DISINFECTI
ON
DR EKADASHI RAJNI
Definition
• Sterilization: Process by which all living microorganisms including
viable spores, are either destroyed or removed from an article,
body surface or medium.
• Disinfection- Process that destroys or removes most if not all
pathogenic organisms but not bacterial spores.
Definition
• Asepsis- Process where 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 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.
Factors influencing efficacy of sterilant/ disinfectant
3. Concentration of the chemical agent.
4. Nature of the disinfectant
5. Duration of exposure
6. Temperature
7. Local environment
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 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
CHEMICAL AGENTS OF STERILIZATION/
DISINFECTION
Classification
Level of Bacterial Tubercle Nonenveloped Fungi Envelope Vegetative
disinfectant spores bacilli 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 (eg. 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 soaking for 10 to 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 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.
• 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 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).
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
1. Hydrogen peroxide (H2O2)
2. Peracetic acid
3. 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 as
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 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.
Plasma sterilization
• 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.
Plasma sterilization
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 most popular
cationic disinfectants in use.
• Quaternary ammonium compounds are characterized by a positively
charged side chain.
• Disrupt microbial membranes and may also denature proteins.
• Examples of quaternary ammonium compounds include-
o Acetyl trimethyl ammonium bromide (cetavlon or cetrimide)
o Benzalkonium chloride
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.
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 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 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 tissues.
• Activity is reduced in presence of organic material such as pus.
• Mechanism of action - Interfere with 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
oDetermined 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.
TESTING OF DISINFECTANTS
• 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.
TESTING OF DISINFECTANTS
• 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.
TESTING OF DISINFECTANTS
• 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
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 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)
