STERILISATION AND DISINFECTION

DEFINITIONS –

 Sterilization: It is the process by which all living microorganisms including

viable spores, are destroyed with reduction of at least 106 log colony forming
units (CFU) of microorganisms and their spores.
 Disinfection: It refers to a process that destroys or removes most if not all
pathogenic organisms but not bacterial spores with reduction of at least 103
log CFU of microorganism but not spores.
 Asepsis: It is a 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): It refers to the reduction of pathogenic
microbes to a level at which items are considered as safe to handle without
protective attire with reduction of at least 1 log CFU of microorganism but not
spores.

FACTORS INFLUENCING EFFICACY OF STERILANT –

efficiency of a sterilant/disinfectant antimicrobial agent is affected by at following

factors:

 Organism load: Larger microbial population requires a longer time to die

than a smaller one.

 Nature of organisms: It greatly influences the efficacy of the disinfectants

 Concentration of the chemical agent and temperature of the physical agent

 Nature of the sterilant/disinfectant: • Microbicidal ability, Rapidity of action,

Residual activity. • Ability to act in presence of organic matter such as pus,
blood, and stool.

 Duration of exposure: More is the exposure time, better is the efficacy.

 pH: Heat kills more readily at an acidic pH

 Biofilm formation: Prevents the entry of disinfectants.

PHYSICAL METHODS –
HEAT STERILISATION –

Mechanism of Action
• Dry heat kills the organisms by Charring, Oxidative damage, Denaturation of
 bacterial protein and Elevated levels of electrolytes (CODE).

• Moist heat kills the microorganisms by denaturation and coagulation of proteins.

Dry Heat (Hot air oven)

• Holding temperature required: 160°C for 2 hours

• Materials sterilized: Hot air oven is best method for sterilization of: ○ Glassware
like glass syringes, petri dishes, flasks, pipettes and test tubes.○ Surgical
instruments like scalpels, scissors, forceps, etc.○ Chemicals such as liquid
paraffin, fats, glycerol, and glove dust powder, etc.

• Sterilization control:

Spores (106) of nontoxigenic strains of Clostridium tetani or Bacillus subtilis

subsp. niger

Thermocouples and Browne’s tube.

Moist Heat at a Temperature below 100oC

• Pasteurization: Used for perishable beverages like fruit and vegetable juices, beer,
and dairy products such as milk.

• Two methods: Holder method (63oC for 30 min) and Flash method

• All nonsporing pathogens are killed except Coxiella burnetii which may survive in
holder method.

• Water bath: Used for disinfection of serum, body fluids, and vaccines

• Inspissation (Fractional sterilization):

• It is a process of heating an article on 3 successive days at 80–85oC for 30 min

• Used for sterilization of egg based (LJ and Dorset’s egg medium) and serum based
media (Loeffler’s serum slope).

Moist Heat at a Temperature of 100oC

• Boiling: Boiling of the items in water for 15 minutes may kill most of the
vegetative forms but not the spores.

• Steaming: Koch’s or Arnold’s steam sterilizers are used to provide temperature of

100°C for 90 minutes. It is useful for those media which are decomposed at
high temperature of autoclave. It kills most of the vegetative forms but not the
spores.

• Tyndallization or intermittent sterilization: Involves steaming at 100°C for 20

 min for 3 consecutive days. It is used for sterilization of gelatin and egg,
serum or sugar containing media. It kills most of the vegetative forms
including spores.

Moist Heat at a Temperature above 100oC (Autoclave)

• Principle: Autoclave functions similar to a pressure cooker. At normal pressure,
water boils at 100°C but when pressure inside a closed vessel increases, the
temperature at which water boils also increases.

• Sterilization conditions: 121°C for 15 min at pressure of 15 psi (most commonly

used). • Uses of autoclave: Autoclave is useful for surgical instruments and culture
media and those materials which cannot withstand the higher temperature of hot air
oven or media

containing water that cannot be sterilized by dry heat. • Sterilization control:

○ Biological indicator-Spores of Geobacillus stearothermophilus (best indicator) ○

Thermocouple and indicators like Browne’s tube, Autoclave tapes:

Filtration
 excellent way to remove the microbial population in solutions of heat-labile
materials like vaccine, antibiotics, toxin, serum and sugar solution as well as
for purification of air in laminar air flow systems.

Depth filters: They are porous filters that retain particles throughout the depth
of the filter, rather than just on the surface. They are used for industrial
applications such as filtration of food and beverage, and chemicals, but are not to
filter bacteria.

 Candle filters made up of diatomaceous earth (Berkefeld filters), unglazed

porcelain (Chamberland filters)

 Asbestos filters (Seitz and Sterimat filters)

 Sintered glass filters

Membrane filters:

 widely used filters for bacterial filtration

 They are porous; retain all the particles on the surface that are smaller than
their pore size.

 Made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene

fluoride

 Pore size: Membrane filters have an average pore diameter of 0.22 μm

  Sterilization control includes Brevundimonas diminuta and Serratia marcescens.

Radiation
Ionising radiations:

 Examples include, X-rays, gamma rays (from Cobalt 60 source), and cosmic
rays.

 Mechanism: It causes breakage of DNA without temperature rise (hence called

as cold sterilization).

 It destroys spores and vegetative cells, but not effective against viruses.

 used for:

 Disposable plastics,

 Catgut sutures, bone and tissue grafts and adhesive dressings, antibiotics and
hormones.

 Advantages of Ionizing radiation:

 High penetrating power, ○ Rapidity of action and○ Temperature is not raised

 Sterilization control: Efficacy of ionising radiation is tested by using Bacillus

pumilus.

Nonionizing radiation:

 Examples of nonionizing radiation include infrared and ultraviolet radiations.

 Examples of nonionizing radiation include infrared and ultraviolet radiations.

 Used for sterilization of clean surfaces in operation theatres, laminar flow

hoods as well as for water treatment.

CHEMICAL METHODS –
ALCHOLS ---

 Used for sterilization of clean surfaces in operation theatres, laminar flow

hoods as well as for water treatment.
  act by denaturing proteins and possibly by dissolving membrane lipids

 Ethyl alcohol is used as surgical spirit (70%) in hand rubs as antiseptics.

 Isopropyl alcohol: Used for clinical thermometers.

ALDEHYDES –

 combine with nucleic acids and proteins and inactivate them,

 probably by crosslinking and alkylating the molecules.

 They are sporicidal and can be used as chemical sterilants.

Formaldehyde: It is best used for:

 Preservation of anatomical specimen

 Formaldehyde gas is used for fumigation of closed areas such as operation

theaters

 Preparation of toxoid from toxin. It is toxic, irritant and corrosive to metals.

Glutaraldehyde

 less irritant and less corrosive

 It is used as 2% concentration (2% cidex) for 20 min.

• It has to be activated by alkalinization before use. Once activated, it remains active

only for 14 days.

Ortho-Phthalaldehyde

 can also be used for sterilization of endoscopes and cystoscopes

 It does not require activation

 Low vapor property

 Better odor

PHENOLIC COMPOUNDS –

Phenolics as disinfectants:

• Cresol, xylenol, Lysol and ortho-phenylphenol are used as disinfectants in

laboratories and hospitals.

• All have the ability to retain activity in presence of organic matter.

• They are toxic and irritant to skin, hence 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.

• they are more active against gram- positive than gram-negative bacteria.

• Chlorhexidine: It is an active ingredient of savlon

• Chloroxylenol: It is an active ingredient of dettol.

• Hexachlorophane: As it can cause brain damage, hence its use as antiseptic is

restricted only to a staphylococcal outbreak.

HALOGENS –

Iodine: It is used as a skin antiseptic and kills microorganisms by oxidizing cell

constituents and iodinating cell proteins, e.g. Tincture of iodine (2%) and Iodophor
(iodine complexed with an organic carrier) e.g. Betadine.

Chlorine: It is the most commonly used disinfectant:

• For municipal water supplies and swimming pools and is also employed in the
dairy and food industries

• As laboratory disinfectant

• As bleaching agent: to remove the stain from clothes. (Common uses of chlorine
are given in table below).

Disadvantages:

•  Organic matter interferes with its action, hence excess chlorine always is add- ed
to water to ensure microbial destruction

• Carcinogenic

• Need daily preparation

•  They are not active against Giardia and Cryptosporidium,

• Sodium hypochlorite is corrosive and should be handled cautiously.

OXIDISING AGENTS—
Hydrogen Peroxide (H2O2)
  Mode of action: It is a chemical sterilant, acts by liberating toxic free hydroxyl
radicals which attack membrane, lipid, DNA, and other cellular components.

 Concentration of H2O2 3–6% is ideal, except for catalase producing organisms

and spores which require 10% of H2O2.

 Used to disinfect ventilator, soft contact lenses, and tonometer biprisms.

Vaporized H2O2 is used for plasma sterilization.

 Advantage: 1. It acts perfect even in presence of organic matter 2. Low toxicity

3. Environmentally safe.

Peracetic Acid
 chemical sterilant; often used in conjunction with H2O2,

 to disinfect hemodialyzers and in plasma sterilization.

 used for sterilizing endoscopes

 it may corrode steel, iron, copper, brass and bronze.

Plasma Sterilization
 cently introduced sterilization device

 used for creating plasma state, so as to maintain a uniform vacuum inside the
chamber.

 Chemical sterilants such as H2O2 alone or a mixture of H2O2 and peracetic acid

 Active agent is Ultraviolet (UV) photons and radicals (e.g., O and OH): Kill
microorganisms and spores.

 Low temperature is maintained (< 50°C), So best for heat labile surgical
instruments.

 Sterilization control: Geobacillus stearothermophilus, Bacillus subtilis subsp.

niger.

HEAVY METAL SALTS –

• Silver sulfadiazine is used on burns surfaces.

• Silver nitrate (1%) solution used for eyes of infants to prevent ophthalmia
neonatorum.
• Copper sulfate is an effective fungicide (algicide) in lakes and swimming pools.

• Mercury salts such as mercuric chloride, thiomersal and mercurochrome were

known antiseptics in past. Thiomersal (merthiolate) is used as preservative in
vaccines and sera.

• 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.

SURFACE ACTIVE REAGENT –

 lower the surface tension between two liquids or between a liquid and a solid.

 Surfactants may act as detergents, wetting agents, and emulsifiers because

they have both polar hydrophilic and nonpolar hydrophobic ends.

Cationic surfactants (Quaternary ammonium compounds):

 They disrupt microbial membranes and may also denature proteins.

 They kill most bacteria (gram-positives are better killed than gram-negatives)
but not M. tuberculosis or spores.

 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

 soaps, have strong detergent but weak antimicrobial properties. They are
active at acidic pH.

The amphoteric surfactants:

 They are active over a wide range of pH, but is reduced in presence of organic
matter.

 E.g. ‘Tego compounds’: Used as antiseptics in dental practice, but cause allergic
reactions.

DYES----

 Aniline and acridine dyes have been used extensively as skin and wound
antiseptics.
Aniline dyes:

 They are more active against gram-positive bacteria than gram-negative and
have no activity against M. tuberculosis.

 They are non-toxic and non-irritant to the tissues.

 Their activity is reduced in presence of organic material such as pus.

 They interfere with the synthesis of peptidoglycan component of the cell wall.

 These dyes are used in the laboratory as selective agents in culture media (e.g.
malachite green in LJ medium)

Acridine dyes:

 include acriflavine, euflavine, proflavine and aminacrine:

 They are affected very little by the presence of organic material.

 More active against gram-positive bacteria but are not as selective as the
aniline dyes.

 They interfere with the synthesis of nucleic acids and proteins in bacterial
cells.

GASEOUS STERLISATION –

Ethylene Oxide (ETO)

Ethylene oxide sterilizer is one of the widely used gaseous chemical sterilants in
present days.

 It has high penetration power, has both microbicidal and sporicidal activity; acts
by combining with cell proteins.

 However, it is highly inflammable, irritant, explosive and carcinogenic. Hence

it is usually supplied in a 10 to 20% concentration mixed with inert gases.

 Sterilization condition: 5 to 8 hours at 38°C or 3 to 4 hours at 54°C.

 Sterilization control: Bacillus globigii.

TESTING OF DISINFECTANTS –
• Phenol coefficient (Rideal Walker) test:

• 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.

• Phenol coefficient of > 1 is taken as satisfactory.

• Drawbacks: ○ Only the phenolic compounds can be assessed ○ It does not assess
disinfectant ability to act in presence of organic matter.

• Chick Martin test: Modified 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.