Sterilization & disinfection

Pharmaceutics 1

DR. wahome

Sterilization & Disinfection Dr. wahome 1

 Sterilization
Specific objectives:
1. To define sterilization and sterility
2. To discuss the methods of sterilization and indicators of sterility
3. To discuss the equipment used in sterilization
4. To discuss the aseptic techniques
5. To give examples of substances that can be dispensed sterile
Sterilization is the process where all the living microorganisms, including bacterial spores are
killed. Sterilization can be achieved by physical, chemical and physiochemical means. A product
is said to be sterile when it is free from all living microorganisms and passes the sterility tests.
Chemicals used as sterilizing agents are chemisterilants.

Disinfection is the process of elimination of most pathogenic microorganisms (excluding

bacterial spores) on inanimate objects. Disinfection can be achieved by physical or chemical
methods. Chemicals used in disinfection are called disinfectants. Different disinfectants have
different target ranges as not all disinfectants can kill all microorganisms. Some methods of
disinfection such as filtration, do not kill bacteria, they separate them out.
Sterilization is an absolute condition while disinfection is not. The two are not synonymous.
Asepsis is the employment of techniques (such as usage of gloves, air filters, UV rays etc) to
achieve microbe-free environment.
Antisepsis is the use of chemicals (antiseptics) to make skin or mucus membranes devoid of
pathogenic microorganisms.
Bacteriostasis is a condition where the multiplication of the bacteria is inhibited without killing
them.
Bactericidal is that chemical that can kill or inactivate bacteria. Such chemicals may be classified
depending on the spectrum of activity, as bactericidal, virucidal, fungicidal, microbicidal,
sporicidal, tuberculocidal or germicidal.

Other Terms Used in Relation to Sterilization

Antiseptics: These are the substances used to inhibit the growth or destroy germs. They are
used on the skin.

Bactericides: These are the substances used to kill bacteria.

Bacteriostatic Agents: These are the agents which inhibit the growth of bacteria. Different
bacteriostatic agents are used against different bacteria.

Disinfectants: These are the agents used to kill bacteria and other infectious agents from the
non-living articles. They are too strong to be applied to the human body.
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Fungicides: These agents kill fungi.

Germicides: These substances kill germs but not necessarily bacterial spores.

Mycocides: These agents kill moulds.

Viricides: These agents kill viruses.

The products or materials which require sterilization include all parenteral preparations like
implants, ophthalmic preparations, surgical instruments, ligatures and sutures, surgical
dressings, gloves etc. The working area where aseptic techniques are to be performed should
also be sterilized.

Physical methods of sterilization:

Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays
in it. It is responsible for spontaneous sterilization in natural conditions. In tropical countries,
the sunlight is more effective in killing germs due to combination of ultraviolet rays and heat. By
killing bacteria suspended in water, sunlight provides natural method of disinfection of water
bodies such as tanks and lakes. Sunlight is not sporicidal, hence it does not sterilize.
Heat: Heat is considered to be most reliable method of sterilization of articles that can
withstand heat. Heat acts by oxidative effects as well as denaturation and coagulation of
proteins. Those articles that cannot withstand high temperatures can still be sterilized at lower
temperature by prolonging the duration of exposure.
Factors affecting sterilization by heat are:
- Nature of heat: Moist heat is more effective than dry heat

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 - Temperature and time: temperature and time are inversely proportional. As
temperature increases the time taken decreases.
- Number of microorganisms: the more the number of microorganisms, the higher the
temperature or the longer the duration required.
- Nature of microorganism: Depends on species and strain of microorganism. The
sensitivity to heat may vary. Spores are highly resistant to heat.
- Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure. Certain heat sensitive articles must be sterilized at lower
temperature.
- Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.
Action of heat:
Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated levels of
electrolytes. The moist heat acts by coagulation, hydrolysis of macromolecules like nucleic acids
and proteins. Moist heat is superior to dry heat in action. Temperature required to kill microbes
by dry heat is more than the moist heat.
Thermal death time is the minimum time required to kill a suspension of organisms at a
predetermined temperature in a specified environment.
Dry heat:
Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame.
Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure. This method too is limited to
those articles that can be exposed to flame. Cracking of the glassware may occur.
Incineration: This is a method of destroying contaminated material by burning them in an
incinerator. Articles such as soiled dressings; animal carcasses, pathological material and
bedding etc should be subjected to incineration. This technique results in the loss of the article,
hence is suitable only for those articles that have to be disposed. Burning of polystyrene
materials emits dense smoke, and hence they should not be incinerated.
Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed
to high temperature (160o C) for 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 a
fan. The heat is transferred to the article by radiation, conduction and convection. The oven

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should be fitted with a thermostat control, temperature indicator, meshed shelves and must
have adequate insulation.
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 and both ends of pipettes must be plugged with
cotton wool. Articles such as petri dishes and pipettes may be arranged inside metal canisters
and then placed. Individual glass articles must be wrapped in kraft paper or aluminum foils.
Sterilization cycle: This takes into consideration the time taken for the articles to reach the
sterilizing temperature, maintenance of the sterilizing temperature for a defined period
(holding time) and the time taken for the articles to cool down. Different temperature-time
relations for holding time are 60 minutes at 160oC, 40 minutes at 170oC and 20 minutes at
180oC. Increasing temperature by 10 degrees shortens the sterilizing time by 50 percent. The
hot air oven must not be opened until the temperature inside has fallen below 60oC to prevent
breakage of glassware.
Sterilization control: Three methods exist to check the efficacy of sterilization process, namely
physical, chemical and biological
Physical: utilizes a temperature chart recorder and a thermocouple.
Chemical: Browne’s tube No.3 (green spot, color changes from red to green)
Biological: 106 spores of Bacillus subtilis var niger or Clostridium tetani on paper strips are
placed inside envelopes and then placed inside the hot air oven. Upon completion of
sterilization cycle, the strips are removed and inoculated into thioglycolate broth or cooked
meat medium and incubated at 37oC for 3-5 days. Proper sterilization should kill the spores and
there should not be any growth.
Advantages: It is an effective method of sterilization of heat stable articles. The articles remain
dry after sterilization. This is the only method of sterilizing oils and powders.
Disadvantages:
- Since air is a poor conductor of heat, hot air has poor penetration.
- Cotton wool and paper may be slightly charred.
- Glasses may become smoky.
- Takes longer time compared to autoclave.

Infra red rays: Infrared rays bring about sterilization by generation of heat. Articles to be
sterilized are placed in a moving conveyor belt and passed through a tunnel that is heated by
infrared radiators to a temperature of 180oC. The articles are exposed to that temperature for a
period of 7.5 minutes. Articles sterilized include metallic instruments and glassware. It is mainly
used in central sterile supply department. It requires special equipments, hence is not
applicable in diagnostic laboratory. Efficiency can be checked using Browne’s tube No.4 (blue
spot).
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Moist heat:
Moist heat acts by coagulation and denaturation of proteins.
At temperature below 100oC:
Pasteurization: This process was originally employed by Louis Pasteur. Currently this procedure
is employed in food and dairy industry.
There are two methods of pasteurization: the holder method (heated at 63oC for 30 minutes)
and flash method (heated at 72oC for 15 seconds) followed by quickly cooling to 13oC.
Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15 sec and
149oC for 0.5 sec. This method is suitable to destroy most milk borne pathogens like Salmonella,
Mycobacteria, Streptococci, Staphylococci and Brucella. However, Coxiella may survive
pasteurization. Efficacy is tested by phosphatase test and methylene blue test.
- Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by
heating in a water bath at 60oC for one hour. Only vegetative bacteria are killed while
spores survive.
- Serum bath: The contaminating bacteria in a serum preparation can be inactivated by
heating in a water bath at 56oC for one hour on several successive days. Proteins in the
serum will coagulate at higher temperature. Only vegetative bacteria are killed while
spores survive.
- Inspissation: This is a technique to solidify as well as disinfect egg and serum containing
media. The medium containing serum or egg are placed in the slopes of an inspissator
and heated at 80-85oC for 30 minutes on three successive days. On the first day, the
vegetative bacteria would die and those spores that germinate by next day are then
killed the following day. The process depends on germination of spores in between
inspissation. If the spores fail to germinate then this technique cannot be considered
sterilization.
At temperature 100oC:
Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses immediately. Certain
bacterial toxins such as Staphylococcal enterotoxin are heat resistant. Some bacterial spores
are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing
activity can be enhanced by addition of 2% sodium bicarbonate.
When absolute sterility is not required, certain metal articles and glasswares can be disinfected
by placing them in boiling water for 10-20 minutes. The lid of the boiler must not be opened
during the period.
Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free
steam at 100oC.
Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with discharge tap open)
can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold the
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articles and a conical lid. The bottom of steamer is filled with water and heated. The steam that
is generated sterilizes the articles when exposed for a period of 90 minutes. Media such as
TCBS, DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium may get
decomposed on autoclaving hence they are exposed to free steaming for 20 minutes for three
successive days. This process is known as tyndallisation (after John Tyndall) or fractional
sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure
and the spores that germinate by next day are killed in subsequent days. The success of process
depends on the germination of spores
At temperature above 100oC:
Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an
autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the
temperature at which the water boils also increases. In an autoclave the water is boiled in a
closed chamber. As the pressure rises, the boiling point of water also raises to 121oC. Exposure
of articles to this temperature for 15 minutes sterilizes them. To destroy the infective agents
associated with spongiform encephalopathies (prions), higher temperatures or longer times are
used; 135oC or 121oC for at least one hour are recommended.
Advantages of steam:
- It has more penetrative power than dry air, it moistens the spores (moisture is essential
for coagulation of proteins).
- Condensation of steam on cooler surface releases latent heat and draws in fresh steam.
Different types of autoclave include simple “pressure-cooker type” laboratory autoclave, Steam
jacketed downward displacement laboratory autoclave and high pressure pre-vacuum
autoclave
Construction and Operation of Autoclave: A simple autoclave has vertical or horizontal
cylindrical body with a heating element, a perforated tray to keep the articles, a lid that can be
fastened by screw clamps, a pressure gauge, a safety valve and a discharge tap.
The articles to be sterilized must not be tightly packed. The screw caps and cotton plugs must
be loosely fitted. The lid is closed but the discharge tap is kept open and the water is heated. As
the water starts boiling, the steam drives air out of the discharge tap. When all the air is
displaced and steam start appearing through the discharge tap, the tap is closed. The pressure
inside is allowed to rise up to 15 lbs per square inch. At this pressure the articles are held for 15
minutes, after which the heating is stopped and the autoclave is allowed to cool. Once the
pressure gauge shows the pressure equal to atmospheric pressure, the discharge tap is opened
to let the air in. The lid is then opened and articles removed.
Articles sterilized include culture media, dressings, certain equipment, linen etc.
Precautions:
- Articles should not be tightly packed.
- The autoclave must not be overloaded.
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 - Air discharge must be complete and there should not be any residual air trapped inside.
- Caps of bottles and flasks should not be tight.
- Autoclave must not be opened until the pressure has fallen or else the contents will boil
over.
- Articles must be wrapped in paper to prevent drenching.
- Bottles must not be overfilled.
Advantage: Very effective way of sterilization, quicker than hot air oven.
Disadvantages:
- Drenching and wetting of articles may occur.
- Trapped air may reduce the efficacy.
- Takes long time to cool

Sterilization control
Physical methods includes automatic process control, thermocouple and temperature chart
recorder.
Chemical methods includes Browne’s tube No.1 (black spot) and succinic acid (whose melting
point is 121oC and Bowie Dick tape. Bowie Dick tape is applied to articles being autoclaved. If
the process has been satisfactory, dark brown stripes will appear across the tape.
Biological method includes a paper strip containing 106 spores of Geobacillus
stearothermophilus.

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Radiation:
Two types of radiation are used, ionizing and non-ionizing.
Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays are high-
energy rays with good penetrative power. Since radiation does not generate heat, it is termed
"cold sterilization".
Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most
effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this
wavelength that the absorption by the microorganisms is at its maximum, which results in the
germicidal effect.
UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA
replication. UV readily induces mutations in cells irradiated with a non-lethal dose.
Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV
radiation are inactivated within seconds. Since UV rays don’t kill spores, they are considered to
be of use in surface disinfection.
UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories,
corridors, etc.

Disadvantages of using UV rays

- Low penetrative power
- Limited life of the UV bulb
- Some bacteria have DNA repair enzymes that can overcome damage caused by UV rays
- Organic matter and dust prevents its reach
- Rays are harmful to skin and eyes
- It does not penetrate glass, paper or plastic.

Ionizing rays: Ionizing rays are of two types: particulate and electromagnetic rays.
Electron beams are particulate in nature while gamma rays are electromagnetic in nature.
High speed electrons are produced by a linear accelerator from a heated cathode. Electron
beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and
pharmaceuticals. Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the
instruments can be switched off.
Disadvantage includes poor penetrative power and requirement of sophisticated equipment.

Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain
radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but
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require longer times of exposure. These high-energy radiations damage the nucleic acid of the
microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used
commercially to sterilize disposable petridishes, plastic syringes, antibiotics, vitamins,
hormones, glasswares and fabrics.
Disadvantages
- Unlike electron beams, they can’t be switched off
- Glassware tends to become brownish
- Loss of tensile strength in fabric
Bacillus pumilus E601 is used to evaluate sterilization process.

Filtration:
Filtration does not kill microbes it separates them out. Membrane filters with pore sizes
between 0.2-0.45 μm are commonly used to remove particles from solutions that cannot be
autoclaved. It is used to remove microbes from heat labile liquids such as serum, antibiotic
solutions, sugar solutions and urea solution.
Applications of filtration
- removing bacteria from ingredients of culture media
- preparing suspensions of viruses and phages free of bacteria
- measuring sizes of viruses
- separating toxins from culture filtrates
- counting bacteria
- Clarifying fluids and purifying hydatid fluid.
Filtration is aided by using either positive or negative pressure using vacuum pumps. The older
filters made of earthenware or asbestos are called depth filters.
Different types of filters are:
1. Earthenware filters: These filters are made of diatomaceous earth or porcelain. They are
usually baked into the shape of candle. Different types of earthenware filters are:
a. Pasteur-Chamberland filter: These candle filters are from France and are made up of
porcelain (sand and kaolin). Similar filter from Britain is Doulton Chamberland filters made with
various porosities, which are graded as L1, L1a, L2, L3, L5, L7, L9 and L11. Doulton filters are P2,
P5 and P11.
b. Berkefeld filter: These are made of Kieselguhr, a fossilized diatomaceous earth found in
Germany. They are available in three grades depending on their porosity (pore size); they are V
(veil), N (normal) and W (wenig). Quality of V grade filter is checked using culture suspension of
Serrtia marcescens (0.75 μm).
c. Mandler filter: This filter from America is made of kieselguhr, asbestos and plaster of Paris.
2. Asbestos filters: These filters are made from chrysotile type of asbestos, chemically
composed of magnesium silicate. They are pressed to form discs, which are to be used only
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 once. The disc is held inside a metal mount, which is sterilized by autoclaving. They are
available in the following grades HP/PYR (for removal of pyrogens), HP/EKS (for absolute
sterility) and HP/EK (for clarifying).
3. Sintered glass filters: These are made from finely ground glass that is fused sufficiently to
make small particles adhere to each other. They are usually available in the form of disc
fused into a glass funnel. Filters of Grade 5 have average pore diameter of 1-1.5 μm. They
are washed in running water in reverse direction, cleaned with warm concentrated sulfuric
acid, and sterilized by autoclaving.
Membrane filters
These filters are made from a variety of polymeric materials such as cellulose nitrate, cellulose
diacetate, polycarbonate and polyester. The older type of membrane, called gradocol (graded
colloidion) membrane was composed of cellulose nitrate. Gradocol membranes have average
pore diameter of 3-10 μm.
The newer ones are composed of cellulose diacetate. These membranes have a pore diameter
ranging from 0.015 μm to 12 μm.
These filters are sterilized by autoclaving.
Membrane filters are made in two ways: the capillary pore membranes have pores produced by
radiation while the labyrinthine pore membranes are produced by forced evaporation of
solvents from cellulose esters.
Disadvantages of depth filters
- migration of filter material into the filtrate
- absorption or retention of certain volume of liquid by the filters
- Pore sizes are not definite and viruses and mycoplasma could pass through.
Advantages of membrane filters
- known porosity
- no retention of fluids
- reusable after autoclaving
- Compatible with many chemicals.
However, membrane filters have little loading capacity and are fragile.
Air filters
Air can be filtered using HEPA (High Efficiency Particle Air) filters. They are usually used in
biological safety cabinets. HEPA filters are at least 99.97% efficient for removing particles >0.3
μm in diameter. Examples of areas where HEPA filters are used include rooms housing severely
neutropenic patients and those operating rooms designated for orthopedic implant procedures.
HEPA filter efficiency is monitored with the dioctylphthalate (DOP) particle test using particles
that are 0.3 μm in diameter.
Chemical methods of disinfection:

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Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces.
Some chemical have very narrow spectrum of activity and some have very wide. Those
chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely
applied over skin and mucus membranes are called antiseptics.
An ideal antiseptic or disinfectant should have following properties:
- Should have wide spectrum of activity
- Should be able to destroy microbes within practical period of time
- Should be active in the presence of organic matter
- Should make effective contact and be wettable
- Should be active in any pH
- Should be stable
- Should have long shelf life
- Should be speedy
- Should have high penetrating power
- Should be 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 and must be available easily
Such an ideal disinfectant is not yet available.
The level of disinfection achieved depends on contact time, temperature, type and
concentration of the active ingredient, the presence of organic matter, the type and quantum
of microbial load. The chemical disinfectants at working concentrations rapidly lose their
strength on standing.

Classification of disinfectants:
1. Based on consistency: Liquid (E.g., Alcohols, Phenols) and Gaseous (Formaldehyde vapor,
Ethylene oxide)
2. Based on spectrum of activity: High level, Intermediate level and Low level
3. Based on mechanism of action:
- Action on membrane (E.g., Alcohol, detergent) or
- Denaturation of cellular proteins (E.g., Alcohol, Phenol)
- Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)
- Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide, Formaldehyde)
- Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

Alcohols:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
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Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than absolute
alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin.
Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces. It is used to
disinfect clinical thermometers.
Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

Aldehydes:
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably
damages nucleic acids. It kills all microorganisms, including spores.
Examples: Formaldehyde, Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of
rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc.
Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with
potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with
0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, unaesthetic equipments etc. An
exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2%
formaldehyde at 40oC for 20 minutes is used to disinfect wool and 0.25% at 60oC for six hours to
disinfect animal hair and bristles.
Disadvantages
- Vapors are irritating (must be neutralized by ammonia)
- has poor penetration
- leaves non-volatile residue
- activity is reduced in the presence of protein
- Gluteraldehyde requires alkaline pH and only those articles that are wettable can be
sterilized.
Phenol
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of
enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene,
chlorhexidine, chloroxylenol (Dettol)

Halogens:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl
groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.

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Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds
(tincture iodine, iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be
combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores
such as povidone-iodine. Iodophores permit slow release and reduce the irritation of the
antiseptic.
For hand washing, iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used
undiluted in pre and postoperative skin disinfection. Chlorine gas is used to bleach water.
Household bleach can be used to disinfect floors. Household bleach is used in a stock dilution of
1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium
hypochlorite is used in serology and virology. It is used at a dilution of 1:10 in decontamination
of spillage of infectious material. Mercuric chloride is used as a disinfectant.
Disadvantages
- They are rapidly inactivated in the presence of organic matter
- Iodine is corrosive and staining
- Bleach solution is corrosive and will corrode stainless steel surfaces.
Heavy metals:
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are
bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g.,
mercurochrome, merthiolate)
Applications:
 1% silver nitrate solution can be applied on eyes as treatment for opthalmia
neonatorum (Crede’s method). This procedure is no longer followed.
 Silver sulphadiazine is used topically to help to prevent colonization and infection of
burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000.
 Merthiolate at a concentration of 1:10000 is used in preservation of serum.
 Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic and readily inactivated by organic matter.

Surface-active agents:
Mode of action: They have the property of concentrating at interfaces between lipid containing
membrane of bacterial cell and surrounding aqueous medium. These compounds have long
chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they
contain both of these, they concentrate on the surface of membranes. They disrupt membrane
resulting in leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents
containing negatively charged long chain hydrocarbon are called anionic detergents. These
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include soaps and bile salts. If the fat-soluble part is made to have a positive charge by
combining with a quaternary nitrogen atom, it is called a cationic detergent.
Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and
benzalkonium chloride act as cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They
are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter.
Pseudomonas can metabolize cetrimide, using them as a carbon, nitrogen and energy source.

Dyes:
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial
nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and brilliant green, Acridine dyes
such as acriflavin and aminacrine.
Acriflavine is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial
properties. A related dye, ethidium bromide, is also germicidal. It intercalates between base
pairs in DNA. They are more effective against gram-positive bacteria than gram-negative
bacteria and are more bacteriostatic in action.

Applications: They may be used topically as antiseptics to treat mild burns. They are used as
paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in
certain selective media.

Hydrogen peroxide:
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen
peroxide produces hydroxyl-free radical that damages proteins and DNA.
Application: It is used at 6% concentration to decontaminate the instruments and equipments
such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising
wounds and ulcers. Strong solutions are sporicidal.
Disadvantages:
- Decomposes in light
- broken down by catalase
- proteinaceous organic matter drastically reduces its activity.

Ethylene oxide (EO):

Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl, amino-, carboxyl- and
hydroxyl- groups.

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Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet
ethereal odor, readily polymerizes and is flammable.
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is
highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or
dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well
absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles,
rubber, plastics, syringes, disposable petridishes, complex apparatus like heart-lung machine,
respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

Beta-propiolactone (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and hydroxyl-
groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is a condensation
product of ketane with formaldehyde.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is used to
sterilize biological products. It is more efficient in fumigation that formaldehyde. It is used to
sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.

Physico-chemical method:
Mode of action: A physio-chemical method adopts both physical and chemical method. Use of
steam formaldehyde is a physio-chemical method of sterilization, which takes into account
action of steam as well as that of formaldehyde.
Saturated steam at a pressure of 263 mm has a temperature of 70oC. The air is removed from
the autoclave chamber and saturated steam at sub-atmospheric pressure is flushed in.
Formaldehyde is then injected with steam in a series of pulses, each of 5-10 minutes. The
articles are held at this holding temperature for one hour. Formaldehyde is then flushed by
inflow of steam.
Disadvantages: Condensation of formaldehyde occurs and induction of large volume of
formaldehyde wets the steam resulting in loss of latent heat.
Sterilization control: using paper strips containing 106 spores of G.stearothermophilus. S.typhi
as well as S.aureus. The phenol coefficient is lower than that given by Rideal Walker method.

Aseptic technique
Aseptic technique is essential for all pathology work and must be thoroughly practiced and
mastered. Cultures must be pure. This means that they must be free of any living microbes
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other than the one required. The presence of unwanted fungi or bacteria is known as
contamination and the microbes responsible are referred to as contaminants. We use aseptic
technique so that we can handle, or manipulate microorganisms without introducing
contaminants into the culture. Aseptic technique also protects the operator from potential
infection from pathogenic organisms. Always use aseptic technique when handling
microorganisms and also when preparing micro-biological media in which to grow these
organisms. All equipment and media to be used during the handling of the microorganism must
be sterile. There is no such thing as mostly sterile There are many different methods of
sterilization. The method you use depends on the equipment you have and what it is you are
sterilizing. As a general rule, the following methods are most appropriate.

Microbiological growth media

Wet heat sterilization, usually using an autoclave although a domestic pressure cooker will do
just as well.
1. Raise the temperature to 121°C and the pressure in the closed chamber to 15 psi for 15-
20 minutes.
2. Do not over fill vessels containing liquid, leave a large space at the top of all bottles to
allow for expansion and boiling of the liquid on heating in the autoclave.
3. Loosen screw caps before autoclaving.

Laboratory glassware
Sterilize as above using wet heat sterilization or dry heat sterilization in an oven at 160°C for 1-2
hours. If you have no autoclave, pressure cooker or oven, you can use certain chemical agents
such as strong acids or alkalis, phenols or ethylene oxide. All chemical methods are potentially
hazardous to the operator and should be avoided where possible.

Small pieces of equipment:

Sterilize glass rods and metal tools by dipping them in 70% ethanol (alcohol) and then flaming
to burn off the alcohol. Sterilize inoculating loops and needles by holding in a flame until red-
hot.

Laboratory benches
Swab the working surface with 70% alcohol or chemical disinfectant to prevent the introduction
of contaminants. Never allow anything that is sterile to come into direct contact with the
bench.

Other methods of sterilization are available.

1. Ultraviolet radiation: can be useful for benches and clean rooms.
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 2. Gamma radiation: is used for the sterilization of plastics in industry.
3. Filtration: using filters of a maximum pore size of 1nm generally used to sterilize small
quantities of liquids that are unstable at high temperatures.

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