effect of moist heat
 Due to denaturation and coagulation of proteins.
April 29, 201816

17. Thermal death time
 Def:
 Minimum time required to kill a suspension of
organisms at a predetermined temperature in a
specified environment.
 Thermal death time is inversely proportional to
temperature.
 TDT is increased in presence of organic
substance, proteins, nucleic acid, starch, gelatin,
sugar, fats, oils.
April 29, 201817

18. Dry heat
April 29, 201818
 Flaming:
 Items: inoculating
loop/ wire, tip of
forceps, searing
spatulas
 Using: bunsen burner
 *inoculating loop is
better dipped in
disinfectant first
before flaming to
prevent spattering.

19.  Incineration
 Items: contaminated cloth, animal carcasses and
pathological material.
 polystyrene will emit black smoke. Hence should be
autoclaved in appropriate container.
April 29, 201819

20.  Hot air oven
 Holding period: 160°C, 1 hour
 Items: glassware, forceps, scissors, scalpels, all-
glass syringes, swabs, liquid paraffin, dusting
powder, fats, grease.
 Materials should be properly arranged to allow free
circulation of air.
April 29, 201820

21. Hot air oven
April 29, 201821

22.  Sterilization control
 Usage of the spores of Clostridium tetani. The
nontoxic bacteria will be cultured to see either it is
growing or not.
 To make sure the sterilization is properly done.
April 29, 201822

23. Moist heat
 Moist heat can be categorized into 3 groups:
 Temperature below 100°C
 Temperature at 100°C
 Temperature above 100°C
April 29, 201823

24. MH: temperature <100°C
 Pasteurisation of milk
 Holding period: 63°C, 30 minutes (holder
method) ; or 72°C, 15-20 minutes.
 Target: all nonsporing pathogens
 Eg: mycobacteria, brucellae, salmonella.
 Coxiella burnetti, relatively heat resistant, may survive the
holder method.
April 29, 201824

25.  Inpissator
 Media like LJ and Loeffler’s serum slope
 Holding period: 80-85°C, half an hour. X 3 days
April 29, 201825

26. Inpissator
April 29, 201826

27. Mh: temperature @100°C
 Boiling
 Not recommended for sterilising but ok for
disinfection.
 Sterilisation may be promoted by addition of 2%
sodium bicarbonate to the water.
 Holding period: 10-30 minutes.
 The lid of sterilizer should not be opened during the
period.
April 29, 201827

28.  Steam @ atmospheric pressure (100°C)
 Used to sterilize culture media.
 Koch or Arnold steamer is used.
 Inexpensive method
 Holding period: 100°C, 20 minutes on three
succesive days (tyndallisation/ intermittent
sterilization)
 Principle: first exposure kills vegetative bacteria and
then the next exposure will kill vegetative bacteria
that matures from the spore.
April 29, 201828

29.  Steam under pressure
 Autoclave/steam sterilizer
 Principle: water boils when its vapour pressure
equals the surrounding atmosphere.
 Thus, when pressure inside closed vessels
increases, the temperature at which water boils
increases too.
 Holding period: varies.
 Temperature: 121 deg C @ 15psi x 15 mins
 Items: dressings, instruments, laboratory ware,
media and pharmaceutical products.
April 29, 201829

30. autoclave
April 29, 201830

31.  Several types of steam sterilizer:
 Laboratory autoclaves
 Hospital dressings sterilizers
 Bowl and instrument sterilizers
 Rapid cooling sterilizers
 Sterilization control-
 Spores of Bacillus stearothermophillus is used.
April 29, 201831

32. Steam sterilizer
April 29, 201832

33. filtration
 Helps to remove bacteria from heat labile liquids
 Items: sera and solutions of sugars or
antibiotics.
 Principle: as viruses pass through the ordinary
filters, filtration can be used to obtain bacteria-
free filtrates of clinical samples for virus isolation.
April 29, 201833

34.  Types of filters
 Candle filters
 Asbestos filters
 Sintered glass filters
 Membrane filters
April 29, 201834

35.  Candle filter
 Types-
 Unglazed ceramic filters
 Diatomaceous earth filters
 Asbestos filters
 Disposable, single-used disc
 Usage is discouraged because of its
 carcinogenic property.
 Eg: Seitz and Sterimat filters
April 29, 201835

36. April 29, 201836
 Sintered glass filters
 has low absorptive
properties
 Brittle and expensive
 Membrane filters
 Made of cellulose
esters or other
polymers
 Usually used for
water purification and
analysis, sterilization
and sterility testing
and preparation of
solutions for
parenteral use.

37. radiation
 2 types of radiation
 Ionising gamma rays, high energy electrons
 Non-ionising infrared, UV
 Non-ionising radiation
 Infrared
 Used for rapid mass sterilization of prepacked items;
 Syringe,
 Catheters
 UV
 Disinfect enclosed area such as entryways, operation
theatres and labs.
April 29, 201837

38.  Ionising radiation
 Gamma rays
 Items: plastics, syringes, swabs, catheters, animal
feeds, cardboard, oils, greases, fabric and metal
foils.
April 29, 201838

39. Chemical agents!!!
April 29, 201839

40. Chemical agents
 Ideal antiseptic/disinfectant should
 Effective against all microorganisms
 Be active in presence of organic matter
 Effective in acid as well in alkaline media
 Have speedy action
 Have high penetrating power
 Stable
 Compatible with other antispetics and disinfectant
 X corrode metals
 X cause local irritation or sensitisation
 X interfere with healing
 X toxic if absorbed into circulation
 Inexpensive and easily available.
April 29, 201840

41. Such an ideal
chemical
disinfectant is yet
to be found!!!
April 29, 201841

42. Disinfectants are characterized into 3 activity levels
according to Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition:
April 29, 201842
 High-Level Disinfection This procedure kills vegetative
microorganisms and inactivates viruses. They are used for
relatively short periods of time (e.g., 10 to 30 minutes).
These chemical germicides are potent sporicides. They
are formulated for use on medical devices, but not on
environmental surfaces such as laboratory benches or
floors.
 Intermediate-Level Disinfection This procedure kills
vegetative microorganisms, including Mycobacterium
tuberculosis, all fungi, and inactivates most viruses. They
are used commonly in laboratories for disinfection of
laboratory benches and as part of detergent germicides
used for housekeeping purposes.
 Low-Level Disinfection This procedure kills most
vegetative bacteria except M. tuberculosis, some fungi,

43. April 29, 201843
Class
es
Alcohols Aldehy
des
Chlorin
e
compou
nds
iodoph
ors
Oxidisi
ng
agents
Phenolic
compoun
ds
QACs
Examp
les
-70%ethyl
alcohol -
isopropyl
Formald
ehyde
(3-8%)
Glutaral
dehyde
(2%)
Bleach Betadin
e
PV-I
Hydroge
n
peroxide
Peraceti
c acid
lysol
Aqueo
us
conc.
70% variable 10% 0.1-
0.2%
3-6% 0.5-3%
Activity
level
I High to
Interme
d
High/CS Interme
d
High/CS Intermed
to low
Low

44. Chemical agents
 Factors that determine the potency of
disinfectants:
 Concentration of the substance
 Time of action
 pH of the medium
 Temperature
 Nature of the organisms
 Presence of extraneous material
April 29, 201844

45. Chemical agents
 Action of chemical agents
 Protein coagulation
 Disruption of cell membrane resulting in exposure,
damage/loss of contents
 Removal of sulfhydryl group essential for normal
functioning of enzyme
 Substrate competition
April 29, 201845

46. April 29, 201846

47. April 29, 201847

48. CA: alcohol
 Frequently used:
 Ethyl alcohol
 Isopropyl alcohol
 Must be used at concentration 60-90%
 Mode of Action
 Absolute ethyl alcohol, a dehydrating agent, is
less bactericidal than mixtures of alcohol and
water
 Protein denaturation also is consistent with
observations that alcohol destroys the
dehydrogenases of Escherichia coli
April 29, 201848

49. April 29, 201849
 Activity:
 Rapidly bactericidal rather than bacteriostatic against
vegetative forms of bacteria
 Also tuberculocidal, fungicidal, and virucidal but do
not destroy bacterial spores
 cidal activity drops sharply when diluted below 50%
concentration
 Pseudomonas aeruginosa, Serratia marcescens, E,
coli and Salmonella
 Staphylococcus aureus and Streptococcus pyogenes
 Isopropyl alcohol (isopropanol) was slightly more
bactericidal than ethyl alcohol for E. coli and S.
aureus

50. April 29, 201850
 Herpes, vaccinia, and influenza virus and
adenovirus, enterovirus, rhinovirus, and
rotaviruses, hepatitis B virus, human
immunodeficiency virus (HIV), rotavirus,
echovirus, and astrovirus
 Uses
 Alcohols have been used effectively to disinfect
oral and rectal thermometers, hospital pagers,
scissors, and stethoscopes

51. April 29, 201851

52. CA: aldehyde
 Formaldehyde:
 Bactericidal, sporicidal and has lethal effect on
viruses.
 Used to preserve anatomical specimens, destroying
anthrax spores on hair and wool.
 Gluteraldehyde:
 Effective against tubercle bacilli, fungi, viruses.
 Less toxic and irritant to eyes, skin
 Used to treat corrugated rubber anaesthetic rubber,
face masks, plastic endotracheal tubes, metal
instruments and polythene tubing.
April 29, 201852

53. April 29, 201853
 Formaldehyde:
 The aqueous solution is a bactericide,
tuberculocide, fungicide, virucide and sporicide
 potential carcinogen
 exposure to low levels in the air or on the skin
can cause asthma-like respiratory problems and
skin irritation, such as dermatitis and itching
 Mode of Action.
 Formaldehyde inactivates microorganisms by
alkylating the amino and sulfhydryl groups of
proteins and ring nitrogen atoms of purine bases

54. April 29, 201854
 Uses
 formaldehyde is a high-level disinfectant

55. April 29, 201855
 Gluteraldehyde
 Glutaraldehyde has gained wide acceptance as a
high-level disinfectant and chemical sterilant
 Aqueous solutions are acidic and generally in this
state are not sporicidal. Only when the solution is
“activated” by use of alkalinating agents to pH 7.5–8.5
does the solution become sporicidal.
 shelf-life of minimally 14 days because of the
polymerization of the glutaraldehyde molecules at
alkaline pH levels. This polymerization blocks the
active sites (aldehyde groups) of the glutaraldehyde
molecules that are responsible for its biocidal activity.

56. April 29, 201856
 Mode of Action.
 The biocidal activity of glutaraldehyde results
from its alkylation of sulfhydryl, hydroxyl,
carboxyl, and amino groups of microorganisms,
which alters RNA, DNA, and protein synthesis.
 Activity
 M. tuberculosis, fungi, and viruses, spores of
Bacillus and Clostridium species, Spores of C.
difficile

57. April 29, 201857
 Uses
 Glutaraldehyde is used most commonly as a
high-level disinfectant for medical equipment
such as endoscopes, spirometry tubing,
dialyzers, transducers, anesthesia and respiratory
therapy equipment, hemodialysis proportioning
and dialysate delivery systems, and reuse of
laparoscopic disposable plastic trocars


58. Ortho-phthalaldehyde (OPA)
April 29, 201858
 high-level disinfectant
 Mode of Action
 OPA and glutaraldehyde interact with amino
acids, proteins, and microorganisms
 OPA appears to kill spores by blocking the spore
germination process
 Microbicidal Activity
 Mycobactericidal, B. atrophaeus spores , P.
aeruginosa

59. April 29, 201859
 Advantages over Gluteraldehyde
 excellent stability over a wide pH range (pH 3–9)
 not a known irritant to the eyes and nasal passages
 does not require exposure monitoring
 barely perceptible odor
 requires no activation
 excellent material compatibility
 Disadvantage
 potential disadvantage of OPA is that it stains proteins
gray (including unprotected skin) and thus must be
handled with caution
 costly

60. April 29, 201860

61. April 29, 201861

62. CA: dyes
 2 groups of dyes:
 Aniline dye
 Acridine dye
 Both are bacteriostatic in high dilution but are of
low bactericidal activity.
 Aniline dye is more active against gram +ve than
gram-ve organisms.
 Used in microbiology labs as selective agents in
culture media.
April 29, 201862

63.  Acridine dye
 Not selective as aniline dye.
 Important dyes:
 Proflavine
 Acriflavine
 Euflavine
 Aminacrine
 Impair the DNA complexes of the organisms and
thus kill or destroy the reproductive capacity of the
cell.
April 29, 201863

64. CA: Halogens
 Iodine
 Skin disinfectant
 Active bactericidal, moderate action on spores.
April 29, 201864

65. April 29, 201865
 Hypochlorites
 the most widely used of the chlorine disinfectants
 They have a broad spectrum of antimicrobial
activity,
 do not leave toxic residues,
 unaffected by water hardness,
 inexpensive and fast acting,
 remove dried or fixed organisms and biofilms
from surfaces, and
 have a low incidence of serious toxicity

66. Mode of Action
April 29, 201866
 Inactivation by chlorine can result from a
number of factors:
 oxidation of sulfhydryl enzymes and amino acids;
 ring chlorination of amino acids;
 loss of intracellular contents;
 decreased uptake of nutrients;
 inhibition of protein synthesis;
 decreased oxygen uptake;
 oxidation of respiratory components;
 decreased adenosine triphosphate production;
 breaks in DNA; and depressed DNA synthesis

67. Activity
April 29, 201867
 Action on 25 viruses
 M. tuberculosis
 Clostridium difficile spores
 S. aureus, Salmonella choleraesuis, and P.
aeruginosa
 Uses
 Floor
 Blood spills
 Needles etc

68. April 29, 201868
 Disadvantages
 Sodium hypochlorite at the concentration used in
household bleach (5.25-6.15%) can produce
ocular irritation or oropharyngeal, esophageal,
and gastric burns
 Other disadvantages of hypochlorites include
corrosiveness to metals in high concentrations
(>500 ppm),
 inactivation by organic matter,
 discoloring or “bleaching” of fabrics,
 release of toxic chlorine gas when mixed with
ammonia or acid (e.g., household cleaning

69. April 29, 201869
• Chlorine
Water supplies, swimming pools and food and dairy
industries.
Along with hypochlorite are bactericidal. Also act on
viruses.

70. CA: Phenols
 Obtained from distillation of coal tar between 170-
270°C.
 Lethal effect:
 Capacity to cause cell membrane damage,
releasing cell contents and causing lysis.
 Low concentration will precipitate proteins.
April 29, 201870

71. April 29, 201871

72. CA: gases
 Types of gases
 Ethylene oxide
 Formaldehyde gas
 Beta propiolactone (BPL)
 Ethylene oxide
 Action is due to its alkylating the amino, carboxyl,
hydroxyl and sulphydryl groups in protein
molecules. Also on DNA and RNA.
 Items: heart-lung machines, respirators, sutures,
dental equipment, books, clothing.
April 29, 201872

73. April 29, 201873

74.  Formaldehyde gas
 Employed for fumigation of OT and other rooms.
 After fumigation, the doors should be sealed and
left unopened for 48 hours.
 BPL
 Product of ketane and formaldehyde with a boiling
point of 163°C.
 Rapid biocidal activity but carcinogenic.
 Capable of killing all microorganisms and is very
active against viruses.
April 29, 201874

75. CA: surface-active agents
 Def
 substance that alter the energy relationship at
interfaces, producing a reduction of surface or
interfacial tension.
 Widely used as wetting agents, detergents and
emulsifiers.
 4 main groups:
 anionic
 Cationic
 Nonionic
 amphoteric
April 29, 201875

76. CA: metallic salts
 Salts of heavy metals have a greater action.
 Eg: salts of silver, copper and mercury
 Protein coagulant and have capacity to combine
with free sulfhydryl group of cell enzymes.
April 29, 201876

77. Hydrogen peroxide
April 29, 201877
 high-level disinfectants
 bactericidal, virucidal, sporicidal, and fungicidal
properties
 Mode of Action.
 Hydrogen peroxide works by producing
destructive hydroxyl free radicals that can attack
membrane lipids, DNA, and other essential cell
components. Catalase, produced by aerobic
organisms and facultative anaerobes that
possess cytochrome systems, can protect cells
from metabolically produced hydrogen peroxide
by degrading hydrogen peroxide to water and
oxygen.

78. April 29, 201878
 Microbicidal Activity
 wide range of microorganisms, including bacteria,
yeasts, fungi, viruses, and spores
 S. aureus, S. marcescens, and Proteus mirabilis,
E. coli, Streptococcus species, and
Pseudomonas species, Bacillus species, VRE,
TB, rhinovirus, multidrug-resistant M.
tuberculosis, poliovirus and HAV
 Synergistic sporicidal effects of hydrogen
peroxide (5.9%–23.6%) and per acetic acid

79. April 29, 201879
 Use
 effective disinfectant when used on inanimate
surfaces

80. Per acetic acid
April 29, 201880
 Peracetic, or peroxyacetic, acid is characterized
by rapid action against all microorganisms
 lacks harmful decomposition products (i.e., acetic
acid, water, oxygen, hydrogen peroxide),
enhances removal of organic material, and leaves
no residue
 It remains effective in the presence of organic
matter and is sporicidal even at low temperatures

81. April 29, 201881
 Mode of Action.
 denatures proteins, disrupts the cell wall
permeability, and oxidizes sulfhydryl and sulfur
bonds in proteins, enzymes, and other
metabolites
 Microbicidal Activity.
 Peracetic acid will inactivate gram-positive and
gram-negative bacteria, fungi, and yeasts,
mycobacteria, bacterial spores
 Used in Plasma sterilisation

82. Quaternary Ammonium Compounds
(QACs)
April 29, 201882
 Quaternary ammonium compounds are widely
used as disinfectants
 The quaternaries are good cleaning agents, but
high water hardness and materials such as cotton
and gauze pads can make them less microbicidal
because of insoluble precipitates or cotton and
gauze pads absorb the active ingredients,
respectively
 As with several other disinfectants (e.g.,
phenolics, iodophors) gram-negative bacteria can
survive or grow in them

83. April 29, 201883
 Some of the chemical names: alkyl dimethyl
benzyl ammonium chloride, alkyl didecyl dimethyl
ammonium chloride, and dialkyl dimethyl
ammonium chloride.
 The newer quaternary ammonium compounds
(i.e., fourth generation), referred to as twin-chain
or dialkyl quaternaries (e.g. didecyl dimethyl
ammonium bromide and dioctyl dimethyl
ammonium bromide), purportedly remain active
in hard water


84. April 29, 201884
 Mode of Action.
 The bactericidal action of the quaternaries has been
attributed to the inactivation of energy-producing enzymes,
denaturation of essential cell proteins, and disruption of the
cell membrane
 Microbicidal Activity
 Fungicidal, bactericidal, and virucidal against lipophilic
(enveloped) viruses; they are not sporicidal and generally
not tuberculocidal or virucidal against hydrophilic
(nonenveloped) viruses
 multidrug-resistant S. aureus, vancomycin-resistant
Entercoccus, P. aeruginosa
 Use
 environmental sanitation of noncritical surfaces, such as
floors, furniture, and walls

85. Thank you!
April 29, 201885