Chapter 3 Sterilization and Disinfection

STERILIZATION AND
DISINFECTION
Learning objectives
At the end of the session, the students will know
• Definition of sterilization, disinfection, asepsis,
decontamination.
• Various physical and chemical methods of sterilization
• Disinfection testing and
• Spaulding classification of medical devices
Essentials of Medical Microbiology

Definition
• Sterilization: Process by which all living microorganisms
including viable spores, are either destroyed or removed from
an article, body surface or medium.
o Reduction of at least 106 log CFU of microorganisms.
• Disinfection- Process that destroys or removes most if not all
pathogenic organisms but not bacterial spores.
o Reduction of at least 103 log CFU of microorganism.

Definition
• Asepsis- Process where the chemical agents (called antiseptics)
applied to body surfaces (skin) will kill or inhibit the pathogenic
microorganisms (and also commensals) present on skin.
• Decontamination (or sanitization)- Reduction of pathogenic
microbial population to a level at which items are considered as
safe to handle without protective attire.

Factors influencing efficacy of sterilant/ disinfectant
1. Organism load: Larger microbial population requires a

longer time to be destroyed.
2. Nature of organisms:
o Prions (highest resistance) > Cryptosporidium oocysts > Bacterial spores >
Mycobacteria > Other parasite cysts (e.g. Giardia) > Small non-enveloped viruses >
Trophozoites > Gram-negative bacteria > Fungi > Large non-enveloped viruses >
Gram-positive bacteria > Enveloped viruses.

3. Concentration of the chemical agent or the temperature

of heat sterilization.
4. Nature of the sterilant /disinfectant
5. Duration of exposure
6. Temperature

7. Local environment
o Heat kills more readily at an acidic pH.
o Biofilm are also a good example that prevents the
entry of disinfectants to act on the microorganisms
that are embedded inside the biofilm.

Classification of sterilization methods
1.Sunlight 2.Drying
3.Heat
Dry heat- Flaming, Incineration, Hot air oven
Moist heat-
 Temperature below 1000C- pasteurization, water bath and inspissation
 Temperature at 1000C- e.g. boiling, steaming and tyndallisation
 Temperature above 1000C- e.g. autoclave
4.Filtration – depth filters and membrane filters
5.Radiation
Ionizing radiation-γ rays, X-rays and cosmic rays
Non-ionizing radiation- Ultraviolet (UV) and infrared rays
6. Ultrasonic vibration
Classification of disinfection methods
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
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

PHYSICAL AGENTS OF STERILIZATION/
DISINFECTION
Method Principle
Sunlight Active microbicidal effect due to its content of ultraviolet rays.
Drying 70-80% of the weight of the bacterial cell is due to water. Drying,
therefore has a deleterious effect on many bacteria.
Dry heat Kills the organisms by charring, denaturation of bacterial protein,

oxidative damage and by the toxic effect of elevated levels of
electrolytes.
Moist heat Kills the microorganisms by denaturation and coagulation of proteins.

Dry heat sterilization
Flaming Items are held in the flame of a Bunsen burner either for
long time or short time.
o Longer time exposure - for inoculating wires or loops, tips of forceps.
o For shorter period - mouth of test tubes.
Incineration Used for the disposal of waste materials.
o It burns (sterilizes) the anatomical waste and microbiology waste by
providing a very high temperature 870 to 1,200°C - converting the
waste into ash, flue gas, and heat.

Hot air oven (Dry heat Sterilizer)
• Most widely used method of sterilization by

dry heat.
• It is electrically heated and is fitted with a
fan to ensure adequate and even
distribution of hot air in the chamber.
• It is also fitted with a thermostat which
maintains the chamber air at a chosen
temperature.
• Holding temperature and time of 160°C for
2 hours.
Hot air oven (Dry heat Sterilizer)
• Materials sterilized:
o Glassware - syringes, petri dishes, flasks, pipettes and test tubes.
o Surgical instruments - scalpels, forceps, etc.
o Chemicals such as liquid paraffin, fats, glycerol, oil, and glove
powder, etc.

Hot air oven - precautions
• Overloading of hot air oven should be avoided.

• Material should be arranged in a manner so that free circulation of air is
maintained.
• Material to be sterilized should be dried completely.
• Cotton plugs should be used to close the mouths of test tubes, flasks, etc.
• Paper wrapping of the items should be done.
• Any inflammable material like rubber (except silicone rubber) should not be
kept inside the oven.
• Oven must be allowed to cool for two hours before opening the doors,
since the glassware may crack by sudden cooling.
Hot air oven - Sterilization control
• Biological indicator: Spores (106)

o Bacillus atrophaeus - to check the effectiveness of
sterilization by dry heat. Earlier, non-toxigenic strain of
Clostridium tetani was used.
o Spores should be destroyed if the sterilization is done
properly.
• Physical control: For example, digital displays on the
equipment displaying temperature and time.
Moist Heat sterilization -
Below 100°C
Pasteurization • Method used for control of microorganisms from beverages like

fruit and vegetable, juices, beer, and dairy products, such as milk.
o Holder method (63°C for 30 mins).
o Flash method (72°C for 20 seconds followed by rapid cooling to
13° or lower).
o All nonsporing pathogens, including mycobacteria, brucellae and
salmonellae are killed except Coxiella burnetii which being
relatively heat resistant may survive in holder method.

Below 100°C
Water bath • Used for disinfection of serum, body fluids and vaccines.
• Bacterial vaccines are disinfected at 60°C for 1 hour.
• Serum or heat labile body fluids can be disinfected at 56°C
for one hour.

Below 100°C
Inspissation • Process of heating an article on 3 successive days at 80–85ฐC for

(fractional 30 minutes.
sterilization) • Working principle:
o first exposure - kills all the vegetative forms.
o Intervals between the heating the remaining spores
germinate into vegetative forms which are then killed on
subsequent heating.
• Uses: Sterilization egg-based medium – Lowenstein Jensen
medium and Dorset’s egg medium; serum-based media—
Loeffler’s serum slope.

Moist Heat sterilization
Moist heat at a temperature at 100°C
Boiling • Boiling of the items in water for 15 minutes kills most of the
vegetative forms.
• But do not kill the spores - not suitable for sterilization of surgical
instruments.
Koch’s or • useful for those media which are decomposed at high
Arnold’s steam temperature of autoclave.
sterilizer • Articles exposed to steam (100°C) at atmospheric pressure for 90
minutes.
Most of the vegetative forms are killed by this method
except thermophiles and spores.

at 100°C
Tyndallisation • Involves steaming at 1000C for 20minutes for 3 consecutive days.

or intermittent • Principle is similar to that of inspissation, except that here, the
sterilization temperature provided is 1000C, instead of 800C.
(named after • Used for sterilization of gelatin and egg, serum or sugar
John Tyndall) containing media which are damaged at higher temperature of
autoclave.

above 100°C
• Principle of Autoclave:
o Autoclave functions similar to a pressure cooker and follows
the general laws of gas.
o Water boils when its vapour pressure equals that of the
surrounding atmosphere.
o When the atmospheric pressure is raised, the boiling
temperature is also raised.
o At normal pressure, water boils at 100°C but when pressure
inside a closed vessel increases, the temperature at which
water boils also increases.
Components of Autoclave
• Autoclave comprises of three parts

 Pressure chamber
Lid
Electrical heater.

Horizontal autoclave
and Vertical autoclave

Sterilization Conditions
• Autoclave can be set to provide higher temperatures by

adjusting the pressure provided to the vessel.
o 121⁰C for 15 minutes at pressure of 15 lbs psi.
o 126 ⁰ C for 10 minutes at pressure of 20 psi.
o 133 ⁰ C for 3 minutes at pressure of 30 psi.

Uses of Autoclave
• Surgical instruments
• Culture media
• Autoclavable plastic containers
• Plastic tubes and pipette tips
• Solutions and water
• Biohazardous waste
• Glassware (autoclave resistible).
Precautions to be taken
• Autoclave should not be used for sterilizing waterproof

materials
• Materials are loaded in such a way that it allows efficient
steam penetration (do not overfill the chamber)
• Material should not touch the sides or top of the chamber
• Clean items and the wastes should be autoclaved separately
• olyethylene trays should not be used as they may melt and
cause damage to the autoclave.
Types of Autoclaves
• Gravity displacement type autoclave: most

commonly used.
• Positive pressure displacement type autoclave
• Negative pressure (vacuum) displacement type.

Sterilization control
• Biological indicator: Spores of Geobacillus stearothermophilus

(formerly called Bacillus stearothermophilus). Spores are killed in 12
minutes at 121°C
• Chemical indicators
o Class I - external pack control, (e.g. autoclave tape)
o Class II - equipment control (Bowie-Dick test)
o Class IV/V - internal pack control.
• Physical control: For example, digital displays on the equipment
displaying temperature, time and pressure.
Filtration
• Filtration is an excellent way to remove the microbial
population in solutions of heat-labile materials like vaccine,
antibiotics, toxin, serum and sugar solution.
• Types of Filters
1. Depth Filters.
2. Membrane Filters

Depth Filters
• Depth filters are porous filters composed of random mats
of metallic, polymeric, or inorganic materials.
• Advantages - Retain a large mass of particles before
becoming clogged, flow rate of the fluid is high and low
cost
• Disadvantages- Not suitable for filtration of solution
containing bacteria.
• Depth filters - candle filters (Berkefeld filters), unglazed
porcelain (Chamberlain filters),asbestos filters (Seitz and
Sterimat filters) and sintered glass filters
Membrane Filters
• Most widely used filters for bacterial filtration.
• Porous; retain all the particles on the surface that are smaller
than their pore size.
• Composed of cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride, or other synthetic materials
• Pore size:
o 0.22 µm - Most commonly used (removes most of the
bacteria, allowing the viruses to pass through)
o 0.45 µm - Used to retain coliforms.
o 0.8 µm - Used to remove airborne microorganisms in clean
rooms.
Filtration of Liquid
• To sterilize sera, sugar and antibiotic solutions.
• Separation of toxins and bacteriophages from bacteria.
• To obtain bacteria free filtrates of clinical samples for virus
isolation
• Purification of water—e.g. testing of water samples for Vibrio
cholera or typhoid bacilli.

Filtration of Air
• Simple e.g. - surgical masks (that allow air in but keep
microorganisms out).
• Two important air filters that are used in biological safety cabinets
and laminar airflow systems):
o HEPA filters (High-efficiency particulate air filters) removes
99.97% of particles that have a size of 0.3 µm or more.
o ULPA filters (Ultra-low particulate/penetration) removes from
the air at least 99.999% of dust, pollen, mold, bacteria and any
airborne particles with a size of 0.12 µm or larger.
Sterilization Control
• The sterilization control of membrane filters includes

Brevundimonas diminuta and Serratia marcescens.

Ionizing radiation (cold sterilization)
• Include, X-rays, gamma rays (from Cobalt 60 source), and cosmic rays.
• Mechanism: It causes breakage of DNA without temperature rise.
• Uses
o Disposable rubber or plastic syringes, infusion sets and catheters.
o Catgut sutures, bone and tissue grafts and adhesive dressings as well as antibiotics
and hormones.
o Irradiation of food (permitted in some countries).
• Advantages of ionizing radiation:
o High penetrating power, Rapidity of action and Temperature is not raised
• Sterilization control: Efficacy of ionizing radiation is tested by using Bacillus pumilus.

Non-ionizing Radiation
• Includes infrared and ultraviolet radiations.

• Lethal but do not penetrate glass, dirt films, water; hence their use is
restricted.
• Recommended dose - 250–300 nm wavelength of UV rays, for 30
minutes
• Uses – For disinfection of clean surfaces in operation theaters, laminar
flow hoods as well as for water treatment.
• Because UV radiation burns the skin and damages eyes, hence the area
should be closed and UV lamps must be switched off immediately after
use.
Ultrasound (Ultrasonic) Waves
• High-frequency ultrasonic and sonic sound waves

disrupt bacterial cells.
• Not reliable, hence is not used now a days.

CHEMICAL AGENTS OF STERILIZATION/
DISINFECTION

Classification
Level of Bacterial Tubercle Nonenvelope Fungi Envelope Vegetative
disinfectant spores bacilli d viruses d viruses bacteria
Low level No No No +/- Yes Yes
disinfectant
Intermediate No Yes Yes Yes Yes Yes
level
disinfectant
High level May be Yes Yes Yes Yes Yes
disinfectant
Chemical Yes Yes Yes Yes Yes Yes
sterilant

Various groups of disinfectants
• Alcohols
• Aldehydes
• Phenolic compounds
• Biguanide
• Halogens
• Oxidizing agents
• Heavy metal salts
• Surface active agents
• Dyes
Alcohols
• Most widely used disinfectants and antiseptics. e.g. ethanol and

isopropanol (70 to 80%)
• Mechanism of action: Bactericidal and fungicidal but not
sporicidal; some enveloped viruses (e.g. HIV) are also destroyed.
• Ethyl alcohol is used as surgical spirit (70%) in hand rubs as
antiseptics.
• Isopropyl alcohol- Clinical thermometers and small instruments
are disinfected by soakingEssentials
for 10 to Microbiology
of Medical 15 minutes.
Aldehydes
• Mechanism of action: combine with nucleic acids,
proteins and inactivate them, probably by cross linking
and alkylating the molecules.
• Also sporicidal (used as chemical sterilants).

Formaldehyde
• Dissolved in water or alcohol before use.
• Formaldehyde is toxic and irritant when inhaled, as
well as it is corrosive to the metals.
• Best used for- i)preservation of anatomical specimen,
ii) formaldehyde gas is used for fumigation of closed
areas such as operation theatres, iii)preparation of
toxoid from toxin
Glutaraldehyde
• Less toxic, less irritant and less corrosive, hence is best used
to sterilize hospital and laboratory equipments such as
endoscopes and cystoscopes.
• Used as 2% concentration (2% cidex).
• Disinfects objects within about 20 min but may require as
long as 12 hours to kill spores.
• Available in inactive form, has to be activated by alkalinisation
before use. Once activated, it remains active only for 14 days.
Ortho-Phthalaldehyde
• (0.55%) solution can also be used for sterilization of
endoscopes and cystoscopes.
• Advantages over glutaraldehyde
o Does not require activation
o Low vapour property
o Better odour
o More stable during storage
o ↑Mycobactericidal activity.
Phenolic compounds
• Phenol (carbolic acid) was the first widely used antiseptic and
disinfectant (Joseph Lister – 1867).
• Phenol and its derivatives (phenolics) are produced by distillation of
coal tar between temperatures of 170°C and 270°C.
• Mechanisms: Phenolics act by denaturing proteins and disrupting cell
membranes
• Advantages: They have tuberculocidal activity, some are effective in
the presence of organic materials.

Phenolics as disinfectants
• Cresol, xylenol, Lysol and ortho-phenylphenol are used as
disinfectants in laboratories and hospitals.
• Lysol is a commercial preparation made of a mixture of phenolics.
• All have the ability to retain activity in presence of organic matter.
• Toxic and irritant to skin, hence they are only used as disinfectants
but not as antiseptics.

Phenolics as antiseptics
• Certain phenolics are less irritant to skin, persist in skin for longer
period and are widely used as antiseptics.
• In general they are more active against Gram positive than Gram
negative bacteria.
• Chlorhexidine - Active ingredient of savlon (chlorhexidine and
cetrimide)
• Chloroxylenol- Active ingredient of dettol.
• Hexachlorophane– As it can cause brain damage, hence its use as
antiseptic is restricted only in response to a staphylococcal outbreak.
Halogens
• Among the halogens, iodine and chlorine have
antimicrobial activity.
• They exist in free state, and form salt like compounds
with sodium and most other metals.

Iodine
• Used as a skin antiseptic and kills microorganisms by oxidizing cell
constituents and iodinating cell proteins.
• At higher concentrations, it may even kill some spores.
• Tincture of iodine is a preparation of iodine (2%) in a water-
ethanol solution of potassium iodide.
• Iodophor is prepared by complexing iodine with an organic carrier.
– Used as pre-operative antiseptic as well as disinfectant in
laboratories (Wescodyne and Betadine).
Uses of chlorine
Property
Common Actions Used in places
responsible
Bleaching Laundry and home Oxidation
Disinfection Hospitals Coagulation
Odor Control Toilets Precipitation

Chlorination of drinking and
Water tanks Septication
process water
Swimming pool and boiler pH
Elimination of slime and algae
water Adjustment

Chlorine
• Preparations- It may be available as
o Chlorine gas
o Sodium hypochlorite (household bleach, 5.25%)
o Calcium hypochlorite (bleaching powder)
• Mechanism of action- All preparations yield hypochlorous
acid (HClO) which causes oxidation of cellular materials
and destruction of vegetative bacteria and fungi, but not
spores.

Disadvantages of chlorine
• Organic matter interferes with its action
• Carcinogenic
• Daily preparation
• Not active against Giardia and Cryptosporidium.
• Sodium hypochlorite is corrosive and should be handled
cautiously.

Oxidising agents
• Hydrogen peroxide (H2O2)

• Peracetic acid
• Plasma sterilization

Hydrogen peroxide (H2O2)
• Strong oxidizer, is used as high level disinfectant as well as chemical
sterilant.
• Mode of action- Break down of H2O2, liberates toxic free hydroxyl
radicals which are the active ingredients, attack membrane, lipid ,
DNA, and other cellular components.
• Concentration- H2O2 is effective against most organisms at
concentration of 3-6%, while catalase producing organisms and spores
require higher concentration (10%) of H2O2.

Hydrogen peroxide (H2O2)
• Use-H2O2 is used to disinfect ventilator, soft contact lenses, and
tonometer biprisms. Vaporized H2O2 is used for plasma sterilization.
• Advantage
o H2O2 does not coagulate blood or does not fix tissues to surfaces, and
in fact it may enhance removal of organic matter from equipments.
o Low toxicity to man
o Environmentally safe
o Neither carcinogenic nor mutagenic.

Peracetic acid
• Powerful oxidising agent, even more active than H2O2.
• Concentrations of <1% are sporicidal even at low temperature.
• Uses - It is a high level disinfectant and chemical sterilant;
often used in conjunction with H2O2.
o Disinfect hemodialyzers.
o Sterilizing endoscopes.
• May corrode steel, iron, copper, brass and bronze.

Plasma sterilization
• Plasma-It refers to a gaseous state consisting of ions and free
electrons and neutral uncharged particles (such as O and OH).
• Plasma sterilizers- Special device is used for creating plasma
state (commercial brands such as Sterrad and Plazlyte). Such
devices maintain a uniform vacuum inside the chamber.
• Chemical sterilants such as H2O2 alone or a mixture of H2O2 and
peracetic acid are used in these devices to induce the plasma
state.
• Active agent- Ultraviolet (UV) photons and radicals
• Low temperature is maintained (<500C) throughout the
process which preserves the integrity of heat labile items
• It is used for sterilization of surgical instruments.
• Sterilization control- Efficacy of plasma sterilization is
tested by using Bacillus stearothermophilus, Bacillus
subtilis subsp.niger.


Heavy metal salts
• Mechanism of action- Heavy metals combine with

bacterial cell proteins, often with their sulphydryl
groups, and inactivate them.
• They may also precipitate cell proteins.
• Many heavy metals are more bacteriostatic than
bactericidal.

Heavy metal salts
• Silver sulfadiazine is used on burns surfaces.

• Silver nitrate (1%) solution is often added to the eyes of infants to prevent
ophthalmia neonatorum. It is now replaced by erythromycin in many
hospitals.
• Copper sulphate is an effective fungicide (algicide) in lakes and swimming
pools.
• Mercury salts such as mercuric chloride, thiomersal and mercurochrome
were known antiseptics and antifungal agents, but are not used now days.
• Thiomersal (merthiolate) is used as preservative in vaccines, sera and other
immunoglobulin preparations.
Surface active agents
• Surfactants (or surface active agents) are the compounds that lower
the surface tension (or interfacial tension) between two liquids or
between a liquid and a solid.
• Classified into:
o Anionic
o Cationic
o Non-ionic
o Amphoteric compounds, of which the cationic detergents are
effective disinfectants.
Cationic surfactants
• Cationic surfactants-Quaternary ammonium compounds
are the most popular cationic disinfectants in use.
• Quaternary ammonium compounds are characterized by
a positively charged quaternary nitrogen and a long
hydrophobic aliphatic chain.
• Disrupt microbial membranes and may also denature
proteins.

Cationic surfactants
• Destroys most bacteria (Gram positives are better killed
than Gram negatives) but not M. tuberculosis or spores.
• Stable, nontoxic but are inactivated by acidic pH,
organic matter, hard water and soap.
• Cationic detergents are often used as disinfectants for
food utensils and small instruments and as skin
antiseptics.
Surface active agents
• Examples of quaternary ammonium compounds

include-
o Alkyltrimethylammonium salts
o Acetyl trimethyl ammonium bromide (cetavlon or cetrimide)
o Benzalkonium chloride
o Cetylpyridinium chloride

Anionic surfactants
• E.g. common soaps, have strong detergent but weak

antimicrobial properties.
• These agents are most active at acidic pH.

Amphoteric surfactants
• Possess detergent properties of anionic and antimicrobial activity
of cationic compounds.
• Active over a wide range of pH but the activity is reduced in
presence of organic matter.
• Examples include ‘Tego compounds’ which are the commercial
name of a series of disinfectants whose active ingredient is DDAG
(dodecyl-di-aminoethyl-glycine).
o Used as antiseptics in dental practice, but are known to cause
allergic reactions.
Dyes
• Aniline and acridine are the two groups of dyes.
• Used extensively as skin and wound antiseptics.

Aniline dyes
• Include crystal violet, gentian violet, brilliant green and malachite
green.
• More active against Gram positive bacteria than Gram negative
bacteria and have no activity against M. tuberculosis.
• Non-toxic and non-irritant to the tissues.
• Activity is reduced in presence of organic material such as pus.
• Mechanism of action - Interfere with the synthesis of peptidoglycan
component of the cell wall.
• Used in the laboratory as selective agents in culture media
Acridine dyes
• Include acriflavine, euflavine, proflavine and aminacrine.
• Affected very little by the presence of organic material.
• More active against Gram positive bacteria than Gram
negative bacteria but are not as selective as the aniline
dyes.
• Mechanism of action - Interfere with the synthesis of
nucleic acids and proteins in bacterial cells.

Gaseous sterilization
• Ethylene oxide (EtO)

• Low temperature steam formaldehyde
• Betapropiolactone (BPL)

Ethylene oxide (EtO)-
• Most widely used gaseous chemical sterilants in present days.
• Mechanism of action-EtO has both microbicidal and sporicidal
activity; acts by combining with cell proteins.
• High penetration power- The main advantage of EtO is, it rapidly
penetrates packing materials, even plastic wraps.
• Highly inflammable, irritant, explosive and carcinogenic. Hence, it
is unsuitable for fumigation.

Ethylene oxide (EtO)
• Ethylene oxide sterilizer is a special
equipment where the sterilization is
carried out.
• As pure EtO is explosive, hence it is
usually supplied in a 10 to 20%
concentration mixed with inert gases like
either CO2 or dichloro-difluoro-methane.

Ethylene oxide (EtO)
• Sterilization condition- Three factors that influence the rate of
sterilization are- EtO concentration, humidity, and temperature.
• At EtO concentration of 700 mg/litre and 40 to 50% relative
humidity, sterilization is achieved in 5 to 8 hours at 38°C or 3 to 4
hours at 54°C.
• Extensive aeration of the sterilized materials is necessary to remove
residual EtO because it is so toxic.
• Sterilization control- Bacillus globigi is used as biological indicator
to check the effectiveness of sterilization.
Ethylene oxide (EtO) - Uses
• Sterilization of many heat sensitive items such as:
o Disposable plastic petri dishes
o Syringes
o Heart-lung machine components
o Sutures
o Catheters
o Respirators
o Dental equipments.
Low temperature steam formaldehyde
• Widely used for fumigation of operation theatres, wards and
laboratories.
• No longer preferred, and is being replaced by modern
methods of fumigation.
• Room should be sealed for 48 hours.
• Gas is irritant and toxic when inhaled. Hence, after completion
of disinfection, the effect of irritant vapours should be nullified
by exposure to ammonia vapour.
Betapropiolactone (BPL)
• BPL gas (0.2%) is active against all microorganisms
including spores.
• It has a low penetrating power and is carcinogenic,
hence not used for fumigation.
• Used for inactivation of vaccines.

TESTING OF DISINFECTANTS
• Phenol coefficient (Rideal Walker)test
o Determined by the dilution of the disinfectant in question
which sterilizes the suspension of Salmonella Typhi in a
given time divided by the dilution of phenol which
sterilizes the suspension in the same time.
o Phenol coefficient is >1, the test disinfectant is said to be
more effective than phenol.

• Phenolic coefficient of a test disinfectant=
Highest dilution of the test disinfectant that kills S.Typhi in a given time
--------------------------------------------------
Highest dilution of phenol that kills S.Typhi in the same time

• The drawbacks of Rideal Walker test are:
o Only the phenolic compounds can be assessed.
o Does not assess the ability of the disinfectant to act in
presence of organic matter.

• Chick Martin test-It is a modification of Rideal and walker test
in which the disinfectants act in the presence of organic matter
(e.g. dried yeast, feces, etc.) to simulate the natural conditions.
• Capacity (Kelsey-Sykes) test-It tests the capacity of a
disinfectant to retain its activity when repeatedly used
microbiologically.

• In-use (Kelsey and Maurer) test
o In- use test is used to determine whether an actively
used solution of disinfectant in a clinical setting is
microbiologically contaminated.
o It should be routinely performed in the hospital once in
every 3 months.

SPORICIDAL AGENTS
1. Ethylene oxide 5. O-Phthalic acid

2. Formaldehyde 6. Peracetic acid
3. Glutaraldehyde 7. Autoclave
4. Hydrogen peroxide 8. Hot air oven
9. Plasma sterilization

Indicators monitoring the sterilization process
• Physical or mechanical indicator
o Digital displays of the sterilizer equipment which
verify the parameters of sterilization cycle are met
or not.
o Parameters - Temperature, time and pressure, etc.

Chemical indicator
• Monitor the attainment of one or more parameters
required for a satisfactory sterilization process.
• Use heat or chemical sensitive materials which undergo a
color change if the sterilization parameter (e.g. time,
steam quality and temperature)
• There are of 6 type I to VI

Chemical indicator
Indicator Function
Type I (process indicator or • Used on external surface of each pack, to indicate that the
exposure indicator or pack has been directly exposed to the sterilant.
external pack control) • Does not assure sterility
Type II (Bowie-Dick test) • Used only for pre-vacuum autoclave (steam sterilizer).
• Must be used daily in empty cycle before the first load.
• Used for equipment control, i.e. it checks the efficacy of
air removal, air leaks and steam penetration and ensures
that the autoclave is functioning well

Chemical indicator
Indicator Function
Type III • Single parameter indicator, obsolete now
Type IV (Internal • Used inside each pack.

pack control • Designed to measure any two of the critical variables: time, steam
indicator). quality and temperature.

Chemical indicator
Indicator Function
Type V (Internal • Used inside each pack.
pack control • Measures all three variables: time, steam quality and temperature.
indicator) • Should be used for packs containing critical items.
• Considered as equivalent to biological indicators
Type VI • Cycle specific; the stated values correspond to the critical variables
(Emulating that the sterilizer manufacturer has defined for that cycle of
indicator) sterilization process

Biological indicator (BI)
• BI is the best indicator, because here the bacterial spores are

used to check the effectiveness of sterilization.
• Spores are highly resistant and will be destroyed only when the
effective condition is achieved.
o Geobacillus stearothermophilus for autoclave and hydrogen
peroxide gas plasma and liquid acetic acid sterilizer
o Bacillus atrophaeus for ethylene oxide sterilizer and dry heat

Biological indicator (BI)
• Indication: BI must be used at least weekly (once a day if a

sterilizer is used frequently, e.g. several loads per day) and
must be used for any load containing implants.
• Types: Various types of BI are available which give result in
a specific time frame; next generation BI (48 hours), rapid
read-out BI (24 minutes to 4 hours) and spore-strips (7
days, obsolete now).

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 , Formaldehyde gas > UV > BPL
Preservation of anatomical specimen, woolen blanket
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

Common disinfectants and their spectrum of action
Germicide & their Level of Bacteria & Fungi Un-enveloped M. Spore Inactivated by
concentrations disinfectant enveloped viruses tuberculosis organic
viruses matter
Glutaraldehyde (2%) High/CS + + + + + -
Formaldehyde (3-8%) High/CS + + + + + -
H2O2 (3-25%) High/CS + + + + + +/-

Chlorine (100-1000 ppm of High + + + + +/- +
free chlorine)
Isopropyl alcohol (60-95%) Intermediate + + +/- + - +/-
Phenol (0.4-5%) Intermediate + + +/- + - -
Iodophore (30-50ppm of Intermediate + + + +/- - +
free iodine)
Quaternary ammonium Low + +/- - - - +
compounds (0.4-1.6%)
Chlorhexidine gluconate Low + +/- +/- +/- +/- +
(2-4%

Common chemical disinfectants used in the hospital and their applications

Common chemical disinfectants used in the hospital and their applications

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)

Spaulding’s classification of medical devices
Medical device Definition Examples Recommended
Sterilization
/disinfection
Critical device Enter a normally sterile Surgical Instruments, cardiac and Heat based sterilization,
site urinary catheters, implants, eye & Chemical sterilant or
dental instruments high level disinfectant
Semi-critical Come in contact with Respiratory therapy equipments, High level disinfectant
device mucous membranes or anesthesia equipments,
minor skin breaches endoscopes, laryngoscope,
rectal/vaginal /esophageal probes
Non-critical Comes in contact with BP cuff, ECG electrodes, bedpans, Intermediate level
devices intact skin crutches, stethoscope, thermometer or low level disinfectant
Non-critical Less direct contact with Surfaces of medical equipments, Low level disinfectant
environmental patient examination table, computers
surfaces

CENTRAL STERILE SUPPLY DEPARTMENT
• Central Sterile Services Department (CSSD) is an integrated place in

hospitals that performs sterilization of medical devices, equipment
and consumables; that are used in the operating theater (OT) of
the hospital and also for other aseptic procedures.
• The processing area of CSSD consists of four unidirectional zones
starting from an unsterile area to a sterile area separated by
physical barrier.
• Decontamination area → Packaging area → Sterilization area →
Sterile storage area
CENTRAL STERILE SUPPLY DEPARTMENT
1. Decontamination area
2. Packaging area
3. Sterilization area
4. Sterile storage area

Chapter 3 Sterilization and Disinfection

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Chapter 3 Sterilization and Disinfection

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STERILIZATION AND

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

1. Organism load: Larger microbial population requires a

Essentials of Medical Microbiology

3. Concentration of the chemical agent or the temperature

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Dry heat Kills the organisms by charring, denaturation of bacterial protein,

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Most widely used method of sterilization by

Essentials of Medical Microbiology

• Overloading of hot air oven should be avoided.

• Biological indicator: Spores (106)

Pasteurization • Method used for control of microorganisms from beverages like

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Inspissation • Process of heating an article on 3 successive days at 80–85ฐC for

Essentials of Medical Microbiology

Moist heat at a temperature at 100°C

Essentials of Medical Microbiology

Tyndallisation • Involves steaming at 1000C for 20minutes for 3 consecutive days.

Essentials of Medical Microbiology

• Autoclave comprises of three parts

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Autoclave can be set to provide higher temperatures by

Essentials of Medical Microbiology

• Autoclave should not be used for sterilizing waterproof

• Gravity displacement type autoclave: most

Essentials of Medical Microbiology

• Biological indicator: Spores of Geobacillus stearothermophilus

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• The sterilization control of membrane filters includes

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Includes infrared and ultraviolet radiations.

• High-frequency ultrasonic and sonic sound waves

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Most widely used disinfectants and antiseptics. e.g. ethanol and

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Disinfection Hospitals Coagulation

Odor Control Toilets Precipitation

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Hydrogen peroxide (H2O2)

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

Essentials of Medical Microbiology

• Mechanism of action- Heavy metals combine with