Sterilization techniques

MICRO-304
25th March 2021
Sterilization
• Sterilization is the complete removal of microorganisms from
an object or surfaces.
• Sterilization is obtained when microorganisms are subjected
to antimicrobial agents for sufficient time and at optimum
conditions.

Why is Sterilization performed?

To prevent
• contamination in sterile products
• transmission of disease causing pathogens
• from unwanted microbes in cultures and research studies
• spreading hazardous microbes to spreading to environment
 Other important terms

• Disinfection: process of destruction of all pathogens from

non-living surfaces by lowering the number to the level that
it is no longer harmful to the health.
• Disinfectants: antimicrobial reagents used to destroy
microbes from non-living surfaces.
• Antiseptic: Chemical used on living tissues to prevent the
growth of microorganisms
• Aseptic: characterized by absence of microbes
Methods of sterilization

• How microbes can be killed?

a) Denaturation of proteins
b) Interruption of DNA synthesis/repair
c) Disruption of cell membranes
• Two types of methods
a) Physical methods
b) Chemical methods
Methods of sterilization
• Physical methods
a) Heat; dry and moist heat
b) Radiations
c) Filtration
d) Sonic waves
e) High pressure sterilization
f) Sun light
• Chemical methods
Ethylene oxide, formaldehyde, nitrogen dioxide, ozone.
Hydrogen peroxide, glutaraldehyde, hypochlorite.
Physical methods
• Dry heat
a) Red heat
b) flaming
c) incineration
d) hot-air oven
• Red heat: Materials are held in the
flame of a Bunsen burner till they
become red hot e.g, Inoculating wires
or loops, Tips of forceps and Needles
• Flaming: Materials are passed through the flame of a Bunsen burner
without allowing them to become red-hot e.g. Glass slides, Scalpels, Mouths
of culture tubes.
• Incineration: Materials are reduced to ashes by burning. e.g, Soiled
dressings, animal carcasses, bedding and pathological materials.
Hot-air oven
• Electrically heated and fitted with a fan
to even distribution of air in the
chamber.

• Heat at 180º for half hour , 170º for 1

hr., or 160º C for 2 hrs.

• Glassware like glass syringes, Petri

dishes, pipettes and test tubes, Surgical
instruments like scalpels, scissors,
forceps and Chemicals like liquid
paraffin, fats etc. can be sterilized
Procedure
• Wrap washed and air-dried glassware in paper in
the form of bundle. Secure with thread.

• Place in hot air oven chamber as they don’t touch

the sides of oven.

• Lock door, turn on the electricity, adjust

temperature and time.

• Turn off the oven, allow temperature to fall, remove

the tray.

• Allow to cool.
Advantages

• A dry heat cabinet is easy to install and has relatively low operating costs

• It penetrates materials

• It is nontoxic and does not harm the environment

• it is noncorrosive for metal and sharp instruments.

Disadvantages

• Time consuming method because of slow rate of heat penetration and

microbial killing.

• High temperatures are not suitable for most materials.

 Demonstration video
https://www.youtube.com/watch?v=vvFDypILkTA
 Moist heat
At Temp <1000C
Pasteurization
63° C (145° F) maintained for 30 minutes
72° C (162° F) for 15 seconds
Used for milk, ice cream, yogurt, and fruit juices Heat-tolerant
microbes survive
At temp=1000C
Boiling, tyndalization, steam sterilization

At temp> 100◦C
Autoclaving
 At Temp=1000C
Boiling
• for 10 – 30 minutes may kill most of vegetative forms but
spores with stand boiling
Tyndallisation
• Steam at 100C for 20 minutes on three successive days. Used
for egg , serum and sugar containing media.
Steam sterilizer
• Steam at 100°C for 90 minutes.
• Used for media which are decomposed at
high temperature.
 At temp >1000C (Autoclave)

Steam above 100°C has a better killing power than dry heat.
Bacteria are more susceptible to moist heat.

Moist heat principle

Destroys microorganisms by the irreversible denaturation of

enzymes and structural proteins. The temperature at which
denaturation occurs varies inversely with the amount of water
present.
Autoclave
Uses steam under pressure to kill
harmful bacteria, viruses, fungi, and
spores on items that are placed inside a
pressure vessel
121°C , 15 psi , 15 minutes

Uses
culture media
autoclavable containers
plastic tubes
pipette
Schematic diagram of autoclave
Procedure
• Pour distilled water in autoclave to recommended point

• Place the flask etc. in the holding pan

• Tightly close the lid

• Turn on the electric heater

• Close both the vent and allow steam to generate up-to the set

requirements

• turn off the source and allow the pressure and temperature to fall.

• Open the clamps of the lid and remove the holding pan.
 Demonstration video
• https://www.youtube.com/watch?v=0dlDf8HiiQw
Radiation
Ionization radiation
X-rays, Gamma rays, cosmic rays
Used for sterilization of disposable materials.

Non-ionizing
Infrared rays
• Used for rapid mass sterilization of syringes and catheters.
UV light
• Used to disinfect operating rooms, nurseries, cafeterias.
Ultraviolet radiation is used for disinfecting enclosed areas such
as bacterial laboratory, inoculation hood, laminar flow and
operation theatres.
• Damages skin, eyes in humans.
Filtration
Filtration of liquids and gases. This method is commonly used for
sensitive pharmaceuticals and protein solutions in biological
research.

• A filter with pore size 0.2 µm will effectively remove bacteria.

• If viruses must also be removed, a much smaller pore size

around 20 nm is needed.
Sound waves
Sonicator (>20KHz)

Pressure sterilization
Pascalization in food (87000,psi)

Sun light
Gaseous sterilization
• Gaseous sterilization is a more effective technique as gases
can pass through a tiny orifice and provide more effective
results.

• Besides, gases are commonly used along with heat

treatment which also facilitates the functioning of the gases.

• However, there is an issue of release of some toxic gases

during the process which needs to be removed regularly
from the system.
 Ethylene oxide

• Disinfect different types of equipment and surfaces because of its

wide range of compatibility with different materials.

• Used for disposable medical devices.

• The mechanism of antimicrobial action of this gas is assumed to

be through the alkylation of sulphydryl, amino, hydroxyl, and
carboxyl groups on proteins and imino groups of nucleic acids.

• EO treatment is usually conducted at the temperature range of

30-60°C for several hours which aids in the activity of the gas.
Formaldehydes
• This gas is obtained by heating formalin (37%w/v) to a
temperature of 70-80°C.

• Broad-spectrum biocidal activity

• Sterilization of reusable surgical instruments, specific

medical, diagnostic and electrical equipment, and the
surface sterilization of powders.

• As a result of the low penetrating power, its use is often

limited to paper and cotton fabrics.
Nitrogen dioxide
• Nitrogen dioxide is a rapid and effective sterilant that can be
used for the removal of common bacteria, fungi, and even
spores.

• NO2 has a low boiling point (20°C) which allows a high vapor
pressure at standard temperature.

• The biocidal action of this gas involves the degradation of

DNA by the nitration of phosphate backbone, which results
in lethal effects on the exposed organism as it absorbs NO2.
Ozone

• Ozone is a highly reactive industrial gas that is commonly

used to sterilize air and water and as a disinfectant for
surfaces.

• Ozone has a potent oxidizing property

• Ozone is an unstable and reactive gas, it has to be produced

on-site, which limits the use of ozone in different settings
Liquid sterilization
• Although liquid sterilization is not as effective as gaseous sterilization,
it is appropriate in conditions where a low level of contamination is
present.

Hydrogen peroxide
• Hydrogen peroxide is a liquid chemical sterilizing agent which is a
strong oxidant and can destroy a wide range of microorganisms.

• It is useful in the sterilization of heat or temperature-sensitive

equipment like endoscopes. In medical applications, a higher
concentration (35-90%) is used.
Glutaraldehyde

• Glutaraldehyde is an accepted liquid sterilizing agent which

requires comparatively long immersion time.

• For the removal of all spores, it requires as long as 22 hours of

immersion time.

• The presence of solid particles further increases the immersion

time.

• Less penetration power

• The use of glutaraldehyde is thus limited to certain surfaces with

less contamination.
Hypochlorite
• Hypochlorite solution, which is also called liquid bleach

• Submerging devices for a short period in liquid bleach might

kill some pathogenic organisms but to reach sterilization
submersion for 20-24 hours is required.

• It is an oxidizing agent and thus acts by oxidizing organic

compounds which results in the modification of proteins in
microbes which might ultimately lead to death.