Sterilization and Disinfection

STERILIZATION &
DISINFECTION
DR R SATHYAJITH
19:09 PMDR R SATHYAJITH

STERILIZATION
• Latin sterilis means unable to produce offspring or
barren
• Process by which an “article, surface or medium is
freed of all living organisms either in vegetative or
spore state”
• When done using a chemical agent, it is referred to as
‘Sterilant’
• Generally, it implies physical process rather than

DISINFECTION & ANTISEPTICS
• Disinfection is the killing, inhibition or removal of all
pathogenic organisms or organisms capable of
causing infections.
• Antiseptics are chemical disinfectants that can be
safely applied to the skin & mucous membrane. They
are used to prevent infections by killing or inhibiting
pathogenic growth.
• Antiseptics also reduce total microbial population and
are less toxic as disinfectants.

COMMON TERMS
• Bactericidal agents or germicides are those that can
kill bacteria.
• Bacteriostatic agents – prevent multiplication of
bacteria, which may remain alive. A chemical which is
bactericidal at a particular concentration may only be
bacteriostatic at higher dilutions
• Decontamination is the process of rendering an article
or area free of danger from contaminants, including
microbial, chemical, radioactive & other hazards.
• Cleaning - A process that removes foreign material

STERILIZATION AND DISINFECTION
• Importance -
Safety in the laboratory.
The patient safety depends on using proper methods
of sterilization to prepare instruments, needles, IV
fluids.
The accuracy and validity of microbiological tests.
9:09 PM 5DR R SATHYAJITH

METHODS OF STERILIZATION
• Physical methods
1.Sunlight
2.Drying
3.Heat
4.Filtration
5.Radiation
6.Ultrasonic & sonic
vibrations
• Dry heat
a)Incineration
b)Red heat
c)Flaming
d)Hot air sterilizers
e)Microwave ovens
• Moist heat
a)Pasteurization
b)Boiling
c)Steam under normal pressure
d)Steam under pressure69:09 PMDR R SATHYAJITH

SUNLIGHT
• Role in spontaneous sterilization under natural
conditions
• Has appreciable bactericidal properties
• Bacteria suspended in water are readily destroyed on
exposure.
• Disinfectant action primarily due to UV rays & also
heat rays.
• Under natural conditions sterilizing power varies
according to circumstances.
• UV rays are screened out by glass or impurities/

DRYING
• Moisture is essential for growth of bacteria
• 80% of bacterial cell weight is water
• Drying hence has a deleterious effect
• Unreliable & of theoretical interest only
• Spores are not affected

REFRIGERATION
• Refrigeration 5o C
• Freezing -20o C
• Ordinary refrigeration has a bacteriostatic effect, but
does not kill, metabolic rate of some microbes is so
reduced that they cannot reproduce or synthesize
toxins
• Growth of pathogenic bacteria slows down at
refrigerator temperature & can be used to preserve
microorganisms 99:09 PMDR R SATHYAJITH

FILTRATION
• Filtration is commonly the mechanical operation which
is used for the separation of microbes from fluids
(liquids or gases) by interposing a medium through
which only the fluid can pass.
• Different from Sieving/ Adsorption 109:09 PMDR R SATHYAJITH

FILTRATION - USES
• Heat sensitive solutions – For sterilization of
pharmaceuticals, ophthalmic solutions, culture media,
oils, antibiotics etc.
• Separation of bacteriophages & bacterial toxins from
bacteria
• Isolating organisms which are scanty in fluids
• Concentration of bacteria from liquids
• Isolation of viruses – pore size of 0.22mcm most virus
& Mycoplasma pass through.

FILTERS - TYPES
•Earthware filters
•Asbestos filters
•Sintered glass filters
•Membrane filters
•Syringe filters
•Vacuum & ‘In-line’
filters
• Pressure filters
• Air filters

EARTHWARE/CANDLE FILTERS
• Available in different grades of porosity
• Used for purification of water – Industrial & Drinking
• Fluid is forced by suction or pressure from inside to
outside or vice versa
• After use sterilized by scrubbing with stiff brush -
boiling – autoclaving
Unglazed ceramic filters (Chamberland & Doulton)
Compressed diatomaceous earth filters (Berkefeld &
Mandler)

ASBESTOS FILTER
• Disposable, single use discs
• Filter disk is made up of asbestos (Mg trisilicate)
• High adsorbing capacity
• Alkalinize filtered fluid.
• Carcinogenic potential of asbestos has discouraged its
use
• E.g. Seitz filter, Carlson & Sterimat 169:09 PMDR R SATHYAJITH

SEITZ FILTER

SINTERED GLASS FILTER
• Sintered glass is a glass mesh used for filtration
• Prepared by heat fusing finely powdered glass
particles
• Pore size can be controlled by particle size of the
glass powder
• Easy to clean
• Low absorption properties
• Cons - Fragile & expensive

SINTERED GLASS FILTER

MEMBRANE FILTER
• Consists of polymeric material like cellulose nitrate,
cellulose diacetate, polycarbonate & polyester.
• Made as disks from 13-293mm diameter & 0.015-
12mcm porosities
• Average pore diameter (APD) 0.22mcm MC used in
sterilization

MEMBRANE FILTER

MEMBRANE FILTER
• Uses
Water purification & analysis
Sterilization & sterility testing
Preparation of sterile solutions for parenteral use
Bacterial count of water – Known amount of water is
filtered & the membrane disk is placed upside down
on a culture medium & incubated & colonies are
counted to know the viable count 239:09 PMDR R SATHYAJITH

SYRINGE FILTERS
• Membrane of 13-25mm diameter is fitted into syringe
like holders of stainless steel or polycarbonate
• For sterilization, the fluid is forced through the disk
membrane by pressing the piston of the syringe.

SYRINGE FILTERS

VACUUM & IN-LINE FILTERS
• Membrane diameter of 25 & 45mm are used
• In-line filter holder of Teflon or stainless steel &
aluminium
• Vacuum holders of borosilicate glass, polycarbonate
or stainless steel
• Used for sterilizing large volumes of liquids

VACUUM FILTERS

IN-LINE FILTERS

PRESSURE FILTERS
• Large membranes 100-293mm in diameter or filter
cartridges are used
• Housed in pressure filter holders
• Used for very pure water for lab use
• If fitted with Teflon filter, then autoclavable with filter
in situ

PRESSURE FILTERS

AIR FILTERS
• HEPA – High efficiency particulate filters
Large volumes of air can be rapidly freed from
infection by passage through them
Remove fine particles of size 0.3mcm or larger
Widely used in laminar air flow (LAF)
cabinets/chambers
Available in different efficiencies from 95.0% to
99.997% depending on the need
Do not filter out gasses, chemical vapors and odor
molecules

HEPA IN LAF SAFETY CABINET
9:09 PM 32
Supply
HEPA
filter
Exhaust
HEPA
filter
High-velocity
air barrier
Safety
glass
viewscree
n
DR R SATHYAJITH

HEPA
• First used to remove radioactive contaminants from
the air in the development of the atomic bomb
• Working with hazardous items such as the HIV virus,
TB bacilli & other lethal bacteria and viruses for the
sake of research requires HEPA filtration
• Medical-use HEPA filtration systems also incorporate
high-energy ultra-violet light units to kill off the live
bacteria and viruses trapped by the filter media

RADIATION - NONIONIZING
• 2 types – IR & UV
• Effectiveness of UV related to its wavelength
• Most effective is 240-280nm range
• Produced artificially by mercury vapor lamp
• Unlike ionizing radiation, energy of UV is lower &
penetration power is poor
• Does not penetrate glass, dirt films, water etc.

RADIATION – NONIONIZING UV

• Microbial sensitivity to UV radiation
 Bacterial spores are more resistant than vegetative
cells
 Viruses are inactivated & are more sensitive than
spores
 HIV not inactivated
• Practical uses
Disinfect water
Disinfect closed areas like OT, wards, labs &

UV light sterilization offers great potential for
tackling HAI’s or Healthcare Associated Infections
transmitted through the surrounding environments of
the healthcare institutions.
 Effective against – MRSA, VRE, C. difficile, A.
baumannii, ESBL, Influenza virus A.
 Disadvantages – Uninterrupted line of sight required
– Production of ozone

RADIATION - NONIONIZING
• Hazards of UV – Burns skin & damages eyes
– Human exposure should be avoided
• Infrared
IR can be considered a form of hot air sterilization
Used for rapid mass sterilization of prepacked items
like syringes & catheters

RADIATION – NONIONIZING IR
• Bacti – Cinerator (IR
Sterilizer)
• Sterilizes needles, loops,
and mouth of culture tube,
small bottle in 5-7 seconds
at optimum sterilizing
temperature above 800° C
• Maximum temperature
towards 930º C.

RADIATION – NONIONIZING IR

RADIATION – IONISING
• Very high penetrating power
• Highly lethal to all cells – Humans/ Bacteria
• Causes structural defects in microbial DNA synthesis
leading to cell death
• Bacterial spores are resistant
• E.g. X-rays, Gamma rays & cosmic rays.

RADIATION – IONISING GAMMA

RADIATION – IONISING RADIATION
• Cold sterilization
No appreciable increase in temperature
Commercial plants use gamma rays emitted from
radioactive elements like Cobalt 60
• Uses – Sterilizing in pharmacy & medicine (Abx,
hormones, sutures, vaccines) / packaged disposable
articles ( IV lines, catheter, syringes, Gloves)

ULTRASONIC & SONIC VIBRATIONS
• Have bactericidal properties
• Effects variable on different bacteria
• Unreliable & of no practical value in medicine
• Sometimes used for fine cleaning to remove
soil from joints, crevices, lumens and other
difficult to clean areas

ULTRASONIC STERILIZERS

HEAT
• Most reliable
• Universally applicable
• Method of choice – whenever applicable
• 2 types – Dry & Moist
• Materials damaged by heat – Sterilized at low
temperature or longer periods or repeated cycles
• Recommended minimum holding time does not
include the time taken to reach the specified
temperature 509:09 PMDR R SATHYAJITH

DECIMAL REDUCTION TIME (DRT)
• DRT also called D-value refers to the time required at
a certain temperature to kill 90% of the organisms
being studied
• Thus after a colony is reduced by 1 D, only 10% of the
original organisms remain.
• E.g. if a organism is reduced by 90% after exposure to
temperatures of 300° F for 20 minutes, the D-value
would be written as D300F = 20 minutes
• Sterilization-resistant organism are given a unique D-
value

Z VALUE
• Used in thermal death time calculations
• The z-value of an organism is the temperature, in
degrees (Fahrenheit or Celsius), that is required for
the thermal destruction curve to move one log cycle
• Simply, temperature required for one log reduction in
the D-value

Z VALUE

D-VALUE & Z-VALUE
• The Z-value is 10, D-value is 4.5 minutes at 150°F,
what is the D-value for 160°F ?
• The new D-value is 0.45 minutes
• This means that each 10°F increase in temperature will
reduce our D-value by 1 log.
• Conversely, a 10°F decrease in temperature will
increase our D-value by 1 log. So, the D-value for a
temperature of 140°F would be 45 minutes.

HEAT – FACTORS INFLUENCING
• Nature of heat
 Dry or Moist
• Temperature & Time
 Time required is inversely proportional to
temperature
 Thermal death time (TDT) – Minimum time required
to kill a suspension of organisms at a predetermined
temperature in a specified environment 569:09 PMDR R SATHYAJITH

HEAT – FACTORS INFLUENCING
• Number of microbes
 More number of microbes there are to begin with, longer
it takes to eliminate the entire population
• Characteristics of organisms
 Vegetative bacteria & virus are more susceptible & spores
are more resistant.
• Nature of contaminated material
 Presence of organic substances, proteins, nucleic acid,
starch, fats & oils, increase the TDT
 High acid/ alkaline pH increases bacterial killing 579:09 PMDR R SATHYAJITH

HEAT - MOA
• Dry heat
 Protein denaturation
 Oxidative damage
 High electrolyte level toxicity
• Moist heat
 Coagulation & Denaturation of enzymes/ structural
proteins

RED HEAT - FLAMING
• Red heat - Item to sterilized are held almost vertically
in a Bunsen flame until red hot along their whole
length, almost up to the tip of their metal holder.
• Items sterilized by red heat –
 Inoculating wire loops
 Points of forceps
• Flaming – Only exposed to the flame for a few
seconds & not heating to red hot. E.g Scalpel blades,
glass slides, mouth of culture tubes 599:09 PMDR R SATHYAJITH

RED HEAT

FLAMING

DRY HEAT - INCINERATION
• Excellent & safe method
• Material destroyed completely – reduced to ashes
• Very high temperatures reached
• Used for – Pathological wastes, surgical dressing,
animal carcasses, plastics like PVC/ polythene
• Avoid – Materials made of polystyrene as they emit
black dense smoke

INCINERATOR

DRY HEAT – HOT AIR OVEN

• Most widely used method by dry heat
• Used - When can withstand high temperatures & when
affected by contact with steam
• Parts – Electric heater, Fan to provide forced air
circulation, Temperature indicator, Control thermostat
& timer, Open mesh shelving, Heating coils on
wall/floor & adequate wall insulation – Inner layer
poor conductor & outer metallic

• Preparation of load
 No overloading – to allow free circulation of air
 Articles should be thoroughly clean & perfectly dry
 Test tubes & flasks – Should be wrapped in paper
 Avoid – Rubber except silicon, Cotton plugs, Plastics

• Sterilizing cycle
 Sterilization hold time – It is set to 160o C for 2hours
or 170o C for 1hour or 180o C for 30min
 Cutting instruments like used in ophthalmic surgery
should be sterilized at 150o C for 2hours
 Oils, glycerols & dusting powder holding time
recommended is 150o C for 1hour.

HOT AIR OVEN - USES
• Cooling takes several hours – Open the door after
chamber & load have cooled below 80o C or else
glassware will crack
 Glassware such as tubes, flasks, all glass syringes,
petri dishes, pipettes.
 Metal instruments like forceps, scissors & scalpels
 Nonaqueous materials & powders, oils & greases in
sealed containers, swab sticks placed in test tubes 699:09 PMDR R SATHYAJITH

HOT AIR OVEN - STERILIZATION CONTROLS
• Biological control
An envelope containing filter paper strip impregnated
with 106 spores of Bacillus atrophaeus (formerly
Bacillus subtilis subsp niger) is inserted
 Non toxigenic strains of Clostridium tetani can be
also used
 After sterilization, strips are removed & inoculated
into tryptone soy broth/Cooked Meat media
 Incubated anaerobically at 37o C for 5days 709:09 PMDR R SATHYAJITH

BACILLUS ATROPHAEUS

• Chemical indicators
 Browne’s tubes No. 3 containing red solution is
inserted
 Color change from red to green indicates proper
sterilization
• Thermocouples
 May be used periodically

HOT AIR OVEN VS AUTOCLAVE
• Safe – No water used & no pressure build up inside
• Smaller – But as effective as autoclave
• Cheaper – Suitable for small laboratory
• Rapid – also higher temperatures reached
• Dry heat penetrates more slowly

DRY HEAT – MICROWAVE OVEN
• Heating effect if not uniform
• Not reliable method for strerilization

MOIST HEAT
• Temp below 100o C
 Pasteurization
 Inspissation
 Water bath
 Vaccine preparation
• Temp at 100o C
Boiling
Koch & Arnold
steamer
Tyndallization
• Temp above 100oC
 Autoclave

PASTEURIZATION
• Used in dairy industry for milk
• All non sporing pathogens like Mycobacteria, Brucella
& Salmonella are destroyed
• Coxiella burnetii is relatively heat resistant & may
survive holder method
• All f/b rapid cooling to <13o C

PASTEURIZATION

PASTEURIZATION
• Holder method – 60o C for 30min
• Flash method/ High temperature short term (HTST) –
72o C for 15-20sec
• Higher-Heat Shorter Time (HHST) – 90o C for 0.5sec
• Ultra high temperature (UHT) sterilization – 140-150o
C for 1-3sec, Shelf stable
Source - International dairy food association (IDFA) guidelines

INSPISSATION
• Literally means the process of thickening by
dehydration
• Also called fractional sterilization
• Used when heating high-protein (Egg) containing
culture media like Lowenstein-Jenson & Loeffler’s
serum slope.
• Heated at 80o C for 30min (2hrs*) on 3 successive
days
• Arnold’s inspissator used* Handbook of Media for Clinical Microbiology By James W. Snyder, Ronald M. Atlas

INSPISSATOR

WATER BATH
• Washing or rinsing laundry or eating utensils at 70-
80o C for few minutes will kill most non sporing
microorganisms

BOILING
• Boiling at 100o C for 10-30min kill all vegetative
forms
• Sporing bacteria require prolonged periods of boiling
• Hard water should not be used
• Adding 2% NaHCo3 increases efficacy
• Not recommended for sterilization, only means of
disinfection
 Disinfection of surgical instruments before sterilizing
 Disinfection in field conditions & when sterility not

BOILING

TYNDALLIZATION
• Fractional/ Intermittent sterilization
• Boiling & exposure to steam at 100o C for 20min for 3
consecutive days
• Used for heat sensitive culture media
• 1st day – Vegetative cells, 2nd day – Early germinating
spores, 3rd day – Late germinating spores
• Failure to kill some anaerobic spores & thermophiles.
• Works for broth media that supports the growth of
spores & not water

KOCH & ARNOLD STEAMER

MOIST HEAT - AUTOCLAVE
• Process of sterilization using saturated steam under
high pressure above 100o C
• Principle – water boils when its vapor pressure equals
that of surrounding atmosphere.
• When pressure inside a closed vessel increases, the
temperature at which water boils also increases
• Steam condenses to water & gives up its latent heat to
the cooler surface it comes in contact with. Energy
available from this latent heat is considerable

AUTOCLAVE - PARTS
• Vertical/ Horizontal
• Stainless steel/ Gun metal
• Screw clamps to fasten lid
• Airtight by washer
• Heated by gas/ electricity

AUTOCLAVES - TYPES
1.Steam jacketed autoclave:
Double walled chamber in which steam at 121 degree
is introduced from an external source.
Steam enters the jacket from which it enters the
chamber migrating downwards replacing the air which
is expelled through a discharge tap at the bottom.
2. Flash autoclaves:
It sterilizes at 134 degree for 3 – 5 minutes.
Used in operation rooms in emergency situations.9:09 PM 89DR R SATHYAJITH

AUTOCLAVE PROCEDURE

AUTOCLAVE

AUTOCLAVE PROCEDURE
1.Sufficient water is put in the cylinder.
2.Articles are kept above the perforated shelf
3.Lid is screwed tight
4.Autoclave is heated
5.Discharge tap is kept open
6.Safety valve is adjusted to required pressure
7.Steam-air mixture is allowed to escape freely till all
air is displaced 929:09 PMDR R SATHYAJITH

AUTOCLAVE PROCEDURE
8. Discharge tap closed
9. Steam pressure rises inside
10. Safety valve opens when it reaches the desired set
level (15psi)
11. Excess steam escapes
12. Holding period calculated from now (15min)
13. Heater turned off when holding period over.
14. Cool the autoclave till pressure inside equals
atmospheric pressure
15. Discharge tap is open & later lid is opened & items

AUTOCLAVE - PRECAUTIONS
• Air must be allowed to escape from the chamber
because the temperature of air-steam mixture is lower
than that of pure steam
• If the discharge tap is opened when pressure inside is
high, liquid media will boil violently & spill from the
container & can also explode
• If discharge tap is opened after pressure inside has
fallen below atmospheric pressure, excessive water
would have evaporated & lost from the media
• Material to be arranged to ensure free circulation of
steam inside the chamber

AUTOCLAVE - USES
• MOA – Bacterial proteins coagulates & condensed
water ensures moist condition fro killing
• Sterilizing culture media, aqueous solutions, rubber
materials, dressing materials, gowns, dressing, linen,
gloves, instruments & pharmaceutical products
• For all materials that are water containing, permeable
or wettable
• Useful for materials unable to withstand higher
temperature of hot air. 959:09 PMDR R SATHYAJITH

AUTOCLAVES - CONTROLS
• Filter paper strip impregnated in 104 spores of
Geobacillus stearothermophilus (ATCC 7953) used
• Placed in least accessible & coolest part
• Inoculated in TSB & incubated at 56o C for 5days
• It withstands 121o C for upto 12min

9:09 PM 97
Cap that
allows
steam to
penetrate
Flexible
plastic
vial
Crushable
glass
ampule
Nutrient
medium
containing pH
color indicator
Endospore strip
After autoclaving,
flexible
vial is squeezed to
break
ampule and release
medium onto spore
strip.
Yellow
medium
means spores
are
viable;
autoclaved
objects are not
sterile.
Red medium
means spores
were
killed; autoclaved
objects are
sterile.
DR R SATHYAJITH

• Self adhesive sterilisation
indicator tape with bands that
turn dark brown. Supplied in 19
or 25mm widths.
• Paper sterilizer control strips
with a heat sensitive colored dot
that changes from yellow to
purple.

AUTOCLAVE VS HOT AIR OVEN
• More lethal – action of moist heat
• Quicker in heating up articles
• Greater penetration of porous material like cotton
plugs, paper & cloth wrappers, surgical linen, hollow
apparatus.

REFERENCES
• Seymour S. Block: Disinfection, sterilization and
preservation, Williams &Wilkins (ISBN 0812113640)
• CDC guideline for disinfection and sterilization in
healthcare facilities
http://www.cdc.gov/hicpac/pdf/guidelines/Disinfection_Nov_2008.pdf
• Textbook of microbiology, Surinder kumar, Jaypee
• Textbook of microbiology, Ananthanarayan &
Panicker, 9th Ed 9:09 PM 100DR R SATHYAJITH

THANK YOU

Sterilization and Disinfection

More Related Content

What's hot

Viewers also liked

Similar to Sterilization and Disinfection

Recently uploaded

Sterilization and Disinfection

Editor's Notes