Sterilization techniques

• Sterilization is the process by which all living cells, viable

spores, viruses, and viroids are either destroyed or
removed from an object or habitat.
• A sterile object is totally free of viable microorganisms,
spores, and other infectious agents.
• When sterilization is achieved by a chemical agent, the
chemical is called a sterilant.
• Disinfection is the killing, inhibition, or removal of
microorganisms that may cause disease. The primary goal is
to destroy potential pathogens, but disinfection also
substantially reduces the total microbial population.
• Disinfectants are agents, usually chemical, used to carry
out disinfection and are normally used only on inanimate
objects.
• Sanitization is closely related to disinfection. In sanitization,
the microbial population is reduced to levels that are
considered safe by public health standards.
• Antisepsis is the prevention of infection or sepsis and is
accomplished with antiseptics.
 Microbial death
• A microbial population is not killed instantly
when exposed to a lethal agent.
• Population death, like population growth, is
generally exponential or logarithmic—that is, the
population will be reduced by the same fraction
at constant intervals .
• If the logarithm of the population number
remaining is plotted against the time of exposure
of the microorganism to the agent, a straight line
plot will result.
• When the population has been greatly reduced,
the rate of killing may slow due to the survival of
a more resistant strain of the microorganism.
Microbial death
 Conditions Influencing the
Effectiveness
of Antimicrobial Agent Activity
• Population size.
• Population composition.
• Concentration or intensity of an antimicrobial
agent.
• Duration of exposure.
• Temperature.
• Local environment.
 Physical methods
• The four most frequently employed physical
agents are heat, low temperatures, filtration,
and radiation.
• Heat:
– Fire and boiling water have been used for
sterilization and disinfection since the time of the
Greeks, and heating is still one of the most
popular ways to destroy microorganisms.
– Either moist or dry heat may be applied.
• Effectiveness was expressed in terms of thermal
death point (TDP), the lowest temperature at
which a microbial suspension is killed in 10
minutes.
• Thermal death time (TDT) is the shortest time
needed to kill all organisms in a microbial
suspension at a specific temperature and under
defined conditions.
• The decimal reduction time is the time required
to kill 90% of the microorganisms or spores in a
sample at a specified temperature.
• The F value is the time in minutes at a specific
temperature needed to kill a population of cells or
spores.
Autoclave
Relation between pressure and
temperature
Effect of size on sterilization
Conditions for moist heat killing
Sterility testing
Hot air oven
Low temperature
Filteration
 Seitz filter
• Made of compressed asbestos fibers
• used in microbiological work to sterilize fluids
such as serum and to filter off bacteria
• permits the passage of viruses.
Membrane filters
Filteration
HEPA
• Radiation
• Desiccation
• Osmotic pressure
• High pressure
 Chemical agents
• The disinfectant must be effective against a wide variety of
infectious agents.
• It should be effective in high dilutions.
• It should be active in the presence of organic matter.
• Although the chemical must be toxic for infectious agents, it
should not be toxic to people.
• It should not be corrosive for common materials.
• The disinfectant should be stable upon storage, odourless or
with a pleasant odour.
• The disinfectant should be soluble in water and lipids for
penetration into microorganisms.
• It should have a low surface tension so that it can enter cracks
in surfaces.
• It should be relatively inexpensive.
 Phenolics

• Phenolics act by denaturing proteins and disrupting cell

membranes.
• Phenolics are tuberculocidal, effective in the presence of
organic material, and remain active on surfaces long after
application.
• They have a disagreeable odor and can cause skin irritation.
 Alcohols

• They act by denaturing proteins and possibly by dissolving

membrane lipids.
 Halogens
• The halogens iodine and chlorine are important antimicrobial
agents.
• Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and iodinating cell proteins. At higher
concentrations, it may even kill some spores.
• Tincture of iodine: 2% or more iodine in a water-ethanol
solution of potassium iodide.
• Iodophors are water soluble, stable, and nonstaining, and
release iodine slowly to minimize skin burns and irritation.
 Chlorine
• Chlorine is the usual disinfectant for municipal water supplies
and swimming pools and is also employed in the dairy and
food industries.
• It may be applied as chlorine gas, sodium hypochlorite, or
calcium hypochlorite, all of which yield hypochlorous acid
(HClO) and then atomic oxygen.
• The result is oxidation of cellular materials and destruction of
vegetative bacteria and fungi, although not spores.
• Chlorine is also an excellent disinfectant for individual use
because it is effective, inexpensive, and easy to employ. Small
quantities of drinking water can be disinfected with halazone
tablets.
• Halazone (parasulfone dichloramidobenzoic acid) slowly
releases chloride when added to water and disinfects it in
about a half hour. It is frequently used by campers lacking
access to uncontaminated drinking water.
 Aldehydes
• Aldehydes are highly reactive molecules that combine with
nucleic acids and proteins and inactivate them, probably by
crosslinking and alkylating molecules.
• They are sporicidal and can be used as chemical sterilants.
Heavy metals
 Silver
• 1% silver nitrate solution is used to treat neanatal opthalmia.
• Silver impregnated dressings are useful against antibiotic
resistant bacteria.
• Plastic food containers infused with silver nanoparticles to keep
food fresh longer.
• Silver infused athletic shirts and socks, which minimize odors.
• Silversulfadiazille is available as a topical cream for use on
burns. Silver can also be incorporated into indwelting catheters,
which are a common source of hospital infections, and in wound
dressings.
• Surfacille is a relatively new antimicrobial for application to
surfaces, either animate or inanimate. It conlains water-insoluble
silver iodide in a polymer carrier and is very persistent, lasting at
least 13 days.
• When a bacterium contacts the surface, the cell's outer membrane
is recognized, and a lethal amount of silver ions are released .
• Mercuric chloride has a very broad spectrum
of activity; its effect is primarily bacteriostatic.
• Its use is now limited because of their toxicity,
corrosiveness, and ineffectiveness in organic
matter.
• Copper sulfate is used chiefly to destroy green
algae (algicide).
 Quaternary Ammonium Compounds
• Quaternary Ammonium Compounds (Quats) The most widely used
surface-active agents are the cationic detergents.
• Their cleansing ability is related to the positively charged portion- the
cation-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.
• Their chemical mode of action is unknown, but they probably affect the
plasma membrane. They change the cell's permeability and cause the loss
of essential cytoplasmic constituents, such as potassium.
• Two popular quats are benzalkonium chIoride, and cetylpyridinillm
cllIoride.
• They are strongly antimicrobial, colorless, odorless, tasteless, stable, easily
diluted, and nontoxic, except at high concentrations.
• Organic matter interferes with their activity, and they are rapidly
neutralized by soaps and anionic detergents.
 Chemical sterilization
• Ethylene oxide is both microbicidal and sporicidal
and kills by combining with cell proteins.
• It is a particularly effective sterilizing agent because
it rapidly penetrates packing materials, even plastic
wraps.
• It is toxic and explosive in its pure form, so it is
usually mixed with a nonflammable gas, such as
carbon dioxide and supplied in concentrations of
10-20%.
• A clean object can be sterilized if treated for 5 to 8
hours at 38°C or 3 to 4 hours at 54°C when the
relative humidity is maintained at 40 to 50% and the
EtO concentration at 700 mg/liter.
• Extensive aeration of the sterilized materials is
necessary to remove residual EtO because it is toxic.
• Betapropiolactone is occasionally employed as a sterilizing
gas. In the liquid form it has been used to sterilize vaccines
and sera.
• BPL decomposes to an inactive form after several hours and is
therefore not as difficult to eliminate as EtO.
• It also destroys microorganisms more readily than ethylene
oxide but does not penetrate materials well and may be
carcinogenic.
• For these reasons, BPL has not been used as extensively as
EtO.
Chemical agents
Antibiotics