Sterilization

and
Disinfection
 Principles of Sterilization and
Disinfection

• Disinfectant: Typically chemical agents

that are applied to inanimate objects

• Antiseptics: Typically chemical agents

that are applied to living tissues
• Sterilization: The killing or removal of
all living cells, viable spores, viruses in a
material or on an object

• Sterility: there are no living organisms

in or on an object

• Disinfection: The reduction of the

number of pathogenic microorganisms to
the point that they pose no danger of
disease
•Phenol or carbolic acids are standard disinfectant since 1867.
•With phenol other disinfectants are compared under same conditions,
the result of this comparison is called phenol coefficient.
•Coefficient less than one means the disinfectant is less effective
Disinfectant and Antiseptic Response of
Staphylococcus aureus
Mercury
compound
Disinfectant and Antiseptic Response of
Escherichia coli
 Disinfectant selection

• Be fast acting even in the presence of organic substances,

such as those in body fluids.

• Be effective against all types of infectious agents without

destroying tissues or acting as a poison if ingested.

• Easily penetrate material to be disinfected without damaging

or discoloring the materials.

• Be easy to prepare and stable even when exposed to light,

heat or other environmental factors.

• Be inexpensive and easy to obtain and use.

• Not have an unpleasant odour.

Mechanisms of action of chemical agents

•Reactions that affects proteins- with KMnO4, boric acids,

heaving metal, alkyating agents, halogens etc

•Reactions that affects membranes- surfactants (soap

solutions/ detergents)

•Reactions that affect other cell - components-alkyalting

agents, certain dyes (crystal violet), endproducts of fermentations

•Reactions that affect viruses- alkyalting agents e.g.

elthelene oxide, nitrous acid, hydroxyl amine (mutagens), alcohols,
detergents, dyes (acrydine orange-intercalating agents)
 Denaturing Proteins
•Denaturation: Alteration of the functional structure of protein is called denaturation.

•In denaturation process, hydrogen and disulphide bonds are disrupted and functional
shape of the molecule destroyed.

•Denaturation is bacteriocidal when it permanently alters the protein so that the

normal structure of the protein will not be restored.
 Action of a Surfactant
•Surfactants are the substances that dissolve membrane lipids.
•These are soluble compounds that reduce surface tension, just as soap
or detergents breaks up the grease molecule.

•These include alcohols, detergents and quaternary ammonium

compounds (benzalkonium chloride) which dissolve lipids.

The negatively charged

oxygen attracts the positively
charged sites of the water
molecule to make the
insoluble substances soluble
Structural Formulas of Common
Disinfectants
Heavy Metal Effect on Bacterial Growth
•Selenium
•Mercury
•Silver
•Copper
Tincture means the
compounds dissolved in
alcohol to exert greater
germicidal action
Biodecontamination
Unit—Hydrogen
Peroxide
Biodecontamination
Unit—Ethylene
Oxide
 Physical Antimicrobial agents
Principles and applications of heat killing:

•It is the preferred method of sterilization for all the material those are not
damaged by it.

•It rapidly penetrates thick materials not easily penetrated by chemical

agents.

•Thermal death point is the temperature that kills all the bacteria in a 24-
hour-old broth culture at neutral pH in 10 minutes.

•Thermal death time is the time required to kill all the bacteria in a
particular culture at a specified temperature.

•Decimal Reduction Time (DRT) or D-value, is the length of time needed

to kill 90% of the organisms in a given population at a specified
temperature (the temperature is indicated by a subscript: D80º C
Sterilization—Hot Air Oven
Model Autoclave
Small Countertop Autoclave
 Autoclaving
• If water is heated under pressure, its boiling point
is elevated, so temperatures above 100oC can be
reached

• Pressure: 15 lbs per square inch (psi)

• 15 – 20 minutes at 121oC

• Prions are highly resistant and must be sterilized

longer and at higher temperature (134oC for 18
min)
Large Automatic Hospital Autoclave
 Ionizing Radiation
• X rays and gamma rays are forms of ionizing
radiation

• So named because it can dislodge electrons from

atoms creating ions

• Damages DNA and produces peroxides (powerful

oxidizing agents in cells)

• Deinococcus radiodurans: Able to survive 1000X

the amount of radiation that would kill a human
(bioremediation of radioactive contaminated sites)
Checking for Sterility
 Pasteurization
• A process invented by Pasteur to destroy microbes
that caused wine to sour, does not achieve sterility

• Kills pathogens: Salmonella and Mycobacterium

• Milk is pasteurized by heating it to 71.6oC for at least

15 seconds (flash method)

• Milk is pasteurized by heating it to 62.9oC for 30

minutes (holding method)
 Preservation
by Drying
Examples: Peas, beans,
raisin and other fruits

Bacteria such as Treponema

pallidum, which causes
Syphilis, are extremely
sensitive to drying and it dies
immediately on dry surface
Lyophilization—Stoppering Tray Dryer
Freeze drying
Lyophilization—
Manifold Dryer
 The Control of Microbial Growth
• A definite proportion of the organisms die in
a given time interval

• The fewer organisms present, the shorter the

time needed to achieve sterility

• Microorganisms differ in their susceptibility

to antimicrobial agents

• The most susceptible phase for most

organisms is the logarithmic growth phase
• Germicide: An agent capable of killing
pathogens and nonpathogens but not
necessarily endospores

• Bacteriostatic agent: An agent that

inhibits the growth of bacteria

1. Bactericide
2. Bacteriostatic
The Use of Physical Methods in Control
of Microbial Growth
• Heat and other physical agents are
normally used to control microbial
growth and sterilize objects:

1. Heat
2. Low Temperatures
3. Filtration
4. Radiation
 Heat
• One of the most popular ways to destroy
microbes (flame or boiling)

• Moist or Dry heat may be used

• Exposure to boiling water for 10 minutes is

sufficient to destroy vegetative cells and
eukaryotic spores

• Steam sterilization (autoclaving) is necessary to

destroy the bacterial endospore
 Radiation
• Ultraviolet radiation (260nm): is quite lethal in
destroying microbes but it does not penetrate
glass, dirt films, or water

• Ionizing radiation (IR): An excellent sterilizing

agent because of its ability to penetrate deep into
objects

• IR will destroy bacterial endospores and both

prokaryotic and eukaryotic vegetative cells
 UV Radiation—Serratia marcescens

Unexposed

Exposed
Microwave Sterilization
 Filtration

• Can be used to sterilize substances that are

destroyed by heat (drugs, serum, vitamins,
sucrose)

• To separate viruses from bacteria

(manufacture of vaccines)

• To collect microorganisms from air and

water samples (water quality testing)
Filtration Sterilization
Staphylococcus epidermidis on surface of
0.22mm Millipore Membrane Filter