STERILIZATION AND DISINFECTION OF ARTICLES

DEFINITION

Sterilization
It is defined as the process where all the living microorganisms,
including bacterial spores are killed. Sterilization can be achieved by
physical, chemical and physiochemical means. Chemicals used as
sterilizing agents are called chemisterilants.

Disinfection
It 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, 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.

METHOD OF STERILIZATION AND DISINFECTION

physical and chemical method
Physical sterilization

MOIST HEAT:

Moist heat acts by coagulation and denaturation of proteins.

At temperature below 100 degree C:
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 63o
C for 30 minutes) and flash method (heated at 72o C for 15 seconds)
followed by quickly cooling to 13o C. Other pasteurization methods
include Ultra-High Temperature (UHT), 140o C for 15 sec and 149o C 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 60o C for one hour. Only
vegetative bacteria are killed and spores survive.
Serum bath: The contaminating bacteria in a serum preparation can be
inactivated by heating in a water bath at 56o C for one hour on several
successive days. Proteins in the serum will coagulate at higher
temperature. Only vegetative bacteria are killed and 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 80-85o C 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 100 degree C:

Boiling: Boiling water (100o C) kills most vegetative bacteria and
viruses immediately. Certain bacterial toxins such as Staphylococcal
enterotoxin are also 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 100 degree C: Instead of keeping the articles in boiling water,
they are subjected to free steam at 100o C. 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 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 100degree C:

Autoclave: Sterilization can be effectively achieved at a temperature
above 100o C using an autoclave. Water boils at 100o C 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. At a pressure of 15
lbs inside the autoclave, the temperature is said to be 121o C. 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; 135o C or 121o C 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, condensation
of steam draws in fresh steam.
Different types of autoclave: Simple “pressure-cooker type” laboratory
autoclave, Steam jacketed downward displacement laboratory autoclave
and high pressure pre-vacuum autoclave
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
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 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 should be fitted with a thermostat control, temperature indicator,
meshed shelves and must have adequate insulation.
Articles sterilized:
Metallic instruments (like forceps, scalpels, scissors), glasswares (such as
petri-dishes, pipettes, flasks, all-glass syringes), swabs, oils, grease,
petroleum jelly and some pharmaceutical products.
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.

Infra red rays:

Infrared rays bring about sterilization by generation of heat. Articles to be

sterilized are placed in a moving conveyer belt and passed through a tunnel
that is heated by infrared radiators to a temperature of 180o C. The articles
are exposed to that temperature for a period of 7.5 minutes. Articles
sterilized included 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 .

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". In some
parts of Europe, fruits and vegetables are irradiated to increase their shelf
life up to 500 percent.

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 include 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 doesn't
penetrate glass, paper or plastic.

Ionizing rays:

• Ionizing rays are of two types, particulate and electromagnetic rays.

o Electron beams are particulate in nature while gamma rays are
electromagnetic in nature.
• Highspeed 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 require longer
time 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 petri dishes, plastic
syringes, antibiotics, vitamins, hormones, glasswares and fabrics.
• Disadvantages include; unlike electron beams, they can’t be switched
off, glasswares tend to become brownish, loss of tensile strength in
fabric. Gamma irradiation impairs the flavour of certain foods.
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 can't be autoclaved.
• It is used to remove microbes from heat labile liquids such as serum,
antibiotic solutions, sugar solutions, urea solution.
• Various applications of filtration include 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

Chemical Methods of Disinfection

When chemicals are used to destroy all life forms they are
called chemical sterilants or biocides;however, these same chemicals
used for shorter periods are disinfectants. Disinfectants are chemicals that
kill microorganisms and are used on inanimate objects. Chemicals used on
living tissue (skin) are called antiseptics.

Classification of disinfectants:

1. Based on consistency

a. Liquid (E.g., Alcohols, Phenols)

b. Gaseous (Formaldehyde vapor, Ethylene oxide)

2. Based on spectrum of activity

a. High level
b. Intermediate level
c. Low level

3. Based on mechanism of action

a. Action on membrane (E.g., Alcohol, detergent)

b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
c. Oxidation of essential sulphydryl groups of enzymes (E.g.,
H2O2, Halogens)
 d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,
Ethylene Oxide, Formaldehyde)
e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

Chemical disinfectants can be classified into four groups based on their

microbicidal activity;
. Low-level disinfectants
•Intermediate-level disinfectants
•High-level disinfectants
•Chemical sterilant

Chemical disinfectants comprise many classes, including the following:

1.Alcohols: They are among the most widely used disinfectants and
antiseptics. Ethyl or isopropyl alcohol is non-sporicidal (does not kill
spores) and evaporates quickly. Alcohols are best used on the skin as
an antiseptic (surgical spirit). Clinical thermometers and small
 instruments can be disinfected by soaking in isopropyl alcohol for 10-
15 minutes.
2.Aldehydes: Formaldehyde, glutaraldehyde, and ortho-
phthalaldehyde are commonly used aldehydes. They are sporicidal
and can be used as chemical sterilants. Formaldehyde is used to
preserve anatomical specimens and for fumigation of closed areas,
such as operation theaters. They are generally not used as a surface
disinfectants because of their irritating fumes.
3.Halogens: The halogens, especially chlorine and iodine, are
frequently used as disinfectants as they possess antimicrobial activity.
They exist in a free state and form a salt with sodium and most other
metals.
• Chlorine: It is the most commonly available disinfectant. Chlorine is
most often used in the form of sodium hypochlorite (NaOCl), the
compound known as household bleach. CDC recommends that
tabletops be cleaned following blood spills with a 1:10 dilution of
bleach. It is also used for municipal water supplies, swimming pools,
dairy and food industries, etc.
• Iodine: Iodine compounds are widely employed antiseptics. Iodine is
prepared either as tincture with alcohol or as an iodophor coupled
with a neutral polymer, for example, povidone-iodine.
Heavy metals: Salts of heavy metals, such as mercury, silver, zinc,
and copper were widely used in the past as germicides but their use
has been replaced by less toxic chemicals over the period. Eyedrop
solution containing 1% silver nitrate is still instilled in the eyes of
newborns to prevent ophthalmia neonatorum (an infection with
Neisserigonorrhoeae).
“Heavy metals containing mercury is no longer recommended
because mercury is toxic to the environment”.
 3.Quaternary ammonium compounds (Quats): Quaternary
ammonium compounds such as benzalkonium chloride, are used to
disinfect bench-tops or other surfaces in the laboratory. However,
organic materials, such as blood, may inactivate heavy metals or
quaternary ammonium compounds, thus limiting their utility.

4.Phenolics: Phenolics, such as the common laboratory disinfectant

amphyl, are derivatives of carbolic acid (phenol). The addition of detergent
results in a product that cleans and disinfects at the same time, and at a
concentration between 2%-5% these products are widely used for cleaning
benchtops.

PHYSIO-CHEMICAL METHOD:

Mode of action: A physio-chemical method adopts both physical and

chemical method. Use of steamformaldehyde 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 70o C. 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

TESTING OF DISINFECTANTS:

A disinfectant must be tested to know the required effective dilution, the

time taken to effect disinfection and to periodically monitor its activity. As
disinfectants are known to lose their activity on standing as well as in the
presence of organic matter, their activity must be periodically tested.

Different methods are:

1. Koch’s method
2. Rideal Walker Method
3. Chick Martin test
4. Capacity use dilution test (Kelsey-Sykes test)
5.In-usetest

Koch’s method:
Spores of Bacillus anthracis were dried on silk thread and were
subjected to action of disinfectants. Later, it was washed and transferred
to solid medium.

Rideal Walker method:

This method relies on the estimation of phenol coefficient. Phenol
coefficient of a disinfectant is calculated by dividing the dilution of test
disinfectant by the dilution of phenol that disinfects under predetermined
conditions.
Both the phenol and the test disinfectant are diluted from 1/95 to
1/115 and their bactericidal activity is determined against Salmonella
typhi suspension. Subcultures are performed from both the test and phenol
at intervals of 2.5, 5, 7.5 and 10 minutes.
The plates are incubated for 48-72 hours at 37°C. That dilution of
disinfectant which disinfects the suspension in a given time is divided by
that dilution of phenol which disinfects the suspension in same time gives
its phenol coefficient.

Chick Martin test:

This test also determines the phenol coefficient of the test disinfectant.
Unlike in Rideal Walker method where the test is carried out in water, the
disinfectants are made to act in the presence of yeast suspension (or 3%
dried human feces).
Time for subculture is fixed at 30 minutes and the organism used to
test efficacy is S.typhi as well as S.aureus. The phenol coefficient is lower
than that given by Rideal Walker method.

Capacity use dilution test (Kelsey-Sykes test):

Inoculum of four different test organisms, namely Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris
are added to the disinfectant in three successive.
Dried yeast is included to simulate presence of organic matter. The
method can be carried out under 'clean' or 'dirty' conditions.
The dilutions of the disinfectant are made in hard water for clean
conditions and in yeast suspension for dirty conditions. Test organism
alone or with yeast is added at 0,10 and 20 minutes interval.
The contact time of disinfectant and test organism is 8 min. The
disinfectant is evaluated on its ability to kill microorganisms or lack of it
and the result is reported as a pass or a fail and not as a coefficient.
The capacity test of Kelsey and Sykes gives a good guideline for the
dilution of the preparation to be used. Disadvantage of this test is the fact
that it is rather complicated.
 In-use test:
The routine monitoring of disinfectant in use can be done by the ‘in
use’ test of Maurer. This test is intended to estimate the number of living
organism in a vessel of disinfectant in actual use.
The disinfectant that is already in use is diluted 1 in 10 by mixing 1
ml of the disinfectant with 9 ml of sterile nutrient broth. Ten drops of the
diluted disinfectant (each 0.02 ml) is placed on two nutrient agar plates.
One plate is incubated at 37o C for 3 days while the other is held at room
temperature for 7 days.
The number of drops that yielded growth is counted after incubation.
If there growth in more than five drops on either plate, it represents failure
of disinfectant.