2. HISTORY
• 1862 Invention of the Autoclave
• 1880 The first indicator : a potato
• 1906 Creation of the FDA
• 1925 Waxy pellets that melt at 121oC
• 1932 (ATI) CI with lead sulphite
passes from black to white
• 1940 (ATI) CI Chromium TriChloride
passes from purple to green
DR.T.V.RAO MD 2
3. HISTORY (CONT)
• 1960-70 Development of the Dvalue concept
using heat resistant spores for
sterilisation + Fvalue + Zvalue
• 1965 Proposal by Sweden of the SAL for a
definition of sterility
• 1979 Proposal by Canada of a legal
definition of sterility
DR.T.V.RAO MD 3
4. 4
TERMINOLOGY IN STERILIZATION
• Sterilization – a process that destroys all viable
microbes, including viruses and endospores;
microbicidal
• Disinfection – a process to destroy vegetative
pathogens, not endospores; inanimate objects
• Antiseptic – disinfectants applied directly to
exposed body surfaces
• Sanitization – any cleansing technique that
mechanically removes microbes
DR.T.V.RAO MD
5. 5
TERMINOLOGY IN STERILIZATION
• Degermation – mechanically removing microbes
form surface (skin) such as surgical hand
scrubbing, or wiping skin with alcohol prior to
venipuncture
• Sepsis – bacterial contamination
• Asepsis – absence of significant contamination
• Bactericidal (microbicidal) - -cidal means kill
• Bacteriostatic (micro biostatic) - -static means
inhibition of growth and multiplicationDR.T.V.RAO MD
6. AIM OF STERILIZATION - ASEPSIS
Asepsis is the practice to reduce or eliminate
contaminants (such as bacteria, viruses, fungi,
and parasites) from entering the operative field
in surgery or medicine to prevent infection.
Ideally, a field is "sterile" — free of
contaminants — a situation that is difficult to
attain. However, the goal is elimination of
infection, not sterility.
• http://en.wikipedia.org/wiki/Asepsis
DR.T.V.RAO MD 6
7. HOW STERILIZATION WORKS
• Cell wall maintains integrity of cell
• When disrupted, cannot prevent cell from bursting due to
osmotic effects
• Cytoplasmic membrane contains cytoplasm and controls
passage of chemicals into and out of cell
• When damaged, cellular contents leak out
• Viral envelope responsible for attachment of virus to target cell
• Damage to envelope interrupts viral replication
• So…non enveloped viruses have greater tolerance of harsh
conditions
DR.T.V.RAO MD 7
8. METHODS
• Physical
• Heat
• Filtration
• Irradiation
• Quarantine
• Chemical
• Choice of method depends
on practical issues such
as ease of use or material
compatibility
• Proctoscope need not be as
free of contamination as an
artificial heart valve
• Cleaning of objects
needed before attempt at
sterilization
DR.T.V.RAO MD 8
9. • Strength of the killing agent
• Time that the agent has to act
• Temperature of environment
• rate of microbe death
doubles with every 10˚C rise
in temp.
• Type of microbe
• Environment around the area to
be decontaminated
• Number of microbes to be killed
FACTORS INFLUENCING ABILITY TO
KILL MICROBES
DR.T.V.RAO MD 9
11. FACTORS THAT INFLUENCE EFFICACY
OF DISINFECTION/STERILIZATION
Contact time
Physico-chemical environment (e.g. pH)
3 Presence of organic material
4 Temperature
5 Type of microorganism
6 Number of microorganisms
7 Material composition
DR.T.V.RAO MD 11
13. DISINFECTION AND STERILIZATION
EH Spaulding believed that how an object will be
disinfected depended on the object’s intended use.
CRITICAL - objects which enter normally sterile tissue or the
vascular system or through which blood flows should be
sterile.
SEMICRITICAL - objects that touch mucous membranes or
skin that is not intact require a disinfection process (high-
level disinfection[HLD]) that kills all microorganisms but
high numbers of bacterial spores.
NONCRITICAL -objects that touch only intact skin require
low-level disinfection.
DR.T.V.RAO MD 13
17. Physical Methods of Microbial Control:
Heat: Kills microorganisms by denaturing their
enzymes and other proteins. Heat resistance varies
widely among microbes.
Thermal Death Point (TDP): Lowest temperature at which
all of the microbes in a liquid suspension will be killed in ten
minutes.
Thermal Death Time (TDT): Minimal length of time in which
all bacteria will be killed at a given temperature.
Decimal Reduction Time (DRT): Time in minutes at which
90% of bacteria at a given temperature will be killed. Used in
canning industry.
DR.T.V.RAO MD 17
18. • 100 oC at 1 ATM
• 121 oC at 2 ATM (1 barr)
• 132 oC at 3 ATM (2 barr)
• 80 oC at 0.5 ATM (Mont Blanc)
• 70 oC at 0.35 ATM (Mont
Everest)
• 40 oC at 0.02 ATM
(Mechanical vacuum)
BOILING TEMPERATURE IS LINKED TO
PRESSURE
DR.T.V.RAO MD 18
19. BOILING
• Kills vegetative cells of bacteria and fungi, protozoan
trophozoites, and most viruses within 10 minutes at sea
level
• Temperature cannot exceed 100ºC at sea level; steam
carries some heat away
• Boiling time is critical
• Water boils at lower temperatures at higher elevations;
requires longer boiling time
• Endospores, protozoan cysts, and some viruses can
survive boilingDR.T.V.RAO MD 19
20. PHYSICAL METHODS: HEAT
• Pasteurisation
• First used with milk: 72°C for 20
seconds
• Heating to 80°C for 1 minute will
kill most vegetative organisms
• Examples: bed-pan washer,
proctoscope
• Dry heat (hot air oven)
• used on waxes, oils (wet heat
usually preferred)
• Incineration
• the ultimate sterilization
• used for disposal of hospital waste
• Wet heat
• Boiling
• limited use as spores may be
resistant, boilers may be misused
• Low temperature steam disinfection
(75°C for 30 mins)
• Used for e.g. ventilator tubing
• Autoclaving
• High-temperature steam plus
pressure (same principle as
pressure cooker)
DR.T.V.RAO MD 20
21. MOIST HEAT
• Used to disinfect, sanitize, and sterilize
• Kills by denaturing proteins and destroying cytoplasmic
membranes
• More effective than dry heat; water better conductor of heat than
air
• Methods of microbial control using moist heat
• Boiling
• Autoclaving
• Pasteurization
• Ultrahigh-Temperature Sterilization
DR.T.V.RAO MD 21
22. MOIST HEAT
• Pasteurization
Definition: a process in which fluids are heated at
temperatures below boiling point to kill pathogenic
microorganisms in the vegetative state without altering
the fluid’s palatability.
Conditions: 62℃, 30min or 71.7℃, 15sec
Significance: kills vegetative pathogens
Applications: milk, beer
DR.T.V.RAO MD 22
23. AUTOCLAVING
• Pressure applied to boiling water prevents steam from escaping
• Boiling temperature increases as pressure increases
• Autoclave conditions – 121ºC, 15 psi, 15 minutes
DR.T.V.RAO MD 23
27. Other Physical Methods of Microbial
Control:
Filtration: Removal of microbes by passage of a liquid
or gas through a screen like material with small pores.
Used to sterilize heat sensitive materials like vaccines,
enzymes, antibiotics, and some culture media.
High Efficiency Particulate Air Filters (HEPA): Used in
operating rooms and burn units to remove bacteria from air.
Membrane Filters: Uniform pore size. Used in industry and
research. Different sizes:
0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain
spirochetes, mycoplasmas and viruses.
0.01 um Pores: Retain all viruses and some large proteins.
DR.T.V.RAO MD 27
28. Physical Methods of Microbial
Control:
Radiation: Three types of radiation kill microbes:
1. Ionizing Radiation: Gamma rays, X rays, electron beams, or
higher energy rays. Have short wavelengths (less than 1
nanometer).
Dislodge electrons from atoms and form ions.
Cause mutations in DNA and produce peroxides.
Used to sterilize pharmaceuticals and disposable medical
supplies. Food industry is interested in using ionizing radiation.
Disadvantages: Penetrates human tissues. May cause genetic
mutations in humans.
DR.T.V.RAO MD 28
30. Physical Methods of Microbial
Control:
Radiation: Three types of radiation kill microbes:
Microwave Radiation: Wavelength ranges from 1 millimeter
to 1 meter.
Heat is absorbed by water molecules.
May kill vegetative cells in moist foods.
Bacterial endospores, which do not contain water, are not
damaged by microwave radiation.
Solid foods are unevenly penetrated by microwaves.
Trichinosis outbreaks have been associated with pork cooked
in microwaves.
DR.T.V.RAO MD 30
35. • Major Categories
• Phenols
• Alcohols
• Halogens
• Oxidizing agents
• Surfactants
• Heavy Metals
• Aldehydes
• Gaseous Agents
• Antimicrobics
CHEMICAL METHODS OF MICROBIAL
CONTROL
DR.T.V.RAO MD 35
36. FACTORS INFLUENCING ANTIMICROBIAL
ACTIVITY
• the concentration and kind of an agent used
• the length of exposure to the agent
• the temperature at which the agent is used
• the number of microorganisms present
• the kinds of microorganisms present
• the nature of the material bearing the microorganism
DR.T.V.RAO MD 36
37. PHENOL AND PHENOLICS
• Intermediate- to low-level disinfectants
• Denature proteins and disrupt cell membranes
• Effective in presence of organic matter and remain
active for prolonged time
• Commonly used in health care settings, labs, and
homes (Lysol, triclosan)
• Have disagreeable odor and possible side effects
DR.T.V.RAO MD 37
38. • Intermediate-level
disinfectants
• Denature proteins and
disrupt cytoplasmic
membranes
• Evaporate rapidly – both
advantageous and
disadvantageous
• Swabbing of skin with 70%
ethanol prior to injection
ALCOHOLS
DR.T.V.RAO MD 38
39. ISOPROPYL ALCOHOL (70%)
ISOPROPYL ALCOHOL (70%)
• Powerful disinfectant and antiseptic
• Mode of action: denatures proteins,
dissolves lipids and can lead to cell
membrane disintegration
• Effectively kills bacteria and fungi
• But does not inactivate spores!
DR.T.V.RAO MD 39
40. • Intermediate-level
antimicrobial chemicals
• Believed that they damage
enzymes via oxidation or by
denaturing them
• Iodine tablets, iodophores
(Betadine®), chlorine
treatment of drinking water,
bleach, chloramines in
wound dressings, and
bromine disinfection of hot
tubs
HALOGENS
DR.T.V.RAO MD 40
41. • Peroxides, ozone, and per acetic
acid kill by oxidation of microbial
enzymes
• High-level disinfectants and
antiseptics
• Hydrogen peroxide can disinfect
and sterilize surfaces of objects
• Ozone treatment of drinking
water
• Per acetic acid – effective
sporocide used to sterilize
equipment
OXIDIZING AGENTS
DR.T.V.RAO MD 41
42. SURFACTANTS
• Surface active” chemicals that reduce surface tension of
solvents to make them more effective at dissolving solutes
• Soaps and detergents
• Soaps have hydrophilic and hydrophobic ends; good
degerming agents but not antimicrobial
• Detergents are positively charged organic surfactants
• Quats – colorless, tasteless, harmless to humans, and
antimicrobial; ideal for many medical and industrial application
• Low-level disinfectants
DR.T.V.RAO MD 42
43. HEAVY METALS
• Ions are antimicrobial because they alter the 3-D shape of
proteins, inhibiting or eliminating their function
• Low-level bacteriostatic and fungistatic agents
• 1% silver nitrate to prevent blindness caused by
N. gonorrhoeae
• Thimerosal (mercury-containing compound) used to preserve
vaccines
• Copper controls algal growth in reservoirs, fish tanks, swimming
pools, and water storage tanks; interferes with chlorophyll
DR.T.V.RAO MD 43
44. • Denature proteins and
inactivate nucleic acids
• Glutaraldehyde both
disinfects (short exposure)
and sterilizes (long
exposure)
• Formalin used in
embalming and disinfection
of rooms and instruments
ALDEHYDES
DR.T.V.RAO MD 44
45. PROBLEMS WITH STERILITY
• Lack of understanding of risk/process
• physicians introducing new products (borrowed,
samples)
• Multidose vials
• What is sterile vs not
• Lack of understanding of components of
process
• MDs, technologists have less training than nurses
(anaesthesiology, imaging, urology)
DR.T.V.RAO MD 45
46. GASEOUS AGENTS
• Ethylene oxide, propylene oxide, and beta-
propiolactone used in closed chambers to
sterilize items
• Denature proteins and DNA by cross-linking
functional groups
• Used in hospitals and dental offices
• Can be hazardous to people, often highly
explosive, extremely poisonous, and are
potentially carcinogenicDR.T.V.RAO MD 46
47. • Yes.
• Plasmas are currently
employed in many
industries to
accomplish both highly
effective, and delicate
sterilization.
• Not future technology!
Plasmas are used today!
• But, how do they work?
CURRENT STERILIZATION
MEANS: PLASMAS?
DR.T.V.RAO MD 47
48. PLASMA STERILIZATION
• A plasma is a quasi-neutral collection of electrons,
positive ions, and neutrals capable of collective
behavior
• Positive ions = free radicals
• Plasma sterilization operates synergistically via
three mechanisms:
• Free radicals interactions
• UV/VUV radioactive effects
• Volatilization
• Dead microorganisms = sterilization
DR.T.V.RAO MD 48
49. DISADVANTAGES OF PLASMA
STERILIZATION
• Weak penetrating power of the plasma species.
Complications arise in:
• Presence of organic residue
• Packaging material
• Complex geometries
• Bulk sterilization of many devices
• Solutions: Introduce preferentially targeting UV/VUV
radiation of proper wavelength
DR.T.V.RAO MD 49
50. CAN ENVIRONMENTAL CLEANING REDUCE
MRSA TRANSMISSION?
• Setting: ward with endemic MRSA, and
widespread environmental contamination
• Before-after study
• Cleaning time increased by 57 hours per week
• Responsibility for routine cleaning of shared
equipment delineated
• In 6 months post-intervention, number of MRSA
acquisitions decreased fro 30 to 3 per 6 months
Rampling A JHI 2001;49:109DR.T.V.RAO MD 50
51. • Audit and feedback
• New technology
• Hydrogen peroxide
vapour/gas
• UV room
decontamination?
antibacterial surface
coatings?
IMPROVING NON-CRITICAL
ITEM/ENVIRONMENT DISINFECTION
Otter ICHE 2009;30(6):574-7DR.T.V.RAO MD 51
52. THE PROBLEM OF CJD AND TSES
• Creutzfeldt-Jakob syndrome and other transmissible spongiform
encephalopathies caused by highly resistant proteinaceous particles,
prions
• can survive 3 years of environmental exposure and are unusually
resistant to conventional decontamination methods
• Iatrogenic CJD documented in three circumstances
• use of contaminated medical equipment (2 cases)
• use of extracted pituitary hormones (> 130 cases)
• implantation of contaminated grafts from humans (cornea, 3 cases; dura
mater, > 110 cases)
DR.T.V.RAO MD 52
53. INACTIVATION OF PRIONS
• Steam sterilization with NaOH
• Alkaline cleaner (pH 2.2, 1 hr 23°C)
• Copper plus peracetic acid
• Vapourized hydrogen peroxide (Sterrad NX)
Yan ICHE 2004;25:280, Fichet Lancet 2004;384:251, Baier JHI 2004;57:80,
Lemmer J Gen Virol 2004;85:3805; Roger-Kreuz, ICHE 2009;30(8):769-77
Lehman Hosp Infect. 2009;72(4):342-50;DR.T.V.RAO MD 53
54. WHAT IS AN INDICATOR OR
STERILISATION CONTROL ?
• On paper
• Self -contained
• Sealed ampulla (spores + broth)
• Spores suspension
• Tube witness (point of fusion)
It is a device conceived to verify
if the process operated as expected
DR.T.V.RAO MD 54
55. VALIDATION OF BIOLOGICAL
INDICATORS
• The reality :
• We do not use the most resistant organisms
• The predictive behaviour is generally linear only for one
process
• Manufacturers seldom use more “practical” strains
(read : “less linear” strains for more economical,
inoffensive, self resistance and stability)
• There is no such thing as a “universal biological indicator”
• The choice of any particular strain is therefore a manner of
arbitrary choice
DR.T.V.RAO MD 55
58. • Little evidence that
extensive use of
products containing
antiseptic and
disinfecting chemicals
adds to human or
animal health
• The use of such
products promotes the
development of
resistant microbes
EXCESSIVE USE OF CHEMICALS CAN
CREATE RESISTANT MICROBES ???
DR.T.V.RAO MD 58
59. • Avoid cleanrooms when ill
• Frequent bathing and
shampooing
• Avoid cosmetics such as
face powder, hair sprays,
perfumes and aftershave
• Clothing should be clean,
nonfrayed and nonlinting
• Avoid smoking
STERILIZATION IS SUCCESSFUL
WITH PERSONNEL: HYGIENE
DR.T.V.RAO MD 59
60. WHO / WHAT IS IMPORTANT IN PREVENTION OF
INFECTIONS
62. DR.T.V.RAO MD 62
• Programme created by Dr.T.V.Rao MD for
Medical and Paramedical Professionals in
the Developing World
• Email
• doctortvrao@gmail.com
