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Control of Microbial growth
Dr. Hala Al Daghistani
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Terminology
 Sepsis: Characterized by the presence of pathogenic microbes in living
tissues or associated fluids.
 Asepsis: absence of significant contamination.
 Aseptic surgery techniques prevent microbial contamination of
wounds.
 Antimicrobial : chemicals, expected to destroy pathogens but not to
achieve sterilization
 Disinfectant: used on objects (reduce the number of viable
microorganisms)
 Antiseptic: used on living tissue, destroys or inhibits the growth of
microorganisms
 Nosocomial Infection(Hospital Acquired Infection) an infection that
is contracted from the environment or staff of a healthcare facility.
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 Sterilization: A defined process used to render a
surface or product free from viable organisms, including
bacterial spores.
 Biocide: A chemical or physical agent, usually broad
spectrum, that inactivates (kill) microorganisms.
Chemical biocides include hydrogen peroxide, alcohols,
bleach, cycloheximide, and phenols
physical biocides include heat and radiation.
Fungicide, Virucide, Germicide, bactericide
 Sanitization: Lowering of microbial counts to prevent
transmission in public setting (e.g., restaurants & public
rest rooms)
 Antibiotics: Naturally occurring and synthetically
derived organic compounds that inhibit or destroy
selective bacteria, generally at low concentrations.
 Bacteriostatic: Inhibits bacterial reproduction
 Bactericidal: Kills bacteria
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Factors affect the antimicrobial treatment
 Time it takes to kill a microbial population is
proportional to number of microbes.
 Microbial species and life cycle phases (e.g.:
endospores) have different susceptibilities to
physical and chemical controls.
 Organic matter may interfere with heat treatments
and chemical control agents.(heat treatment is efficient
at low pH)
 Exposure time: Longer exposure to lower heat
produces same effect as shorter time at higher
heat.
Actions of Microbial Control Agents
 Disruption of the Cell Membrane permeability(damage
protein or lipids of PM)
 Disruption of the cell Wall synthesis(either
by inhibit the
enzyme involved in cell wall synthesis or interfere with CW building block
synthesis). E.g. penicillin interfere with transpeptidase and prevent
the assembly of PG layer.
 Damage to proteins (break down H- bonds, destruction of three
dimensional structure of a protein , inhibition of translation and transcription
of genetic material).
(
 Damage to nucleic acids include ionizing radiations,
ultraviolet light, and DNA-reactive chemicals. Ultraviolet light,
induces cross-linking between adjacent pyrimidines on one or
the other of the two DNA strands, forming thiamine dimers
 Antimetabolite groups(sulfa drugs interfere with folic acid
synthesis). Antimetabolites are substances that interfere with the
normal metabolism of an organism, thereby causing its death.
Evolution of drug resistance:
* spontaneous mutation
* gene transfer
Antibiotic Resistance
 Mechanisms of antibiotic resistance
1. Enzymatic destruction of drug
2. Prevention of penetration of drug
3. Alteration of drug's target site
4. Rapid ejection of the drug
 Resistance genes are often on plasmids or
transposons that can be transferred between bacteria.
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Resistance to Antibiotics
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Fig 20.20
The disk-diffusion method
Antibiotic Susceptibility test
KIRBY-BAUER TEST
DETERMINES SUSCEPTIBILITY OF AN ORGANISM TO
A SERIES OF ANTIBIOTICS OR CHEMICALS
Disk-diffusion method is used in teaching laboratories to
evaluate the efficacy of a chemical agent. A disk of filter
paper is soaked with a chemical and placed on an agar
plate that has been previously inoculated and incubated
with the test organism
After incubation, if the chemical is effective, a clear zone
representing inhibition of growth can be seen around the
disk (Inhibition Zone)
Physical Methods of Microbial Control
 A) Heat (used for media, food, glass wares)
 Two types (moist and dry heat)
 Heat is very effective (fast and cheap).
 Thermal death point (TDP): Lowest temperature at
which all cells in a culture are killed in 10 min.
 Thermal death time (TDT): Min Time to kill all cells
in a culture at a given temp.
 Decimal Reduction Time (DRT):
Time in minutes in which 90% of a bacterial
population are killed at a given Temp.
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 Moist heat
 A temperature of 100°C (Boiling) will kill bacteria, viruses,
and fungi spores, but not spore forms of bacteria within 10
minutes in laboratory-scale cultures.
 a temperature of 121°C, pressure of 15 lb/sq inches for 15
minutes is used to kill spores. Steam is generally used, both
because bacteria are more quickly killed when moist heat are
used, and because steam provides a means for distributing
heat to all parts of the sterilizing vessel. Autoclave: Steam
under pressure, Most dependable sterilization method
 Prions (infectious proteins) need 134°C and NaOH solution
for 4-5 h.
 Moist heat kill M.O. by denaturing of proteins(breakdown
H bonds that hold 3-dimentional structures).
Pasteurization
 Moist heat method
 Significant number reduction (esp. spoilage and
pathogenic organisms)  does not sterilize
 Denaturation of proteins by breaking H-bonds
 Historical goal(LOUIS PASTEUR): destruction of M.
tuberculosis
 Classic pasterization method: 63C for 30 min
 Flash pasteurization (HTST): 72C for 15 sec.
Most common method (milk pasteurization).
Thermoduric(heat resistant) organisms survive
 Ultra High Temperature (UHT):140C for 4 sec.
(liquid is sprayed with high T –steam under
pressure then rapidly cooled).
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Dry heat sterilization
 For sterilizing materials that must remain dry,
circulating hot air electric ovens are available.
because heat is less effective on dry material, we
use to apply a temperature of 160–170°C for 2 h.
 Flaming of loop
 Incineration of carcasses (Anthrax, Foot and mouth disease, Bird flu)
 Hot-air sterilization
 Dry heat kill M.O. by oxidation
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Filtration
 Used for heat sensitive materials(vaccines, antibiotics)
 Air filtration using high efficiency particulate
air (HEPA) filters. Effective to 0.3 m
 Membrane filters for fluids.
 Pore size for bacteria: 0.22 – 0.45 m
 Pore size for viruses: 0.01 m
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Low Temperature
 Refrigeration(0-7C) (reduce metabolic rate and prevent toxin
production).Freezing forms ice crystals that damage microbial
cells
 Deep freezing(-50,-95C) bacteriostatic effect
 Lyophilization( removing water at low T)
 Three methods used for food and drugs)
Various Other Methods

High pressure in liquids: denatures bacterial proteins
and CH2O and inactivation of the cells, preserves flavor

Desiccation prevents metabolism

Osmotic pressure causes plasmolysis (hypertonic
solution). Molds and yeast can grow in high osmotic pressure
environment.
Radiation
Ionizing radiation (X-rays, -rays, electron beams)
- have short wavelength, penetrate deeply, ionize water forming
OH¯ radicals and other highly reactive molecules
- Salmonella and Pseudomonas are particularly sensitive
 Used for plastic syringes, surgical gloves, suturing materials, and
catheters.
Non ionizing radiation ( UV light)
Most effective wave legnth ~ 260 nm, Effect: thymine dimers (inhibit
correct replication)
Used to limit air and surface contamination. Use at close range to directly
exposed microorganisms. e.g.: germicidal lamps in the lab
used for sterilization of heat sensitive materials: drugs,
vitamins, herbs, suture material
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Chemical Methods of Microbial Control

Few chemical agents achieve sterility.

We should considered the followings:
 The presence of organic matter
 The degree of contact with microorganisms
 The temperature
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Types of Disinfectants
 Phenol = carbolic acid
(historic importance)
 Phenolics: Cresols (Lysol)
- disinfectant
 Bisphenols
 Hexachlorophene
 Used in hospitals
Fig 7.7
 Triclosan (toothpaste,
antibacerial soaps, etc.)
Phenol and derivatives disrupt plasma membranes
(lipids) and lipid rich cell walls
Remain active in presence of organic compounds
Halogens
Chlorine




Oxidizing agent
Widely used as disinfectant
Forms bleach (hypochlorous acid) when added to water.
Broad spectrum, not sporicidal (pools, drinking water)
Iodine
More reactive, more germicidal. Alters protein synthesis and
membranes.
Tincture of iodine (solution with alcohol)  wound
antiseptic
Iodophors combined with an organic molecule  iodine
detergent complex. Occasional skin sensitivity, partially
inactivated by organic debris, poor sporicidal activity.
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Alcohols
 Ethyl (60 – 80% solutions)
and isopropyl alcohol
 Denature proteins, dissolve
lipids
 No activity against spores
and poorly effective against
viruses and fungi
 Easily inactivated by
organic debris
 Also used in hand sanitizers
and cosmetics
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Heavy Metals
Oligodynamic action: Oligodynamic action: the ability of very
small amounts of heavy metals to exert antimicrobial activity
toxic effect due to metal ions combining with sulfhydryl (—SH)
and other groups  proteins are denatured.
 Mercury (HgCl2, used for skin lesions)
 Copper against chlorophyll containing organisms 
Algicides
 Silver (AgNO3): Antiseptic for eyes of newborns
 Zinc (ZnCl2) in mouthwashes
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Surface Acting Ingredients / Surfactants
 Soaps and Detergents
 Major purpose of soap: Mechanical removal and use as
wetting agent
 Definition of detergents
 Acidic-Anionic detergents Anion reacts with plasma
membrane. Nontoxic, non-corrosive, and fast acting.
Laundry soap, dairy industry.
 Cationic detergents  Quaternary ammonium
compounds. Strongly bactericidal against a wide range, but
esp. Gram+ bacteria
Soap
Degerming
Acid-anionic detergents
Sanitizing
Quarternary ammonium compounds
(cationic detergents)
Strongly bactericidal, denature
proteins, disrupt plasma membrane
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Aldehydes (alkylating agents)
 Inactivate proteins by cross-linking
with functional groups
(–NH2, –OH, –COOH, –SH)
 Glutaraldehyde: Sterilant for delicate surgical
instruments
Formaldehyde: Virus inactivation, use for vaccines
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Plasma
 The four state of matter in which a gas is excited by
electromagnetic field to make a mixture of nuclei with
assorted electrical charges and free electrons.
 used for metal or plastic surgical instruments (Tubular
instruments).
 Free radicals in plasma gases are used to sterilize plastic
instruments and destroy microbes
Supercritical Fluids
Supercritical fluids, which have properties of liquid and gas,
can sterilize at low temperatures.
Peroxygens and Other Forms of Oxygen
 Hydrogen peroxide, peracetic acid, benzoyl peroxide, and
ozone exert their antimicrobial effect by oxidizing
molecules inside cells.
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Hydrogen Peroxide: Oxidizing agent
Inactivated by catalase 
Not good for open wounds
Good for inanimate objects; packaging
for food industry (containers etc.)
3% solution is used
Effective against anaerobic bacteria
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Microbial
and
Microbial Control
Characteristics
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Copyright © 2010 Pearson Education, Inc.
Copyright © 2010 Pearson Education, Inc.