Sterilization

Definition
Sterilization (or sterilisation) is the elimination of all transmissible
agents (such as bacteria, prions and viruses) from a surface, a piece of
equipment, food or biological culture medium. This is different from
disinfection, where only (comparatively larger) microorganisms are
removed by a disinfectant.
Wash and Sterilization
The Department maintains an autoclave on one of
the research floors and a Wash-Sterilization
Facility with two part-time staff for large
volumes of glassware used for Teaching.
Arrangements can be made for washing and
sterilization of glassware for research.
Explanation
In general, any instrument that enters an already sterile part of the
body (such as the blood, or beneath the skin) should have a high sterility
assurance level. This includes equipment like scalpels, hypodermic needles
and artificial pacemakers. This is also essential in the manufacture of
many sterile pharmaceuticals.
Heat sterilization is known to have been in used in Ancient Rome, but it
mostly disappeared throughout the Middle Ages where sanitation was not
usually a concern.

Sterilization can be achieved through heat, chemicals, radiation, or

filtration.

Front-loading autoclaves are very common.

Heat sterilization
Autoclaves
A widely-used method for heat sterilization is the autoclave. Autoclaves
commonly use steam heated to 121C (250F), at 103 kPa (15 psi) above
atmospheric pressure, for 15 minutes. The steam and pressure transfer
sufficient heat into organisms to kill them.
Proper autoclave treatment will inactivate all fungi, bacteria, viruses and
also bacterial spores, which can be quite resistant. It will not necessarily
eliminate all prions (discussed later).
To ensure the autoclaving process was able to cause sterilization, most
autoclaves have meters and charts that record or display pertinent
information such as temperature and pressure as a function of times.
Indicator tape is often taped onto packages of products to be
autoclaved. The tape contains a chemical that will change color when the
appropriate conditions have been met. Some types of packaging have
built-in indicators on them.

Biological indicators ("bioindicators") can also be used to independently

confirm autoclave performance. Several simple bioindicator devices are
commercially available based on microbial spores. Most contain pure
strains of the heat resistant microbe Bacillus stearothermophilus which
are among the toughest organisms an autoclave will have to destroy.
Several of these devices have a self-contained growth medium (with or
separate to the spores) and a growth indicator.
After a run in an autoclave, the internal glass ampule in the biological
indicator vial is shattered, allowing the spores into a differential liquid
medium. The vial is then incubated (typically at at 56C (132F)) for a
certain period . If the autoclave destroyed the spores, the medium will
remain its original color. If autoclaving was unsuccessful the B.

sterothermophilus will metabolize during incubation, causing a color

change during the prescribed period of incubation.
Bathing
Likewise, no shower or bath you can take, no matter how hot, sterilizes
bacteria on your skin. You would die from being scalded long before the
water was actually hot enough to do anything but increase the rate of
bacterial growth (again, ignoring the effects of any detergents or soaps).
Most hot tap water is between 43 and 49C (110 and 120F), though some
people set theirs as high as 55C (130F), all of which is considered a
prime range for increasing bacterial growth, rather than sterilization,
and most human beings shower in water considerably cooler, though it
feels scalding hot to them. Humans find water painful at a mere 41 to
42C (106 to 108F), which to many bacteria is just starting to get warm

enough for them to grow quickly; they will keep growing faster, rather
than being killed, up to 55C (130F) or more.
Other Methods
Other heat methods include flaming, incineration, boiling, tindalization,
and using dry heat.
Flaming is done to loops and straight-wires in microbiology labs. Leaving
the loop in a Bunsen burner flame 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 (see Incineration below).
Incineration will also burn any organism to ash. It is used to sanitize
medical and other biohazardous waste before its ash goes to the tip.
Boiling in water for 15 minutes will kill most bacteria and viruses, but
boiling is ineffective against prions and many bacterial spores; therefore
boiling is unsuitable for sterilization. However, since boiling does kill most
bacteria and viruses, it is useful if no better method is available. Boiling
is a simple and familiar enough process, and is an option available to most
anyone most anywhere, requiring only water, enough heat, and a container
that can withstand the heat; however, boiling can be hazardous and
cumbersome.
Tindalization is a cumbersome process designed to reduce the level of
activity of sporolating bacteria that are left by a simple boiling-in-water
method. The process involves boiling for 20 minutes, cooling, incubating
for a day, boiling for 20 minutes, cooling, incubating for a day, boiling for
20 minutes, cooling, incubating for a day, and finally boiling for 20
minutes again. The three incubation periods are to allow spores formed

by bacteria in the previous boiling period to produce the heat-sensitive

bacterial stage, which are killed by the next boiling step. ineffective.
Dry heat can be used to sterilize items, but as the heat takes much
longer to be transferred to the organism, both the time and the
temperature must usually be increased, unless forced ventilation of the
hot air is used. The standard setting for a hot air oven is at least two
hours at 160C (320F). A rapid method heats air to 190C (374F) for 6
minutes for unwrapped objects and 12 minutes for wrapped objects [1]
[2]. Dry heat has the advantage that it can be used on powders and other
heat-stable items that are adversely affected by steam (for instance, it
does not cause rusting of steel objects).
For prion elimination, various recommendations state 121132C(270F)
for 60 minutes or 134C (273F) for at least 18 minutes. The prion that
causes the disease scrapie (strain 263K) is inactivated relatively quickly
by such sterilization procedures; however, other strains of scrapie, as
well as strains of CJD and BSE have shown much more resistance. Using
mice as test animals, one experiment showed that heating BSE positive
brain tissue at 134-138C (273-280F) for 18 minutes resulted in only a
2.5 log decrease in prion infectivity. (The initial BSE concentration in the
tissue was relatively low). To have a significant margin of safety, cleaning
should reduce infectivity 4 logs, and the sterilization method should
reduce it a further 5 logs.
By combining immersion in sodium hydroxide (NaOH 0.09N) for two hours
with one hour autoclaving (121C / 250F), several investigators have
shown complete (>7.4 logs) inactivation. (Note that sodium hydroxide may

corrode surgical instruments, especially if the sodium hydroxide

immersion and autoclaving steps are combined.)
Chemical sterilization
Chemicals are also used for sterilization. Although heating provides the
most effective way to rid an object of all transmissible agents, it is not
always appropriate, because it destroys objects such as most fiber
optics, most electronics, and some plastics.
Ethylene oxide (EO) gas is commonly used to sterilize objects that
cannot survive temperatures greater than 60C such as plastics, optics
and electrics. Ethylene oxide treatment is generally carried out between
30C and 60C with relative humidity above 30% and a gas concentration
between 200mg/l and 800mg/l for at least 3 hours. Ethylene oxide
penetrates very well, moving through paper, cloth, and some plastic films
and is highly effective. Ethylene oxide however is highly flammable, and
requires a longer time to sterilize than any heat treatment. The process
also requires time for aeration post sterilization to remove toxic
residues. Ethylene oxide is widely used and sterilizes around 50% of all
disposable medical devices.
A rapid biological indicator is available for use in EO sterilizers. This
indicator contains Bacillus subtilis, which is a very resistant organism. If
sterilization fails, incubation at 37C will cause a fluorescent change
within four hours, which is read by an auto-reader. After 96 hours, a
visible color change will occur. The fluorescence is emitted when a
particular (EO resistant) enzyme is present, which means that spores are
still active. The color change is brought on by a pH shift due to bacterial

metabolism. The test is suitable for most types of ethylene oxide cycles.
The rapid results mean that if a cycle was found to be ineffective, the
objects treated can be quarantined and physicians quickly advised of
possible contamination.
Ozone is used in industrial settings to sterilize water and air, as well as a
disinfectant for surfaces. It has the benefit of being able to oxidize
most organic matter. On the other hand, it is a toxic and unstable gas
that must be produced on-site, so it is not practical to use in many
settings.
Bleach is another accepted liquid sterilizing agent. Household bleach, also
used in hospitals and biological research laboratories, consists of 5.25%
sodium hypochlorite. At this concentration it is most stable for storage,
but not most active. According to the Beth Israel Deaconess Medical
Center Biosafety Manual (2004 edition), in most cases, it should be
diluted to 1/10 of its storage concentration immediately before use;
however, it should be diluted only to 1/5 of the storage concentration to
kill Mycobacterium tuberculosis. This dilution factor must take into
account the volume of any liquid waste that it is being used to sterilize.
Bleach will kill many organisms immediately, but should be allowed to
incubate for 20 minutes for full sterilization. Bleach will kill many spores,
but is ineffective against certain extremely resistant spores. It is highly
corrosive, even causing rust of stainless steel surgical implements.
The Dry Sterilization Process, DSP, is a process originally designed for
the sterilization of plastic bottles in the beverage industry. It uses
hydrogen peroxide with a concentration of 30-35% and runs under

vacuum conditions. Using the common reference germs for hydrogen

peroxide sterilization processes, endospores of different strains of
bacillus subtilis and bacillus stearothermophilus, the Dry Sterilization
Process achieves a germ reduction of 106...108. The complete cycle time
of the process is 6 seconds. The surface temperature of the sterilized
items is only slightly increased during the process by 10-15. Particularly
due to the high germ reduction and the slight temperature increase the
Dry Sterilization Process is also useful for medical and pharmaceutical
applications.
A similar chemical used to sterilize instruments is peracetic acid (0.2%),
which is used in the Steris system.
Prions show a great deal of resistance to these sterilization chemicals.
Hydrogen peroxide (3%) for one hour was shown to be ineffective in that
it had a less than 3 log reduction. Iodine, formaldehyde, glutaraldehyde
and peracetic acid also fail this test (one hour treatment). Only chlorine,
a phenolic compound, guanidinium thiocyanate, and sodium hydroxide
(NaOH) reduce the titre by more than 4 logs.
Chlorine and NaOH are the most consistent of these. Chlorine is too
corrosive to use on certain objects. Sodium hydroxide has had many
studies showing its effectiveness.
In all, the chemical antiseptics do not work well against prions. An
interesting note is that treatment with aldehydes (e.g., formaldehyde)
have been shown to increase prion resistance.

Radiation sterilization
Methods exist to sterilize using radiation such as X-rays, gamma rays, or
subatomic particles. Gamma rays are very penetrating, but as a result
require bulky shielding for the safety of the operators of the gamma
irradiation facility; they also require storage of a radioisotope, which
continuously emits gamma rays (it cannot be turned off, and therefore
always presents a hazard in the area of the facility). X-rays are less
penetrating and tend to require longer exposure times, but require less
shielding, and are generated by an X-ray machine that can be turned off
for servicing. Subatomic particles may be more or less penetrating, and
may be generated by a radioisotope or a device, depending upon the type
of particle. 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 their 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.
Ultraviolet light (UV, from a germicidal lamp) can also be used for
irradiation, but only on surfaces and some transparent objects (note that
many objects that are transparent to visible light actually absorb UV). It
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, as can be

seen if one forgets a polystyrene foam object in the cabinet with the
germicidal lamp turned on overnight.
Sterile filtration
If the substance to be sterilized is sensitive and would be damaged by
heat, radiation or chemical sterilization, it can be sterilized by
mechanical filtration. This method is for example used for protein
solutions in biological research. 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 nm is needed.
Steam Sterilization
Steam Sterilization; Decontamination of Spices, Herbs, Seeds and other
Organic Materials The Ventilex Continuous Steam Sterilizing System is
most successfully used for the H.T.S.T. (High Temperature / Short
Time) decontamination of spices, herbs and seeds.
Ventilex purchased the rights to market this system from Stork in the
mid 1990's. Stork had previously supplied these systems utilizing the
Ventilex Fluid Bed Dryer/Cooler along with air filters and dust
separators and the control cabinet.
The process is simple: Expose the contaminated material to saturated
steam for a very short time, then dry and cool the product. While the
idea is simple, the practicality of making a continuous system that can
treat powders next to whole spices is unique and makes the system
applicable for a large range of products.

As you can see from the above diagram, the process starts at the self
cleaning Rotary Pressure Valve for product feed into a continuous
autoclave. These valves are really "key" to a successful system. You must
be able to subject the product to saturated steam of relatively high
pressure in order to decontaminate the product adequately. The Rotary
Steam Valves are very heavy duty, and have the unique feature - they
have a self cleaning knife that scrapes the pocket of the rotary valve
each time it is exposed. This assures that no matter what product you
process, that clogging and sticking do not occur.

Cross Section of Rotary Valve

Rotary Steam Valve - High Pressure

Once the product is through the valve, it is exposed to steam, where
temperature and pressure take over for decontamination and in some
cases, sterilization. The product being processed must be exposed to the
temperature and pressure for a determined amount of time for "kills" to
occur. This is decided by the product being processed. Ventilex uses a
self cleaning "shaking" conveyor (patented) in the continuous pressure
vessel to transport the processed product in an exact time. This is
achieved by plug flow and is variable by the frequency of the shaking.
Ventilex supplies a VFD controller with every system as part of a
comprehensive control system, and can automate the controls with a
complete PLC or use the customer's existing DCS.