Control of Microorganisms by Physical and
Chemical Agents
What is the meaning of sterilization?
• It is the complete destruction or removal of all living organism including
viruses, bacteria, fungi and their spores.
• Cleaning : is a process intended to remove foreign material, like dust,
dirt, organic materials including microorganism.
• Disinfection is the destruction of infectious organism( not including
bacterial spores) by using chemical ( disinfectants and antiseptics. It
results in reduction of the contaminating organism.
• Disinfectant are toxic and irritant material and are used for control of
microorganism ( on the floors for example)
• Antiseptic are less irritant and less toxic and used for disinfection of
body surfaces ( living tissues).
• Preservation: is the prevention of multiplication of microorganism in the
formulated products including pharmaceuticals and foods.
Figure 8.1-Microbial Control Methods
Definition of Frequently Used Terms in Microbial control
methods
Sterilization
destruction or removal of all viable organisms from an object or habitat.
Disinfection
killing, inhibition, or removal of pathogenic organisms that may cause disease: substantial
reduction of total population.
Disinfectants
agents, usually chemical, used for disinfection; not necessary kills viable spores
Sanitization
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reduction of microbial population to levels deemed safe (based on public health standards) .
Destruction of most microorganisms ( whether or not pathogenic) on wounds , clothing, or
hard surfaces, through the use of chemicals or heat
Antisepsis
prevention of infection or sepsis. of living tissue by microorganisms using antiseptics.
Antiseptics
chemical agents that kill or inhibit growth of microorganisms when applied to tissue-should
not be toxic as disinfectants to kill host tissues.
Antimicrobial Agents
Agents that kill microorganisms or inhibit their growth e.g
Chemotherapy - chemical agent to kill or inhibit growth of
microorganisms within host tissues.
-cidal agents to kill
-static agents to inhibit growth
•cide
suffix indicating that agent kills
•Germicide
kills pathogens and many nonpathogens but not necessarily
endospores
include bactericides, fungicides, algicides, and viricides
•-static
Suffix indicating that agent inhibits growth
include bacteriostatic ,fungistatic,algistatic and virustatic
The Pattern of Microbial Death
• A microbial population is not killed instantly
• population death usually occurs exponentially as growth rate.
• microorganisms were previously considered to be dead when they
did not reproduce in conditions that normally supported their
reproduction
however we now know that organisms can be in a viable but
nonculturable (VBNC) condition .
• once they recover they may regain the ability to reproduce and
cause infection.
• Decimal Reduction Time (D):
• D is the time required to kill 90% of the microorganisms or spores in
a sample under specified conditions.
• Microorganisms is defined as dead when they don’t grow or
reproduce when inoculated in culture medium.
Conditions Influencing the Effectiveness of Antimicrobial
Agent Activity
 Population size:
larger populations take longer to kill than smaller populations
 Population composition:
microorganisms differ markedly in their sensitivity to antimicrobial agents e.g.
Mycobacterium tuberculosis.
Bacterial spores are much more resistant to Microbial agents

Concentration or intensity of an antimicrobial agent:
usually higher concentrations or intensities kill more rapidly
relationship is not linear
 Duration of exposure:
the longer exposure to antimicrobial agents the more organisms killed.
 Temperature:
higher temperatures usually increase amount of killing
 Local environment:
many factors (e.g., pH, viscosity and concentration of organic matter) can profoundly impact
effectiveness
organisms in biofilms are physiologically altered and less susceptible to many antimicrobial
agents. Organic matter in biofilms protects biofilm microorganisms.
The Use of Physical Methods in Control
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Heat
Low temperatures
Filtration
Radiation
Moist Heat Sterilization
• must be carried out above 100oC which
requires saturated steam under pressure
• Moist heat destroys viruses, bacteria and fungi
• carried out using an autoclave (Fig8.3) also
known as Steam Sterilizer
• effective against all types of microorganisms
including spores
• degrades nucleic acids, denatures proteins,
and disrupts membranes
The Autoclave or Steam Sterilizer
Figure 8.3-Autoclave Steam Sterilizer
Table 8.2
Measuring Heat-Killing Efficiency
• Thermal death time (TDT)
shortest time needed to kill all microorganisms in a
suspension at a specific temperature and under
defined conditions
• Decimal reduction time (D or D value)
time required to kill 90% of microorganisms or spores
in a sample at a specific temperature .
 Pasteurization
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controlled heating at temperatures well below boiling- Louis Pasteur
used for milk, beer and other beverages
process does not sterilize but does kill pathogens present and slow spoilage by reducing the
total load of organisms present.
 Dry Heat Sterilization
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less effective than moist heat sterilization, requiring higher temperatures and longer
exposure times
items subjected to 160-170oC for 2 to 3 hours
oxidizes cell constituents and denatures proteins.
 Filtration
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reduces microbial population or sterilizes solutions of heat-sensitive materials by removing
microorganisms
also used to reduce microbial populations in air
Filtering Liquids
 Depth filters
• thick fibrous or granular materials bonded in
thick layers that remove microorganisms by
physical screening(size), entrapment, and/or
adsorption to the surface of the filter materials.
• Solution contain microorganisms are sucked
under vacuum
 Membrane filters
• porous membranes with defined pore sizes that
remove microorganisms primarily by physical
screening. This has replaced Depth Filters.
Figure 8.5-Membrane Filter Sterilisation
Figure 8.6-Membrane Filter
Filtering Air
• surgical masks used in
hospitals and Labs
• cotton plugs on culture
vessels
• high-efficiency
particulate air (HEPA)
filters used in laminar
flow biological safety
cabinets (remove
99.97% of particles)
Figure 8.7 (a)-Laminar Floor
Ultraviolet (UV) Radiation
• UV (260nm) quite lethal
is limited to surface
sterilization because it
does not penetrate glass,
dirt films, water, and
other substances.
• UV prevent replication
and transcription of
Microbial DNA.
• has been used for water
treatment
Figure 7.9
Ionizing Radiation
• Excellent sterilization agent e.g. Gamma
radiation
• penetrates deep into objects
• destroys bacterial endospores; not always
effective against viruses
• used for sterilization and pasteurization of
antibiotics, hormones, sutures, plastic
disposable supplies, and food
Figure 8.8-Sterilization with Ionization; Radiation machine which uses
Cobalt 60 as a Gamma radiation to sterilize fruits, veg, fish, meat, etc..
Chemical Control Agents- Disinfectants and
Antiseptics
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Phenolics
Commonly used as laboratory and hospital disinfectants
Act by denaturing prptein and disrupting of cell membrane
Tuberculosidal, effective in presence of organic material, and long lasting
disagreeable odor and can cause skin irritation
Alcohol
Bactericidal, Fungicidal, but not sporicidal
Inactive some viruses
Denature Proteins and possibly dissolve membrane lipids
Halogens
Any of five elements: fluorine, chlorine, bromine, iodine and astatine
Iodine and chlorine are important antimicrobial agents
Halogens - Iodine
• skin antiseptic
• oxidizes cell constituents and iodinates proteins
• at high concentrations may kill spores
• skin damage, staining, and allergies can be a problem
iodophore
• is a preparation containing iodine complexed with a solubilizing agent
• Diluted iodophor is often used to sanitize equipment and bottles
Halogens - Chlorine
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oxidizes cell constituents
important in disinfection of water supplies and swimming pools, used in dairy and
food industries, effective household disinfectant
destroys vegetative bacteria and fungi, but not spores
can react with organic matter to form carcinogenic compounds
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Heavy Metals
e.g., ions of mercury, silver, arsenic, zinc, and copper
effective but usually toxic
combine with and inactivate proteins; may also precipitate proteins
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Ammonium Compounds
These are detergents that have antimicrobial activity and are effective disinfectants
organic molecules (cleansing agents) with hydrophilic and hydrophobic ends for food utensils, small
instruments and skin antiseptics.
Because of its positively charged nitrogen, cationic detergents/ammonium compound, are effective
disinfectants.
They disrupt microbial membrane , may denature protein.
kill most bacteria, but not Mycobacterium tuberculosis or endospores
safe and easy to use, but inactivated by hard water and soap
E.g Benzalkonium chloride and Cetylpyridinium
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Aldehydes
highly reactive molecules
sporicidal and can be used as chemical sterilants
combine with and inactivate nucleic acids and proteins
E.g Formal dehyde and glutaraldehyde
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Sterilizing Gases
• used to sterilize heat-sensitive materials such as disposable
petri dishes, syringes, heart lung machine components,
sutures, catheters
• microbicidal and sporicidal
• combine with and inactivate proteins
• E.g. Ethylene oxide gas (EtO) :
gas is commonly used to sterilize objects that are sensitive to
temperatures greater than 60 °C and / or radiation such as
plastics. Ethylene oxide treatment is generally carried out
between 30 °C and 60 °C with relative humidity above 30%
and a gas concentration between 200 and 800 mg/l
Chemical in common use are :
1-Alcolhol: kill vegetative bacteria only e.g. 75% ( absolute alcohol is not
effective because water is important for the denaturation of bacterial
protein). It is used for skin disinfection.
2- Phenol 2% used as a disinfectant in microbial laboratories.
3- ethylene oxide gas ; used for the disinfection of plastic and rubber articles.
4-halogenes: kill vegetative bacteria, spores and viruses.
Iodine: used in alcohol for skin antisepsis ( e.g. betadine).
5- Glutaraldehyde 2 % used for the decontamination of endoscopes.
6-hydrogen peroxide used in wound cleaning.
7-formaldehyde gas used as a disinfectant for fumigation of areas
contaminated by infectious agents
8- Quaternary ammonium salts
9- Heavy metals ( e.g. mercury and silver) they cause denaturation of
enzymes and other essential proteins.`
Figure 8.11-Ethylene Oxide Sterilizer
Chemotherapeutic Agents
• chemicals that can be used internally to kill or
inhibit the growth of microbes within host
cells (covered later in book)
• their selective toxicity allows them to target
the microbe without harming the host
• most are antibiotics, chemicals synthesized by
microbes that are effective in controlling the
growth of bacteria
CHEMOTHERAPEUTIC AGENTS
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Definitions:
Antimicrobial agents are substances that inhibit or kill the growth of microorganisms. Antimicrobial agents
that are produced by microorganisms are naturally occurring chemotherapeutics and were defined by
Waxman as antibiotics.
Examples of naturally occurring chemotherapeutic agents are : penicillins and cephalosporins,
chloroamphenicol, tetracyclines, macrolids, nystatin and griseofulvin.
Examples of synthetics chemotherapeutic agents are: sulphonamides, nitrofuran, isonictinc acid derivatives,
diaminopyrimidines, quinolones and imidazoles.
The chemical substance that used as a chemotherapeutic agent must be a selective toxic. By means toxic
for the microorganism and not toxic to the host.
Antibiotics: An antibiotic is a product produced by a microorganism or a similar substance produced wholly
or partially by chemical synthesis, which in low concentrations, inhibits the growth of other
microorganisms.
The minimum inhibitory concentration (MIC): is the lowest concentration of an antibiotic that inhibit the
growth of an organism.
Bactericidal antibiotics: are those antibiotics that destroy bacteria, like penicillins, cephalosporins.
Bacteriostatic antibiotics: are those inhibit the multiplication of the bacterial cells. Like tetracyclines and
chloroamphenicol
Antibacterial spectrum—Range of activity of an Antibiotic combinations—Combinations of antibiotics
antimicrobial against bacteria. A broadthat may be used (1) to broaden the antibacterial
spectrum antibacterial drug can inhibit a
spectrum for empiric therapy or the treatment of
wide variety of gram-positive and grampolymicrobial infections, (2) to prevent the emergence
negative bacteria, whereas a narrowof resistant organisms during therapy, and (3) to achieve
spectrum drug is active only against a
a
synergistic
killing
effect.
limited
variety
of
Antibiotic synergism—Combinations of two antibiotics
bacteria.
Bacteriostatic activity—The level of
• that have enhanced bactericidal activity when tested
antimicrobial activity that inhibits the
together compared with the activity of each antibiotic.
growth of an organism. This is determined
in vitro by testing a standardized
Antibiotic antagonism—Combination of antibiotics in
concentration of organisms against a series
which the activity of one antibiotic interferes With the
of antimicrobial dilutions. The lowest
activity of the other (e.g., the sum of the activity is less
concentration that inhibits the growth of
than the activity of the individual drugs).
the organism is referred to as the minimum
inhibitory concentration (MIC).
Bactericidal
activity—The
level
of
• Beta-lactamase—An enzyme that hydrolyzes the betaantimicrobial activity that kills the test
lactam ring in the beta-lactam class of antibiotics, thus
organism. This is determined in vitro by
inactivating the antibiotic. The enzymes specific for
exposing a standardized concentration of
penicillins and cephalosporins are the penicillinases and
organisms to a series of antimicrobial
cephalosporinases,
respectively.
dilutions. The lowest concentration that kills
99.9% of the population is referred to as the
minimum
bactericidal
concentration
(MBC).
Susceptibility Tests
1. Broth dilution - MIC test
Minimal Inhibitory Concentration (MIC) vs. Minimal
Bactericidal Concentration (MBC)
Susceptibility Tests
3.Agar diffusion
Antibiotic Mechanisms of Action
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