STERILIZATION

&
DISINFECTION
PRESENTER –DR ANSHU
J.SS. MEDICAL COLLEGE
MYSORE
 Definitions
• Sterilization-defined as the process by which an article,
surface or medium is freed of all micro organisms either in
the vegetative or spore state
• Disinfection-defined as destruction or removal of all
pathogenic organisms
• Decontamination-refers to the process of rendering an article
or area free of danger from contaminants,includin g
microbial,chemical,radioactive & other hazards
• Bactericidal agents or germicides-are chemicals which are
able to kill bacteria
• Bacteriostatic agents-are chemicals that only prevent
the multiplication of bacteria which may, however
remain alive

• Antisepsis-The term “antisepsis” is used to indicate the

prevention of infection,usually by inhibiting the growth
of bacteria in wounds or tissue

• Antiseptics-chemical disinfectants that can be safely

applied on skin or mucous membrane & used to prevent
infection by inhibiting the growth of bacteria
 Applications in microbiology
laboratory
• Sterilization of culture media, reagents & equipments before
their use in laboratory

• Effective decontamination of spillage,surfaces & equipment

after use

• Disinfection of contaminated material before their safe

disposal from laboratory
PHYSICAL METHODS OF STERILIZATION

 HEAT:considered to be the most reliable method for articles

that can withstand heat
 Dry heat Moist heat
 M.O.A.-
 Dry Heat- Protein denaturation,
Oxidative damage,
Toxic effects of elevated levels of
electolytes
 Moist Heat-Coagulation & denaturation of protein
 Thermal death time-Minimum time reqd to kill a suspension of
organisms at a predetermined temperature in a specific
environment
Factors affecting sterilization by heat
• Nature of heat-Moist heat is more effective than dry heat
• Temp&time-Temp is inversely proportional to time.
As temp increases,time taken decreases
• Number of microorganism-More the no. of organisms,higher the
temp.or longer the duration reqd
• Nature of micro-organism-Spores are highly resistant to heat
• Type of material-Heavily contaminated articles req higher temp or
prolonged exposure
Certain heat sensitive articles must be sterilized at lower temp
• Presence of organic material [protein,sugar,oils,fat]-Increases the
time reqd
Dry Heat
METHOD ARTICLE LIMITATIONS
STERILIZED
A)RED HEAT Bacteriological Only for those articles
loops,straight that can be heated to
wires,tips of redness in flame
forceps,searing
spatulas
B)FLAMING Scalpels, •only for articles that
mouth of test tubes, can be exposed to
flasks, glass slides, flame, no guarantee of
killing spores,
coverslips
Cracking of glassware
may occur
C)INCINERATIO Soiled dressings,animal Technique results in loss
N carcasses,pathological of article,only suitable
material,bedding suitable for the articles
that have to be disposed
HOT AIR OVEN
HOT AIR OVEN
• Most widely used method of sterilization by dry heat
Principle-
• Articles to be sterilized are exposed to high temp.for a
duration of one hour in an electrically heated oven
• Since air is a poor conductor of heat ,even distribution of
heat throughout the chamber is achieved by fan
• Heat is transferred to the article by radiation, conduction &
convection
 Sterilization cycle-
Time taken for articles to reach sterilizing temp +
Maintainence of sterilizing temp.for a defined period(holding
time) +
Time taken for articles to cool down
Different temp-time relationship for holding time:
60min at 160 degree celsius
40min at 170 degree celsius
20min at 180 degree celsius

 Increasing temp. by 10 degrees shortens the

sterilizing time by 50%
 Precautions during sterilization process

 Articles to be sterilized must be perfectly dry before placing

them inside to avoid breakage
 Articles must be placed at sufficient distance, so as to allow
free circulation of air in between
 Mouths of flasks,test tubes & both ends of pipettes must be
plugged with cotton wool
 Articles such as petridishes & pipettes may be arranged
inside metal canisters& then placed
 Individual glass articles must be wrapped in kraft paper or
aluminium foil
 Hot air oven must not opened until the temp inside has fallen
below 60*C to prevent breakage of glasswares
Advantages
 Effective method of
sterilization of heat stable
articles
 Articles remain dry after
sterilization
 Only method of sterilizing
oils & powders
Disadvantages
 Hot air has poor penetration
(poor conductor of heat)
 Cotton wool & paper may
get slightly charred
 Glasses may become smoky
 Takes longer time
compared to autoclave
Sterilization control
 Physical - Temperature chart recorder
- Thermocouple

 Chemical- Brownie’s tube no-3 [green spot]

 Biological- 10*6 spores of Bacillus subtilis var niger

 MOIST HEAT STERILIZATION
At temp.below 100*C

METHOD TEMP-TIME USES ORGANISMS AFFECTED

PASTEURIZATIO Holder method- Food & Salmonella,
N 63*CX30min dairy mycobacteria,
Flash method- industr streptococci,
72*C X15-20 sec y staphylococci, brucella-
f/b quickly cooling destroyed
to 13*C  coxiella may survive
VACCINE BATH 60*C X1 hr Contam Only vegetative
inating bacteria are killed
bacteri  spores survive
a in
vaccine
prepara
tion
WATER BATH OR 56*CX1Hr on Contamin  only vegetative
SERUM BATH several ating bacteria are killed
successive bacteria  spores survive
days[proteins in serum
in the serum preparatio
will coagulate n
at higher
temp]
INSPISSATION 80-85*C X30 Egg & Day1-vegetative
Min on 3 serum forms are killed,
successive containing spores germinate
days media [L- by next day & are
J Media killed
&Loeffler’ Success of the
s serum process depends
on germination of
spores
 At temp.100*C
 Boiling:
• Boiling water at 100*C for 10-20 minutes
• Articles-certain metal articles & glasswares
• Most vegetative bacteria and viruses are killed immediately
 Not effective against - Bacterial toxins
- spores
 Steam at 100*C
• Principle :Exposing articles to free steam at 100*C
• Eg-Arnold’s and Koch’s steamer
An Autoclave (with discharge tap open)
• Exposure period- 90 minutes
• Uses-Media such as TCBS, DCA, Selenite broth
 Tyndallisation
OR
Fractional sterilization OR Intermittent sterilization

 Sugar and gelatin in the medium may get decomposed on

autoclaving, hence they are exposed to free steaming for
20min on 3 successive days

 Success of the process depends on germination of spores

At temp.>100*C

AUTOCLAVE / STEAM STERILIZATION

Principle - [steam under pressure]
 Water boils when its vapour pressure equals that of
surrounding atmospheric pressure.
 When pressure inside the closed vessel increases,the temp at
which water boils also increases
 At a pressure of 15Lbs inside autoclave, the temp is said to be
121*C
 Exposure of articles to this temp for 15 minutes sterilizes
them
 Articles sterilized
• Culture media
• Hospital dressings
• Linen
 Advantages of steam:
• More penetrative power
than steam
• Moistens spores[essential
for coagulation of proteins]
Different types of
autoclave:
• Simple pressure cooker
type
• Steam jacketed downward
displacement laboratory
autoclave
• High pressure
pre-vaccum autoclave
Precautions

 Articles should not be tightly packed

 Autoclave must not be overloaded
 Air discharge must be complete and there should not be any
residual air trapped inside
 Caps of bottles & flasks should not be tight
 Autoclave must not be opened until the pressure has fallen
else the contents will boil over
 Articles must be wrapped in paper to prevent drenching
 Bottles must not be overfilled
 Advantages Disadvantages

 Very effective way of  Drenching and wetting of

sterilization articles

 Quicker than hot air oven  Trapped air may reduce

efficacy

 Takes long time to cool

 Sterilization control

 PHYSICAL : Automatic process control

Thermocouple
Temperature chart recorder
 CHEMICAL: Brownie’s tube no 1[black spot]
Succinic acid(Melting point -121*C)
Bowie’s Dick tape(satisfactory
process-dark brown stripes)
 BIOLOGICAL: 10*6 spores of Geobacillus
stearothermophillus
RADIATION
NON-IONISING RADIATION

 U.V. RAYS[200-280nm]:260nm most effective

 M.O.A.-Formation of thymine-thymine dimers
Inhibition of D.N.A replication
 Uses-to disinfect hospital wards,operation theatres,virus laboratories
 Limitation:do not kill spores
 Disadvantages : Low penetrative power
Limited life of U.V. bulb
Some bacteria have D.N.A.repair enzymes
overcome damage
Organic matter and dust prevents its reach
Rays are harmful to skin & eyes
Doesn’t penetrate papers, plastics or glass
INFRA-RED RAYS

 M.O.A.- Generation of heat

 Temp 180*C X 7.5 minutes

 Articles sterilized : metallic instruments, glasswares

 Mainly used in central sterile supply department

 Sterilization control:Brownie’s tube no.4(blue spot)

IONIZING RAYS
[COLD STERILIZATION]
 Particulate [electron beams]
 Syringes, gloves ,dressing
packs ,food & pharmaceuticals
 Few seconds only
 Disadvantages-
 Poor penetrative power,req
sophisticated equipments
 Not widely used in medicine
 Electromagnetic rays[Gamma
rays & X rays]
 Damage nucleic acid of
microorganism
 Used comercially to sterilize
disposable petridishes, plastic
syringes, antibiotics, vitamins &
hormones
 Dose of 2.5 megarads kills all
bacteria. fungi,viruses & spores
 Longer time of exposure
 Disadvantages: can’t be switched
off, glasswares become
brownish, loss of tensile strength
of fabrics
 FILTRATION

 To remove microbes from Types of filter:

heat labile liquids :Serum
 Earthenware filters
Antibiotic solutions
Pasteur-Chamberland
Sugar solution
Berkefeld
Urea solution
Mandler
• Applications: Removing
 Asbestos filters
bacteria from ingredients of
culture media,  Sintered glass filter
• Preparing suspensions of  Membrane filter
viruses,
• Water purification
 AIR FILTERS

 Air can be filtered using HEPA [High efficiency particle air filter]

 99.97%efficient for removing particles >0.3micrometers in

diameter

 Applications:Rooms having severe neutropenic patients,

Operating rooms for orthopaedic implant procedure

 Efficiency tested : Dioctylphthalate (DOP) particle test using

particles that are 0.3 micrometers in diameter
Chemical Methods

 Ideal disinfectant : should have

• Wide spectrum of activity
• Destroy microbes within practical period of time
• Active in the presence of organic matter
• Active at any pH
• Stable
• Long shelf life
• High penetrating power
• Non-toxic, non-allergenic, non-irritative or non-corrosive
• Should not have bad odour

• Should not leave non-volatile residue or stain

• Efficacy should not be lost on reasonable dilution

• Should not be expensive

• Easily available
Factors determining potency of disinfectant

• Concentration of substance
• Time of action
• pH of the medium
• Temperature
• Nature of organism
• Presence of extraneous material
 CLASSIFICATION OF DISINFECTANTS

A)Based on consistency
B)Based on spectrum of activity
 Liquid [Alcohols,Phenol]  High level
 Gaseous [Formaldehyde
vapour,Ethylene oxide]
 Intermediate level

 Low level
C)Based on mechanism of action
• Action on membrane[Alcohol, Detergents]
• Denaturation of cellular proteins[Alcohol, Phenol]
• Oxidation of essential groups of
enzymes[Halogen,H2O2]
• Alkylation of amino, carboxyl & hydroxyl
group[Ethylene oxide,Formaldehyde]
• Damage to nucleic acid [Ethylene
oxide,Formaldehyde]
 Spectrum of activity
Vegetati mycobac spores fungi viruses example
ve cells teria s

High + + + + + Ethylene
level oxide,
Gluterald
ehyde,
Formald
ehyde
Intermed + + - + + Phenolic
iate level s,Haloge
ns
Low level + - - + +/- Alcohols,
Quatern
ary
ammoni
um
compoun
ds
 Alcohols

 Ethyl alcohol & isopropyl alcohol[preferred]

 M.O.A.-Denaturation of bacterial proteins
 Concentration of 60-90% in water
 No action on spores
 Application-Disinfection of clinical thermometers
 Methyl alcohol-Effective against fungal spores,
To treat cabinets & incubators affected by
them
 Aldehydes
Formaldehyde
• Active against amino group in
protein molecule
• Bactericidal,sporicidal,lethal
effects on viruses
• Applications-
 Preserve anatomical specimens
 10% formalin containing
0.5%sodium tetraborate is used
to sterilize metal instruments
Glutaraldehyde
• Specially effective against
tubercle bacilli,fungi & viruses
• Application-
 Corrugated rubber anaesthetic
tubes,face masks,plastic ET
Tube, metal
instruments,cystoscopes,bronc
hoscopes
 Less toxic & irritant to eyes &
skin than formaldehyde
 Dyes
• Aniline dyes Acridine dyes
Brilliant green Proflavine
Malachite green Acriflavine
Crystal violet Euflavine
Aminacrine
• Mainly bacteriostatic, low bactericidal activity
• No activity against tubercle bacilli, hence the use of malachite green in
L-Jmedia
• More effective against gram positive bacteria than gram negative
bacteria
• Use-Skin & wound antiseptics
 Halogens

Iodine Chlorine[hypochlorites]
• Skin disinfectant
• Markedly bactericidal
• Actively bactericidal,with
• Wide spectrum of action
moderate action against
against viruses
spores
• Active against tubercle • Organic chloramine-
antiseptics for dressing
bacilli & viruses
wounds
Phenols

• Lysol,cresol,chlorophenols,chloroxyphenols,chlorhexidine
• M.O.A.-cell membrane damagerelease cell contents lysis
 Precipitate protein
 Inactivation of membrane bound oxidases & dehydrogenases
• Chlorhexidine-skin antiseptic
• Most effective against gram positive organisms
Gases
Ethylene oxide Formaldehyde gas Betapropiolactone

Alkylating the amino, Alkylation of amino Alkylation of carboxyl &

carboxyl,hydroxyl & group hydroxyl groups
sulphydryl gps in protein
molecule Applications- More efficient than
Reacts with DNA & RNA Operation formaldehyde for fumigation
Applications-Heart-lung theatres,sick
machines,ventilators,sutu rooms,laboratories Active against all organisms &
res,dental equipments viruses
Effective against all type carcinogenic
of microorganisms
including viruses and
spores
Mutagenic &
carcinogenic
 Surface active agents

• M.O.A.- Alter the energy relationship at interface producing a

reduction of surface or interfacial tension
• Most important are cationic surface active agents in the form
of quaternary ammonium compounds
[acetyl,trimethylammonium bromide(CETAVLON OR
CETRIMIDE)]
• Bactericidal, more active against gram positive bacteria
• No action on spores, tubercle bacilli,& most viruses
• Disadvantage- Pseudomonas can metabolise cetrimide,using
them as a carbon, nitrogen & energy source
 Metallic salts

• Salts of silver, copper and mercury

• M.O.A.-protein coagulation
• Capacity to combine with free sulphydryl group of cell
enzymes
• Thiomersal,phenylmercury nitrate & mercurochrome-mild
antiseptics
• Marked bacteriostatic action
• Copper salts -fungicides
Testing of Disinfectants

• No single test is available to determine the efficiency of a

disinfectant
• Different methods-
 Koch’s method
 Rideal walker method
 Chick Martin test
 Kelsey-Sykes test [capacity to use dilution test]
 I n-use test
 NEWER METHODS OF
STERILIZATION AND DISINFECTION
Process Agent

Disinfection Ortho-phthaladehyde(Cidex OPA)

Antimicrobial coating(Surfacine)
Superoxide water(Sterilox)

Sterilization Liquid sterilization process(Endoclens)

Rapid readout ethylene oxide biological
indicator(Attest)
New plasma sterilizer(Sterrad 50)
COMPARISON OF NEW AND STANDARD
DISINFECTION & STERILIZATION TECHNOLOGIES
New Standard Advantages Disadv. Future needs

OPA Glutaralehyde Shorter process Stains Additional

time(12vs45min) protein gray studies of
No activation Higher cost antimicrobial
Not a known efficacy
irritant to eyes pr Cost-
nasal passages effectiveness
No vapour ceiling studies
limit Study of
Weak odour effectiveness
in actual
clinical use
Verification
of more
cycles per
solution than
glutaraldehy
d
Surfacine Disinfectants Antimicrobial Cost? Assess microbicidal
(phenolics,quatern persistance activity against
ary ammonium) (>13days) broad spectrum of
Antiseptics May be used pathogens
(alcohol,chlorhexidi
on animate or Demonstration of
ne)
inanimate efficacy to reduce
objects nosocomial
Broad infection
antimicrobial Human safety &
spectrum toxicity data for use
No as an antiseptic
toxigenicity to Demonstrate
mammalian antimcrobial activity
cell in presence of
Transfers organic matter
active agent
(silver) to
microbes on
demand
without
elution
Superoxide High or low Basic Production Cost-effectiveness
water level materials equipment study
disinfectants; (saline & inexpensive
antiseptics electricity)ine due to
xpensive monitoring
End product Decreased
not damaging efficacy in
to presence of
environment organic
Non toxic to matter
biological Limited use
tissues life(must be
freshly
prepared)
Endoclen None Device automatically •Cost? •Cost.effecti
s cleans & sterilizes •Used for veness
Rapid cycle immersible study
time(<30min) in struments •Study of
Tests endoscope for only eff. In actual
channel blockage •No long clinical use
&leaks term •Assessmen
Advantages of storage t of
automated process microbicidal
(consistant exp. to activity
sterilant,
operator convenience)
EO rapid 48-hr spore Rapid(4hr Cost? Cost-
read out readout )reliable Not effectivene
biological assessmen tested with ss study
indicator t of EO and Validation
sterilizatio CO2 of claimed
nn efficacy mixtures 100%
Prevents sensitivity
recall of
released
sterilizatio
n loads
Plasma Hydrogen Use of Cost? Cost-
sterilizer peroxide gas two H2O2 Endoscop effectivene
plasma diffusion es with s study
sterilizer plasma length Study of
stage >40cm or effectivene
cycles is a diameter ss in actual
more <3mm practice
effective cannot be
sterilization processed
process
Reduced
cycle time
Various
sized units
available
Leaves no
toxic
residue
Conclusion
• Various methods of sterilization & disinfection are available
• Use of a particular method depends upon the article to be
sterilized and nature of microorganism
• Newer sterilization and disinfection technologies may
provide significant advantages over existing technologies
• These new technologies hold the promise of improved
patient care
 References

* Practical Medical Microbiology,

Mackie & McCartney,14th edition
* Text book of Microbiology,
Ananthnarayan & Paniker’s,8th edition
* Internet sources
Thank you