Experiment 1

Sterilization Techniques used in Cell and Tissue Culture

tedRr-FOSUOP-2017AUG

Spore is a reproductive structure that is adapted

for dispersal and surviving for extended periods
INTRODUCTION
of time in unfavourable conditions.

2
The concept of asepsis and its role in the Spores form part of the lifecycles of many
prevention of infection was put forward nearly bacteria, plants, algae, fungi and some protozoa.
2 centuries ago. The general principles for asepsis
were laid down by Hungarian obstetrician, Lgnaz
Semmelweiss in Europe in early 1850s and Oliver Disinfection: Disinfection describes a process that
Holmes in USA. These principles were accepted after eliminates many or all pathogenic microorganisms,
Joseph Listers studies on prevention of wound except bacterial spores, on inanimate objects.
infection carried out between 1865-91. Lister,
working on antisepsis, initially used phenol (dilute
carbolic acid) for contaminated wounds, later
applied it in all surgical wounds, also in operating METHODS OF STERILIZATION
room by nebulization of the solution. Further The various methods of sterilization are:
developments occurred with the introduction of
steam sterilization surgical masks, sterile gloves, 1. Physical Method
sterile gowns and drapes etc.
a. Thermal (Heat) methods
b. Radiation method
Sterilization: c. Filtration method
1
Sterilization is the process of killing all 2. Chemical Method
microorganisms (bacterial, viral, and fungal) with the
use of either physical or chemical agents. a. Antibiotics
b. Disinfection
Sterilization is making a substance free from all c. Antisepsis
microorganisms both in vegetative and spore d. Gaseous method
forming states.
 Precautions:

Glass wares should be dry.

Oven should not be over loaded.
Articles are to be arranged in a manner to
allow free circular of air.
Door of the Oven should be opened after it
(A) Physical agents cools down (2Hours).
Materials should be properly arranged to
I. Sunlight allow free circulation of air
II. Drying
III. Heat Mode of Action:
Dry heat coagulates the protein and destroys
Dry heat: flaming, incineration, hot air
micrograms.
oven, burning, flaming
The efficiency with which heat is able to
Moist heat: pasteurization, boiling,
steam under pressure. inactivate microorganisms is dependent upon
Below I 00 c the degree of heat, the exposure time
At 100 c Temperature (c) Holding time
Above 100 c
(in minutes)
IV. Filtration:
160 45
candles, asbestos pads, membranes
170 18
V. Radiation
180 7.5
VI. Ultrasonic and sonic vibrations.
190 1.5

Heat

Dry heat Uses: 1 Metal instruments, glassware, aluminum

Hot air Oven foil, glassware, forceps, scissors, scalpels, all-glass
syringes, swabs, liquid paraffin, dusting powder,
3
It utilizes hot air sterilization. The dry heat fats, grease. non-aqueous thermos stable liquids and
sterilization process is accomplished by conduction, thermos stable powders.
that is where heat is absorbed by the exterior
surface of an item and then passed inward to the
next layer. Eventually the entire item reaches the
proper temperature needed to achieve sterilization.

Process:
The proper time and temperature for oven
sterilization is 180 for 2 hours. Instruments
should be dry before sterilization since water
will interfere with the process.
exposure to hot dry air (l 30-l 70C) for 2-4 hr
in a hot-air oven. All items should be sealed
before sterilization but not in paper, as it
decomposes at 170C.
Materials should be properly arranged to
allow free circulation of air
Advantages:
They do not require water and there is not much
pressure build up within the oven, unlike
an autoclave, making them safer to work with.
Suitable to be use in a laboratory environment.
They are much smaller than autoclaves but can
still be as effective.
There is a thermostat controlling the
temperature.
Uses The benefit of dry heat includes good
penetrability and non-corrosive nature
Dry heat destroys bacterial endotoxins (or
pyrogens) which are difficult to eliminate by
other means and this property makes it
applicable for sterilizing glass bottles which are
to be filled aseptically
Effective method of sterilization of heat stable
articles
The articles remain dry after sterilization
only method of sterilizing oils and powders.
Disadvantages
As they use dry heat instead of moist heat,
some organisms like prions, may not be
killed by them every time.
only to the THERMO STABLE PRODUCTS
Cannot be used for plastic ware, however,
certain plastic wares can also be heat
sterilized (Instructions of the manufacturer
must be read before doing this)
Since air is poor conductor of heat, hot air
has poor penetration.
Cotton wool and paper may get slightly
charred and Glasses may become smoky
Takes longer time compared to autoclave.
Incineration/ flaming/ burning
Flaming is done to loops, forceps, scalpels and
scissors. It is done by leaving the loop or other
instrument in the flame of a Bunsen burner or
alcohol lamp until it glows red, ensures that any
infectious agent gets inactivated. This is commonly
used for small metal or glass objects, but not for
large objects.
(Inoculating loop is better dipped in disinfectant first
before flaming to prevent spattering)
Usually glass objects are dip in 70 % or a higher
concentration of ethanol and merely touch the
object briefly to the Bunsen burner flame, but not
hold it directly to the flame. The ethanol will ignite
and bum off in a few seconds. 70 % ethanol kills
many but not all, bacteria and viruses, and has the
advantage that it leaves less residue on the object
than a use of gas flame. This method works well for
glass spreaders.
Flaming the Loop
Flaming the loop
helps to prevent
contamination of
the bacteria.
When flaming the
loop, make sure
that all of the wire
has been heated to
redness.
Incineration
It is a process that involves the combustion
of organic substances contained in waste
materials.
Items: contaminated cloth, animal carcasses
and pathological material. PVC, polythene can
be dealt.
(However, polystyrene will emit black smoke. Hence
should be autoclaved in appropriate container.)
 Wet Heat
1. Below 100
Pasteurization (for milk)
Louis Pasteur found a practical method of
preventing the spoilage of beer and wine. Pasteur
used mild heating, which was sufficient to kill the
organisms that caused the particular spoilage
problem without seriously damaging the taste of the
product. The same principle was later applied to
milk to produce what we now call pasteurized milk.
The intent of pasteurization of milk was to eliminate
pathogenic microbes. It also lowers microbial
numbers, which prolongs milk's good quality under
refrigeration. Many relatively heat-resistant
(thermoduric) bacteria survive pasteurization, but
these are unlikely to cause disease or cause
refrigerated milk to spoil.
Uses: milk, ice cream, yogurt, beer
Testes to check Pasteurization:
The phosphatase test: (phosphatase is an
enzyme naturally present in milk). If the
product has been pasteurized, phosphatase
will have been inactivated.
Process
Depends on the product
(For example, heating is less efficient in foods that
are more viscous, and fats in food can have a
protective effect on microorganisms.)
Holding period: 63C, 30 minutes (holder
method); Eg: mycobacteria, brucellae,
salmonella.Coxiellaburnetti, relatively heat
resistant, may survive the holder method.
High temperature short-time (HTST):
temperatures of at least 72C, for only 15
seconds; is applied as the milk flows
continuously past a heat exchanger. In
addition to killing pathogens, HTST
pasteurization lowers total bacterial counts,
so the milk keeps well under refrigeration.
72C, 15-20 minutes followed by cooling
quickly to 130c or lower.
ultra-high-temperature (UHT) treatments
(140C for less than a second). It can then be
stored for several months without
refrigeration
Mode of Action: all nonssporing pathogens,
because of the temperature variations, microbe
get killed.
Advantages:
pathogenic (harmful) bacteria and most non-
pathogenic bacteria are killed with minimal
flavor and nutrient loss in the milk
Disadvantage:
any kind of heat treatment some nutrients are
lost.
Water bath: 56 c for 60 min

At temperature 100c:
Boiling: 100c
Kills most vegetative bacteria and viruses
immediately. (but boiling is ineffective against
prions and many bacterial and fungal spores)
Some bacterial spores are resistant to boiling and
survive; hence its 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 mnts.
The lid of the boiler must not be opened
during the period.
Advantages:
simple process
Only common method to sterilize water easily
used for media which cant withstand
autoclaving and relies upon
the germination of spores to form cells that can
be killed at 100C.
Disadvantages:
boiling is ineffective against prions and many
bacterial and fungal spores
Tyndallization
Tyndalllization/ Tyndallization named after John
Tyndall is a lengthy process designed to reduce the
level of activity of sporulation bacteria that are left
by a simple boiling
water method.
This process is also called as Fractional
sterilization or Intermittent sterilization.
Process:
1) Steam heating to 100 C for 15-30 min
Vegetative cells are destroyed but endospores
survive (most of the vegetative bacteria, molds and
mold spores are Killed)
2) Incubate at 30C-37C overnight
Most bacterial endospores germinate
3) Second heat treatment, 100 C, 15-30 min
Germinated endospores are killed.
4) Second incubation at 30C-37 C overnight
Remaining endospores germinate
5) Third heat treatment, 100 C, 60 min
Last remaining germinated endospores are killed
The three incubation periods are to allow heat-
resistant spores surviving the previous boiling
period to germinate to form the heat-sensitive
vegetative (growing) stage, which can be killed by
the next boiling step. This is effective because many
spores are stimulated to grow by the heat shock.
Advantage:
Can use for broth media sterilization
Disadvantages:
The procedure only works for media that can
support bacterial growth
it will not sterilize plain water
ineffective against prions .
This procedure works only for broth media that
support the growth of spore forming
organisms. It is not useful for sterilizing water or
buffers
If the medium contains more microbes, the dead
cells remain as
debris in the medium/broth
Lengthy procedure
Steam sterilization
Steam at atmospheric pressure for 90 min.
Instead of keeping the articles in boiling water, they
are subjected to free steam at 100c.
Process:
Arnolds and kochs steamers.
A steamer is a metal cabinet with perforated
trays to hold the articles. The bottom
steamer is filled with water and heated. The
steam generated sterilizes the articles when
exposed for a period of 90minutes.
Advantages:
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 20mnts for a pressure gauge,
three successive days. a safety valve and a discharge tap.
The articles to be sterilized must not be
tightly packed. 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.
Water boils when its vapour pressure equals
the surrounding atmosphere. Thus, when
pressure inside closed vessels increases, the
temperature at which water boils increases
too.
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 stooped
and the autoclave is allowed to cool.
When pressure gauge shows the pressure
equals to atmospheric pressure, the
Above 100 c discharge tap is opened to let the air in. The
lid is then opened and articles removed.
Autoclave/ pressure cooker

It utilizes hot air that is heavily laden with water Precautions:

vapor and where this moisture plays the most
Articles should not be tightly packed and
important role in the process of sterilization.
must be wrapped in paper to prevent
drenching,
Process:
The autoclave must not be overloaded,
At a pressure of 15lbs inside the autoclave, it
air discharge must be complete and there
commonly uses steam heated to 121 - 134 C
should not be any residual air trapped
(250 - 273 F). To achieve sterility, a holding time
inside,
of at least 15 minutes at 121 C (250 F)
caps of bottles and flasks should not be
3 minutes at 134 C (273 F)
tight,
Additional sterilization time is usually required
autoclave must not be opened until the
for liquids and instruments packed in layers of
pressure has fallen or else the contents will
cloth, as they may take longer to reach the
boil over,
required temperature.
It will not necessarily eliminate all prions. For
prion elimination, various recommendations
state 121 - 132 C (250 - 270 F) for 60 minutes
or 134 C (273 F) for at least 18 minutes.

Mode of Action: Steam pressure coagulates the

protein in any organism and this is aided by the
water vapor that has a very high penetrating
property.

Construction and operation of Autoclave:

A simple autoclave has vertical or horizontal

cylindrical body with
a heating element,
a perforated try to keep the articles,
a lid that can be fastened by screw clamps,
Important to use:
Following sterilization, liquids in a pressurized
autoclave must be cooled slowly to avoid boiling
over when the pressure is released. Modem
converters/ autoclaves operate around this
problem by gradually depressing the sterilization
chamber and allowing liquids to
evaporate under a negative pressure, while
cooling the contents.
For effective sterilization, steam needs to
penetrate the autoclave load uniformly, so an
autoclave must not be overcrowded, and the
lids of bottles and containers must be left
ajar.
During the initial heating of the chamber,
residual air must be removed. Indicators should
be placed in the most difficult places for the
steam to reach to ensure that steam actually
penetrates there.
For autoclaving, as for all disinfection or
sterilization methods, cleaning is critical.
Extraneous biological matter or grime may
shield organisms from the property intended to
kill them, whether it physical or chemical.
Cleaning can also remove a large number of
organisms.
Proper cleaning can be achieved by physical
scrubbing.
This should be done with detergent and warm
water to get the best results.
Cleaning instruments or utensils with organic
matter, cool water must be used because warm
or hot water may cause organic debris to
coagulate. Treatment with ultrasound or
pulsedair can also be used to remove debris.
.
Uses: decontamination of laboratory waste and the
sterilization of laboratory glassware, media, and
reagents, dressings, instruments, laboratory ware,
media and pharmaceutical products
Advantages:
Widely- used method for heat sterilization is
the autoclave.
Proper autoclave treatment will inactivate all
fungi, bacteria, viruses and also bacterial
spores, which can be quite resistant.
most autoclaves have meters and charts that
record or display pertinent information such as
temperature and pressure as a function of
time.
Sterilization can be effectively achieved
Quicker than hot air oven
Disadvantage:
Drenching and wetting or articles may occur
Trapped air may reduce the efficacy and it
takes long time to cool.
Autoclaving is not advisable for
metal instruments because they may rust and
become blunt under these conditions
Some plastic ware melts in the high heat, and
sharp instruments often become dull.
Moreover, many chemicals breakdown during
the sterilization process and oily substances
cannot be treated because they do not mix
with water.
Advantage of WET HEAT METHODS
more penetrative power than dry air
moistens the spores [moister is essential for
coagulation of proteins],
 Radiation
Energy transmitted through space in a variety of
forms is generally called Radiation.
Electromagnetic radiation can interact with
matter in one of 2 general ways:
I. IONIZING RADIATIONS [X-ray and -rays]
II. NON-IONIZING RADIATION [UV light]
Method of sterilization using radiation such as
electron beams, X- rays, gamma rays or subatomic
particles can be practiced.
I. IONIZING RADIATIONS
Ionizing rays are of two types,
particulate and
electromagnetic rays.
-rays
Gamma rays are very penetrating and are
commonly used for sterilization of disposable
medical equipment, such as syringes, needles,
cannulas and IV sets.
X-ray
X- rays, High-energy X- rays are a form of ionizing
energy allowing to irradiate large
packages and pallet loads of medical devices. Their
penetration is sufficient to treat multiple pallet loads
of low-density packages with very good dose
uniformity ratios. X- ray sterilization is an electricity
based process not requiring chemical nor
radioactive material. High energy and high power X-
rays are generated by an X- ray machine that can be
turned off for servicing and when not in use.
II. NON-IONIZING RADIATION [UV light]
Nonionizing radiation has a wavelength longer than
that of ionizing radiation, usually greater than about
1 nm.
IR also non-ionizing radiation
UV light
Ultra violet in-addition (UV, from a germicidal
lamp) is useful only for sterilization of
surfaces and some transparent objects. Many
objects that are transparent to visible
light absorb UV. UV irradiation is' routinely used to
sterilize the interiors of biological
safety cabinets between uses, but is ineffective in
shaded areas, including areas under.
dirt (which may become polymerized after
prolonged irradiation so that it is very difficult to
remove). It also damages many plastics, such as
polystyrene foam.
Electron Beam
Electron beam processing is also commonly used
for medical device sterilization. Electron beams use
an on-off technology and provide a much higher
dosing rate than gamma or X- rays. Due to the
higher dose rate, less exposure time is needed and
thereby any potential degradation to polymers is
reduced. A limitation is that electron
beams are less penetrating than either gamma or x-
rays.
Irradiation with X- rays or gamma rays does not
make materials radioactive. Irradiation with
particles may make materials radioactive, depending
upon the type of particles and them energy, and the
type of target material: neutrons and very high-
energy particles can make materials radioactive, but
have good penetration, whereas lower energy
particles (other than
neutrons) cannot make materials radioactive, but
have poorer penetration.
MODE OF ACTION:
Gamma rays and X rays which have energies
of more than about 10ev is passes through a
cell they create free hydrogen radicals and
some peroxides which in turn can cause
different kinds of intracellular damage.
Ionizing radiation produce relatively little
heat in the materials being irradiated, thus it
is possible to sterilize heat-sensitive
substances in food and pharmaceutical
industries and the process is called Cold
sterilization
 Less energetic radiations like UV light does Requires very qualified personnel
not ionize, it is absorbed quite specifically by
Another potential problem is that UV light
different compounds and cause Mutation by
can damage human eyes, and prolonged
forming DIMERS or engages in a variety of
exposure can cause burns and skin cancer in
chemical reactions not possible for
humans.
unexcited molecules.

UV light damages the DNA of exposed cells

by causing bonds to form between adjacent
pyrimidine bases, usually thymines, in DNA
chains. These thymine dimers inhibit correct
replication of the DNA during reproduction
of the cell.
Subatomic particles may be more or less
penetrating, and may be generated by a
radioisotope or a device, depending upon
the type of particle.

The most lethal type of radiation is

ultraviolet radiation with a wavelength of
260 nm. This is the wavelength most actively
absorbed by DNA.
Acts by destroying DNA or damaging it.
Its efficiency is dependent on the
wavelength, intensity, and duration.
Ionizing Radiation
X-rays, -rays, electron beams has a
wavelength shorter than that of nonionizing
radiation, less than about 1 nm.
Uses: Radiation sterilization is generally
applied to articles in the dry state; including
surgical instruments, sutures, prostheses,
unit dose ointments, plastic syringes and dry
pharmaceutical products. Infrared-Used for
rapid mass sterilization of prepacked items
such as Syringe, Cathaters; Used for
disinfecting enclosed area such as
entryways, operation theatres and labs.Used
for sterilising plastics, syringes, swabs,
catheters, animal feeds, cardboard, oils,
greases, fabric and metal foils.

Advantages:
Microwaves
The food industry has recently renewed its
interest in the use of radiation for food Very little effect on microbes
preservation.
Microwave ovens kill vegetative pathogens
It is useful for disinfecting surfaces, air and by heating
liquids.
Solid foods heat unevenly
Clean process

Dry process

Ensures full exposure of object from

all directions

Disadvantage:

GAMMA radiation requires bulky shielding

for the safety of the operators; they also
require storage of a radioisotope (usually
Cobalt-60), which continuously emits
gamma rays (it cannot be turned off, and
therefore always presents a hazard in the
area of the facility).

Posses threat to humans (radiation)

Lengthy process
 Filtration
Clear liquids that would be damaged by
heat, irradiation or chemical sterilization can
be sterilized by mechanical filtration. This
method is commonly used for sensitive
pharmaceuticals and protein solutions in
biological research.
Filtration is the passage of a liquid or gas
through a screen like material with pores
small enough to retain microorganisms.
A vacuum is created in the receiving flask; air
pressure then forces the liquid through the filter.
Mode of Action:
A filter with pore size 0.2 m will effectively
remove bacteria. If viruses must also be
removed, a much smaller pore size around
20 run is needed.
Solutions filter slowly through membranes
with smaller pore diameters.
Filtration does not kill microbes, it separates
them out.
Filtration is aided by using either positive or
negative pressure using vacuum pumps.
Precautions:
The filtration equipment and the filters
themselves may be purchased as pre-
sterilized disposable units in sealed
packaging, or must be sterilized by the user,
generally by autoclaving at a temperature
that does not damage the fragile filter
membranes.
To ensure sterility, the filtration system
must be tested to ensure that the
membranes have not been punctured prior
to or during use.
To ensure the best results, pharmaceutical
sterile filtration is performed in a room with
highly filtered air (HEPA filtration) or in a
laminar flow cabinet or "flowbox", a device
which produces a laminar stream of HEPA
filtered air.
Different types of filters are:
1. Earthenware filters:
These filters are made up of diatomaceous earth or
porcelain. They are usually baked into the shape of
candle.
2. Pasteur- Chamberland filter:
These candle filters are from France and are made
up of porcelain (sand and kaolin).
3. Berkefeld filter:
These are made of Kieselguhr, a fossilized
diatomaceous earth.
available in three grades depending on their
porosity (pore size); they are: V (veil),
N(normal) and W (wenig).
4. Asbestos filters:
These filters are made from chrysotile type
of asbestos, chemically composed of
Magnesium silicate.
They are pressed to form disc, which are to be used
only once.
The disc is held inside a metal mount, which is
sterilized by autoclaving.
5. Candel filters:
Used for purification of water for industrial and
drinking purposes.
These are manufactured under different grades
of porosity.
6. Sintered glass filters:
Has low absorptive properties
Brittle and expensive.

7. Membrane filters:
These filters are made from a variety of polymeric
materials such as cellulose nitrate, cellulose
diacetate, polycarbonate and polyester.
These membranes have a pore diameter ranging
from 0.015 m to 12 m. These filters are sterilized
by autoclaving.
The advantages of membrane filters are
known porosity
no retention of fluids
reusable after autoclaving and compatible
with many chemicals.
However, membrane filters have little loading
capacity and are fragile.
The disadvantages of depth filters are
migration of filter material into the filtrat
absorption or retention of certain volume of
liquid by the filters pore sizes are not
definite and viruses and mycoplasma could
pass through.
Air Filters:
Air can be filtered using HEPA (High Efficiency
Particle Air) filters.
usually used in biological safety cabinets.
HEPA filters are at least 99.97% efficient for
removing particles >0.3 m 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 m in diameter.
Advantages:
Can be used with thermolabile culture
media
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.
capable of preventing the passage of both
viable and non viable particles
Flexible; can be used in the separation,
concentration and purification of a huge
variety of materials across a wide range
industries.
Microfiltration and ultrafiltration processes
can operate as highly efficient "sieves",
capable of fractionating particle species
according to size.
No phase changes involved, both feed and
product streams remain in the liquid form.
The processes can function effectively at low
temperatures.
Energy requirements are low.
Processes are relatively simple to scale up.
Membranes can be manufactured in a
uniform and highly precise manner.
Disadvantages:
Prions are not removed by filtration.
The high flow rates used in cross-flow feed
can damage shear sensitive materials.
Equipment cost can be high.
If the membrane manufacturing process is
not precisely controlled, membranes with
wide pore size distribution may result, giving
poor separation performance.
Uses: biological fluids such as solutions of
antibiotics, vitamins, tissue extracts, animal serum,
etc. sterilize heat-sensitive materials, such as some
culture media, enzymes, vaccines, and antibiotic
solutions
HEPA Filter:
High- Efficiency Particulate Air [HEPA] Filter has
made it possible to deliver clean air to an enclosure
such as a cubicle or a room.
This type of air filtration together with a system of
Laminar Airflow is now extensively used to produce
dust and bacteria free air
HEPA filters, by definition, remove at least 99.97% of
airborne particles 0.3 micrometers
(m) in diameter. HEP A filters are composed of a
mat of randomly arranged fibers. The
fibers are typically composed of fiberglass and
possess diameters between 0.5 and 2.0 micron.
Key factors affecting function are fiber diameter,
filter thickness, and face velocity. The air
space between HEP A filter fibers is much greater
than 0.3 m. The common assumption that a HEP A
filter acts like a sieve where particles smaller than
the largest opening can pass through is incorrect.
Unlike membrane filters, where particles as wide as
the largest opening or distance between fibers
cannot pass in between them at all, HEPA filters are
designed to target much smaller pollutants and
particles.
 Laminar flow cabinet
A laminar flow cabinet or laminar flow closet or
tissue culture hood is a carefully enclosed bench
designed to prevent contamination of
semiconductor wafers, biological samples, or any
particle sensitive device. Air is drawn through a HEP
A filter and blown in a very smooth, laminar flow
towards the user. The cabinet is usually made of
stainless steel with no gaps or joints where spores
might collect.
Such hoods exist in both horizontal and vertical
configurations, and there are many different
types of cabinets with a variety of airflow patterns
and acceptable uses. Laminar flow cabinets may
have a UV-C germicidal lamp to sterilize the shell
and contents when not in use. (It is important to
switch this light off during use, as it will quickly give
any exposed skin sunburn and may cause cataracts).
Sonication
Sonication is the act of applying sound energy to
agitate particles in a sample, for various purposes.
In the laboratory, it is usually applied using an
ultrasonic bath or an ultrasonic probe, colloquially
known as a sonicator.
Ultrasonic cleaner is the device which penetrates
the high frequency ultrasound energy deep into the
cracks, holes and the recesses to clean and remove
all the traces of tightly fit germs and greasy
substance and pigments which are
touched to clean in a normal way. It is the
technology that covers the whole range of devices
and cleanses with a challenge.
They use the cavitations process, where the
bubbles are created by the sound waves in order to
clean the surface of the products placed in the tank.
The releasing bubbles act in the form of sponge to
remove the settled dirt on the tattoo
equipment, surgical devices and other items which
need to be sterilized immediately after its use.
Basic Difference between Autoclave and Ultrasonic
Cleaner
In general autoclave is a device which uses high
temperature and heat for cleaning and for getting
rid of dirt, bacteria, viruses, etc. On the other hand
Ultrasonic cleaning devices works by utilizing high
frequency sound waves.
As the process is performed with the use of high
frequency electrical energy, it presents the ability to
clean the tenacious material from the core units.
Professionals sometimes recommend using
ultrasonic cleaners first and then
autoclave for cleaning the same item. This way you
can get the most effective sterilization of the item.
 (B) Chemicals agents Bleach will kill many organisms immediately, but for
full sterilization it should be allowed to
Alcohol react for 20 minutes. Bleach will kill many, but not
all spores. It is highly corrosive and may corrode
Ethyl, isopropyl, trichlorobutanol
even stainless steel surgical instruments. Bleach
Aldehydes decomposes over time when exposed
to air, so fresh solutions should be made daily.
Formaldehyde, glutaraldehyde
Action of chemical agents:
Dyes
Protein coagulation
Halogens
Disruption of cell membrane resulting in
Phenols exposure, damage/loss of contents

Surface active agents Removal of sulfhydryl group essential for

normal functioning of enzyme
Metallic salts
Substate competition.
Antibiotic

Gases: Ethylene oxide, formaldehyde,

beta propiolactone Types of Disinfectants

Phenol: Lister was the first to use phenol (carbolic

acid) to control surgical infections in the operating
Chemicals are also used for sterilization. Although room. It is now rarely used as an antiseptic or
heating provides the most reliable way to rid objects
disinfectant because it irritates the skin and has a
of all transmissible agents, it is not always
disagreeable odor.
appropriate, because it will damage heat-sensitive
materials such as biological materials, fiber optics, Bisphenols: Bisphenols are derivatives of phenol
electronics, and many plastics. Low temperature gas that contain two phenolic groups connected by a
sterilizers function by exposing the articles to be bridge.
sterilized to high concentrations (typically 5 10%
v/v) of very reactive gases (alkylating agents such as Hexachlorophene (pHisoHex, prescription),
ethylene oxide, and oxidizing agents such as hospitals, surgeries, nurseries.
hydrogen peroxide and ozone). Liquid sterilants
and high disinfectants typically include oxidizing Triclosan (toothpaste, antibacerial soaps,
agents such as hydrogen peroxide and peracetic acid etc.)
and aldehydes such as glutaraldehyde and more
recently o-phthalaldehyde. While the use of gas and
liquid chemical sterilants/high level disinfectants
avoids the problem of heat damage, users must
ensure that arti9le to be sterilized is chemically
compatible with the sterilant being used. The
manufacturer of the article can provide specific
information regarding compatible sterilants. In
addition, the use of chemical sterilants poses new
challenges for workplace safety. The chemicals used
as sterilants are designed to destroy a wide range of
pathogens and typically the same properties that
make them good sterilants make them harmful to
humans.

Bleach
Chlorine bleach is another accepted liquid sterilizing
agent. Household bleach consists of 5 .25% sodium
hypochlorite. It is usually diluted to 1/10
immediately before use.
Biguanides
Biguanides have a broad spectrum of
activity, with a mode of action primarily
affecting bacterial cell membranes. They are
especially effective against gram-positive
bacteria.
Biguanides are also effective against gram-
negative bacteria, with the significant
exception of most pseudomonads.
Biguanides are not sporicidal but have some
activity against enveloped viruses.
The best known biguanide is chlorhexidin,
which is frequently used for microbial
control on skin and mucous membranes.
Halogens
Chlorine
Oxidizing agent
Widely used as disinfectant
Forms bleach (hypochlorous acid) when
added to water.
Broad spectrum, not sporicidal (pools,
drinking water)
Iodine
It is active against all kinds of bacteria, many
endospores, various fungi, and some
viruses.
Impairs protein synthesis and alters cell
membranes.
Tincture of iodine (solution with
alcohol) wound antiseptic.
An iodophor is a combination of iodine and
an organic molecule, from which the iodine
is released slowly. lodophors have the
Chemical Food Preservatives
antimicrobial activity of iodine, but they do
not stain and are less irritating.
Alcohols
Two of the most commonly used alcohols
are ethanol and isopropanol
Denature proteins, dissolve lipids
No activity against spores and poorly
effective against viruses and fungi
The recommended optimum concentration
of ethanol is 70%
Heavy Metals
Denature proteins
silver nitrate (topical cream)
Mercury (HgCl2, Greeks & Romans
for skin lesions);
copper sulfate (algicide)
zinc (mouthwash, paints
Surfactants
Surface-active agents, or surfactants, can
decrease surface tension among molecules
of a liquid. Such agents include soaps and
detergents.
Quaternary Ammonium Compounds
(Quats):
Cationic Detergents Attached to NH4+
Their cleansing ability is related to the
positively charged portion of the molecule.
Quaternary ammonium compounds are
strongly bactericidal against gram-positive
bacteria and less active against gram-
negative bacteria.
Quats are also fungicidal, amoebicidal, and
virucidal against enveloped viruses. They do
not kill endospores or mycobacteria.
Probably affects the plasma membranes.
Sulfur dioxide
Sorbic acid
Benzoic acid
Calcium propionate
Sodium nitrate and sodium nitrite
Aldehydes
Inactivate proteins by cross-linking with
functional groups (NH2, OH, COOH, SH).
Formaldehyde gas is an excellent
disinfectant. However, it is more commonly
available as formalin, a 37% aqueous
solution of formaldehyde gas.
Glutaraldehyde is used to disinfect hospital
instruments, including endoscopes and
respiratory therapy equipment, but they
must be carefully cleaned first. When used
in a 2% solution (Cidex), it is bactericidal,
tuberculocidal, and virucidal in 10 minutes
and sporicidal in 3 to 10 hours.
GASEOUS METHOD
The chemically reactive gases such as
formaldehyde, (methanol, H.CHO) and
ethylene oxide (CH2)2O possess biocidal
activity. Ethylene oxide is a colorless,
odorless, and flammable gas.
The mechanism of antimicrobial action of
the two gases is assumed to be through
alkylations of sulphydryl, amino, hydroxyl
and carboxyl groups on proteins and amino
groups of nucleic acids.
The concentration ranges (weight of gas per
unit chamber volume) are usually in range of
800- 1200 mg/L for ethylene oxide and 15-
100 mg/L for formaldehyde with operating
temperatures of 45-63C and 70-75C
respectively.
Both of these gases being alkylating agents
are potentially mutagenic and carcinogenic.
They also produce acute toxicity including
irritation of the skin, conjunctiva and nasal
mucosa
Ethylene oxide (EtO) is both microbicidal
and sporicidal and kills by combining with
cell proteins.
Their application requires a closed chamber
similar to a steam autoclave.
Its activity depends on alkylation, that is,
replacing the proteins labile hydrogen
atoms in a chemical group (such as - SH,-
COOH, or - CH2CH10H) with a chemical
radical. This leads to cross-linking of nucleic
acids and proteins and inhibits vital cellular
functions.
It is toxic and explosive in its pure form, so it
is usually mixed with a nonflammable gas,
such as carbon dioxide.
Among its advantages is that it carries out
sterilization at ambient temperatures and it
is highly penetrating.
Oxidizing Agents
Hydrogen peroxide and other oxidizing agents are
active against bacteria, bacterial spores, viruses, and
fungi at quite low concentrations
Ethylene oxide
Action is due to its alkylating the amino,
carboxyl, hydroxyl and sulphydryl groups in
protein molecules.
Also on DNA and RNA.
Items: heart-lung machines, respirators, sutures,
dental equipment, books, clothing.
Formaldehyde gas:
This is widely employed for fumigation of OT
and other rooms.
Formaldehyde is produced by adding 150g
of KMnO4 to 280ml of formalin for every
1000cu.ft of roomvolume, after closing the
windows and other outlets.
After fumigation, the doors should be sealed
and left unopened for 48 hours.
Betapropiolactone:
Product of ketane and formaldehyde with a
boiling point of 163C.
Having rapid bactericidal activity but
carcinogenic.
Capable of killing all microorganisms and is
very active against viruses.
Surface-active agents:
Substance that alter the energy relationship at
interfaces, producing a reduction of surface or
interfacial tension is called surface active agents.
Metallic salts:

Though all salts have a certain amount of

germicidal action depending on their
concentration,salts of heavy metals have a
greater action.

Eg: salts of silver, copper and mercury

These are Protein coagulants and have capacity to

combine with free sulfhydryl group of cell enzymes.

Cefotaxime/ claforan: Inhibits Gram

positive and Gram negative bacteria

Antibiotics

Nisin is often added to cheese to inhibit the

growth of certain endosporeforming
spoilage bacteria. It is an example of a
bacteriocin , a protein that is produced by o
ne bacterium and inhibits another.

Nisin is present naturally in small amounts in

many dairy products. It is tasteless, readily
digested, and nontoxic. Advantages of Chemical Sterilization:

Natamycin (pimaricin) is an antifungal antibiotic relatively painless (usually used with sedation) and
approved for use in foods, mostly cheese quick procedure
Hygroinycine: Antibiotic produced eliminates risk of complications from anesthesia and
by Streptomyces hygroscopicus. It is surgery
an aminoglycoside that kills bacteria, need for post-procedure care and observation is
fungi and higher eukaryotic cells by minimal
inhibiting protein synthesis considered safe in most cases

Disadvantages of Chemical Sterilization:

may not reduce hormone-related diseases such as

testicular cancer or prostate disease to the same
extent that surgical sterilization may do so
allows for some continued testosterone production
that may not decrease unwanted characteristics
such as roaming, marking, or aggression and fighting
to the same extent that surgical sterilization may do
so
side effects, such as vomiting, loss of appetite,
lethargy, and diarrhea, are possible

Carbenicillin/ geopen: Bactericidal

and bacteriolytic antibiotic belonging
to carboxypenicillin subgroup of the
penicillin
Reference:

1. Plant Tissue Culture : Totipotency to

Transgenic; H.P.Sharma; ISBN8177544675,
9788177544671
2. http://www.drkarthikreddy.com/tag/classifi
cation-of-sterilization/
3. Lab manual
4. http://www.veterinary.texas.gov/Sterilizatio
n.php
5. C :
6. http://ultrasonic-cleaners.org/ultrasonic-
cleaner-vs-autoclave.html