Control of Microbial growth

Copyright © 2010 Pearson Education, Inc.

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.

Copyright © 2010 Pearson Education, Inc.

 Sterilization: A defined process used to render a surface or
product free from viable organisms, including bacterial spores.
(Removal of all microbial life)
 Biocide: A chemical or physical agent, usually broad
spectrum, that inactivates microorganisms.
Chemical biocides include hydrogen peroxide, alcohols, bleach,
cycloheximide, and phenols
physical biocides include heat, filtration and radiation.
Fungicide, Sporicide, Germicide.

 Degerming:Removal of microbes from a limited area

 Sanitization: reduces microbial numbers to safe
levels (e.g.: eating utensils)
 Bacteriostatic: Inhibits bacterial reproduction
 Bactericidal: Kills bacteria
Copyright © 2010 Pearson Education, Inc.
 Preservation: The prevention of multiplication of M.O. in
formulated products, including pharmaceuticals and foods.

 Antibiotics: Naturally occurring and synthetically derived

chemical compounds that inhibit or destroy selective bacteria,
generally at low concentrations.

Copyright © 2010 Pearson Education, Inc.

 Effectiveness of Antimicrobial Treatment
Depends on
 Time it takes to kill a microbial population is
proportional to number of microbes.
 Microbial species and life cycle phases (Microbial
characteristics) (e.g.: endospores) have different
susceptibilities to physical and chemical controls.

 Organic matter may interfere with heat treatments

and chemical control agents.
 Exposure time: Longer exposure to lower heat
produces same effect as shorter time at higher
heat.
Copyright © 2010 Pearson Education, Inc.
Actions of Microbial Control Agents

 Disruption of the Cell Membrane

 Disruption of the cell Wall
 Damage to proteins (disruption of the tertiary structure of
a protein or protein denaturation)
 Damage to nucleic acids (include ionizing radiations,
ultraviolet light, and DNA-reactive chemicals (e.g. alkylating
agents and other compounds that react covalently with purine
and pyrimidine bases). Ultraviolet light, induces cross-linking
between adjacent pyrimidines on one or the other of the two
polynucleotide strands, forming pyrimidine dimers (T- Dimer)

Copyright © 2010 Pearson Education, Inc.

 Disruption of Free Sulfhydryl Groups
Enzymes and coenzyme containing cysteine have side
chains terminating in sulfhydryl groups. Such enzymes and
coenzymes cannot function unless the sulfhydryl groups
remain free and reduced. Oxidizing agents and heavy metals
do widespread damage.
 Chemical Antagonism
The interference by a chemical agent with the normal reaction
between a specific enzyme and its substrate is known as
chemical antagonism.
The antagonist acts by combining with some part of the
holoenzyme (the protein apoenzyme, the mineral activator, or
the coenzyme), thereby preventing attachment of the normal
substrate.
Copyright © 2010 Pearson Education, Inc.
 Physical Methods of Microbial Control
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): Time to kill all cells in a
culture
 Decimal Reduction Time (DRT):
Minutes to kill 90% of a population at a given temp.

Copyright © 2010 Pearson Education, Inc.

 A temperature of 100°C (boiling) will kill all but not
spore forms of bacteria within 2–3 minutes in
laboratory-scale cultures.
 a temperature of 121°C, pressure of 15 lb/sq inches
for 15 minutes is used to kill spores (autoclaving).
Steam is generally used, both because bacteria are
more quickly killed when moist and because steam
provides a means for distributing heat to all parts of
the sterilizing vessel.

Copyright © 2010 Pearson Education, Inc.

 For sterilizing materials that must remain dry,
circulating hot air electric ovens are available.
because heat is less effective on dry material, it is
customary to apply a temperature of 160–170°C for
1 hour or more.

 Under these conditions ( excessive temperatures

applied for long periods of time), heat acts by
denaturing cell proteins and nucleic acids and by
disrupting cell membranes.

Copyright © 2010 Pearson Education, Inc.

 Moist Heat Sterilization
 Denatures proteins
 Autoclave: Steam under pressure, Most dependable
sterilization method
 Steam must directly contact material to be sterilized.
 All microorganisms even spore forming bacteria are
killed at 121 C for 15 min.
 Prion destruction:
132C for 4.5 hours

Copyright © 2010 Pearson Education, Inc.

 Pasteurization
 Significant number reduction (esp. spoilage and
pathogenic organisms)  does not sterilize!
 Historical goal: destruction of M. tuberculosis
 Classic holding method: 63C for 30 min
 High temperature, short time (HTST): 72C for
15 sec. Most common method.
Thermoduric organisms survive
 Ultra High Temperature (UHT):
140C for < 1 sec.
Technically not pasteurization because it
sterilizes.

Copyright © 2010 Pearson Education, Inc.

 Tyndalyzation
(intermittent sterilization)
80°C, 1h, 3 days
For spore formers

Copyright © 2010 Pearson Education, Inc.

Dry heat sterilization kills by oxidation
 Flaming of loop
 Incineration of carcasses
 Anthrax
 Foot and mouth disease
 Bird flu

 Hot-air sterilization

Hot-air Autoclave
Equivalent 121˚C, 15
170˚C, 2 hr
treatments min
Copyright © 2010 Pearson Education, Inc.
 Filtration
 Air filtration using high efficiency particulate
air (HEPA) filters. Effective to 0.3 m
 Membrane filters for fluids.
 Pore size for bacteria: 0.2 – 0.4 m
 Pore size for viruses: 0.01 m

Fig 7.4

Copyright © 2010 Pearson Education, Inc.

 Low Temperature
 Slows enzymatic reactions  inhibits microbial growth
 Freezing forms ice crystals that damage microbial cells
 lyophilization

Various Other Methods

 High pressure in liquids denatures bacterial proteins
and preserves flavor
 Desiccation prevents metabolism
 Osmotic pressure causes plasmolysis
Copyright © 2010 Pearson Education, Inc.
 Ionizing Radiation
 X-rays, -rays, electron beams 
production of free radicals and other highly
reactive molecules
 Commonly used Cobalt-60 radioisotope
 Salmonella and Pseudomonas are particularly
sensitive
 Sterilization of heat sensitive materials: drugs,
vitamins, herbs, suture material

Copyright © 2010 Pearson Education, Inc.

Nonionizing Radiation: UV light

 Most effective wave legnth

~ 260 nm
 Effect: thymine dimers
 Actively dividing organisms are more sensitive
because thymine dimers interfere with the
replication
 Used to limit air and surface contamination. Use at
close range to directly exposed microorganisms
 E.g.: germicidal lamps in our lab
Copyright © 2010 Pearson Education, Inc.
 Chemical Methods of Microbial Control

 Few chemical agents achieve sterility.

 Consider presence of organic matter, degree of contact
with microorganisms, and temperature

Copyright © 2010 Pearson Education, Inc.

 Disk-diffusion Method
Disk of filter paper is soaked with a chemical and
placed on an inoculated agar plate; a zone of
inhibition indicates effectiveness.

Fig 7.6

Copyright © 2010 Pearson Education, Inc.

 Types of Disinfectants
 Phenol = carbolic acid
(historic importance)
 Phenolics: Cresols (Lysol)
- disinfectant
 Bisphenols
 Hexachlorophene hospitals,
surgeries, nurseries
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
Copyright © 2010 Pearson Education, Inc.
 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 (e.g. Betadine®). Occasional skin
sensitivity, partially inactivated by organic debris, poor
sporicidal activity.
Copyright © 2010 Pearson Education, Inc.
 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
Copyright © 2010 Pearson Education, Inc.
Table 7.6
Heavy Metals

Oligodynamic action: toxic effect due to metal ions

combining with sulfhydryl (—SH) and other groups
 proteins are denatured.
 Mercury (HgCl2, Greeks & Romans
for skin lesions); Thimerosal
 Copper against chlorophyll containing organisms
 Algicides
 Silver (AgNO3): Antiseptic for eyes of newborns
 Zinc (ZnCl2) in mouthwashes, ZnO in antifungal in
paint
Copyright © 2010 Pearson Education, Inc.
 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  Quarternary ammonium
compounds (Quats). Strongly bactericidal against against
wide range, but esp. Gram+ bacteria

Soap Degerming
Acid-anionic detergents Sanitizing
Quarternary ammonium compounds Strongly bactericidal, denature
(cationic detergents) proteins, disrupt plasma membrane
Copyright © 2010 Pearson Education, Inc.
 Chemical Food Preservatives
Sulfur dioxide
wine
Organic acids
Inhibit metabolism
Sorbic acid, benzoic acid, and calcium propionate
Control molds and bacteria in foods and cosmetics
Sodium nitrate and nitrite prevents endospore germination.
In meats. Conversion to nitrosamine (carcinogenic)

Copyright © 2010 Pearson Education, Inc.

 Aldehydes and Chemical Sterilants
Aldehydes (alkylating agents)
 Inactivate proteins by cross-linking
with functional groups
(–NH2, –OH, –COOH, –SH)
 Glutaraldehyde: Sterilant for
delicate surgical instruments
(Kills S. aureus in 5,
M. tuberculosis in 10 min)
 Formaldehyde: Virus inactivation
for vaccines
Chemical Sterilants for heat sensitive material
 Gaseous sterilants
 Denature proteins
 Ethylene oxide
Copyright © 2010 Pearson Education, Inc.
Plasma
 Luminous gas with free radicals that destroy
microbes
 Use: Tubular instruments, hands, etc.

Copyright © 2010 Pearson Education, Inc.

 Hydrogen Peroxide: Oxidizing agent
Inactivated by catalase 
Not good for open wounds
Good for inanimate objects; packaging for
food industry (containers etc.)
3% solution
Esp. effective against anaerobic
bacteria
Effervescent action (is the escape of
gas from an aqueous solution), may
be useful for wound cleansing
through removal of tissue debris
Copyright © 2010 Pearson Education, Inc.
Microbial
Characteristics and
Microbial Control
Fig 7.11

Copyright © 2010 Pearson Education, Inc.