Control of Microorganisms by

Physical and Chemical Agents

Control of Microorganisms
1. To destroy pathogens and prevent
their transmission
2.
To
reduce
or
eliminate
microorganisms responsible for the
contamination of water, food, and
other substances
Define the following key terms related
to microbial control : sterilization,
sterilant, disinfection, disinfectant,
sanitization, antisepsis, antiseptic,
germicide, bactericide, bacteriostatic

The Terminology of
Microbial Control
Sepsis refers to microbial
contamination
Asepsis is the absence of significant
contamination
Aseptic surgery techniques prevent
microbial contamination of wounds

The Terminology of
Microbial Control
Sterilization: Removing all microbial life.
All living cells, viable spores, and viruses
destroyed or removed. When sterilization is
achieved by a chemical agent, the
chemical is called a sterilant.
(Commercial sterilization: Killing C.
botulinum endospores).
Disinfection: Removing pathogens. Killing,
inhibition, or removal of microorganisms
that may cause disease.

The Terminology of
Microbial Control
Sanitization: reduced of microbial
population to levels that are considered
safe by public health standard.
Antisepsis: Removing pathogens from
living tissue with chemical agents.
Biocide/germicide: Kills microbes.
Germicide kills pathogens but not
necessarily endospores.
Bacteriostasis: Inhibiting, not killing
microbes, but prevent growth.

A Microbial Death Curve

Population Death Rate Is

Constant

Example: Microbial Heat-Killing experiment

Factors that influence action of

antimicrobial agents
1. Number of microbes
2. Nature of microbes in the
population
3. Temperature & pH of environment
4. Concentration or dosage of agent
5. Mode of action of the agent
6. Presence of solvents, organic
matter, or inhibitors
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Cellular targets of
control
1. Cell wall
2. Cell membrane
3. Cellular synthetic processes (DNA,
RNA)
4. Proteins

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Methods of Physical
Control
1.
2.
3.
4.
5.

Heat
Cold temperatures
Desiccation
Radiation
Filtration

15

1. 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 temperature

1. Heat (Cont)
Moist heat use of hot water or steam
Mode of action denaturation of proteins,
destruction of membranes & DNA
Sterilization
Autoclave 15 psi/121oC/10-40min
Disinfection
Pasteurization <100oC: kills Salmonella,
Listeria and overall microbe count
Boiling at 100oC for 30 minutes to destroy nonspore-forming pathogens
Ultra High Temperature (UHT):
140C for < 1 sec.
Technically not pasteurization because it
sterilizes.
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Dry heat using higher temperatures

than moist heat, can also sterilize
dry ovens 150-180oC- coagulate
proteins

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2. Cold temperatures
Microbistatic : slows the growth of
microbes
Refrigeration 0-15oC & freezing <0oC
Used to preserve food, media and
cultures

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3. Desiccation
Gradual removal of water from cells,
leads to metabolic inhibition.
Not effective microbial control
many cells retain ability to grow
when water is reintroduced.

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4. Radiation
1. Ionizing radiation deep penetrating
power, breaks DNA,
gamma rays, X-rays, cathode rays
used to sterilize medical supplies & food
products

2. Nonionizing radiation little

penetrating power to sterilize air,
water & solid surfaces
uv light creates thymine pyrmidines,
which interfere with replication

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Ionizing Radiation
X-rays, -rays, electron beams
dislodge e- from atoms 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
Also used as cold pasteurization of food
Consumer fears!?

Nonionizing Radiation: UV light

Most effective wave legnth
~ 260 nm
Effect: thymine dimers
Actively dividing organisms are more sensitive
because thymine dimers cause . . . .?
Used to limit air and surface contamination.
Use at close range to directly exposed
microorganisms
E.g.: germicidal lamps in OR, cafeteria, and our lab
??

Nonionizing Radiation: Microwave

Wavelength: 1 mm 1m
H2O quickly absorbs energy
release as heat to
environment
Indirect killing of bacteria through
heat
7.5
FigSolid
food heats unevenly, why?

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5. Filtration
physical removal of microbes by
passing a gas or liquid through filter
used to sterilize heat sensitive liquids
& air in hospital isolation units &
industrial clean rooms

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Filtration

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Filtration
HEPA
removes
microbes
>0.3 m
Membra
ne
filtratio
n
removes
microbes
>0.22

Figure 7.4

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

Physical Methods of Microbial

Control
Learning Objectives

Compare the effectiveness of moist

heat (boiling, autoclaving,
pasteurization) and dry heat.
Describe how filtration, low
temperatures, high pressure,
desiccation, and osmotic pressure
suppress microbial growth.
Explain how radiation kills cells.

Chemical Methods of Microbial Control

Few chemical agents achieve sterility.

Consider presence of organic matter, degree of

contact with microorganisms, and temperature

Disinfectants regulated by EPA

Antiseptics regulated by FDA

Use-dilution test
1. Metal rings dipped in test bacteria are dried.
2. Dried cultures of S. choleraesuis, S. aureus,
and P. aeruginosa are placed in disinfectant
for 10 min at 20C.
3. Rings are transferred to culture media to
determine whether bacteria survived
treatment.

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

Chemical control
1.
2.
3.
4.
5.
6.
7.
8.

Halogens
Phenolics
Chlorhexidine
Alcohols
Hydrogen peroxide
Detergents & soaps
Heavy metals
Aldehydes
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Types of Disinfectants
Phenol = carbolic
acid (historic
importance)
Phenolics: Cresols
(Lysol) - disinfectant
Bisphenols
Hexachlorophene
(pHisoHex,
prescription), 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 compoundsP

1. Halogens
Chlorine Cl2, hypochlorites (chlorine
bleach), chloramines
Denaturation of proteins by disrupting disulfide
bonds
Can be sporicidal

Iodine - I2, iodophors (betadine)

Denature proteins
Can be sporicidal
Milder medical & dental degerming agents,
disinfectants, ointments

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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.

2. Phenolics
Disrupt cell membranes &
precipitating proteins; bactericidal,
fungicidal, virucidal, not sporicidal
Lysol
triclosan- antibacterial additive to soaps

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3. Chlorhexidine
Hibiclens, Hibitane
A surfactant & protein denaturant
with broad microbicidal properties
Not sporicidal
Used as skin degerming agents for
preoperative scrubs, skin cleaning &
burns

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4. Alcohols
Ethyl, isopropyl in solutions of 5090%
Act as surfactants dissolving
membrane lipids and coagulating
proteins of vegetative bacterial cells
and fungi
Not sporicidal

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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

Table 7.6

5. Hydrogen peroxide
Weak (3%) to strong (25%)
Produce highly reactive hydroxyl-free
radicals that damage protein & DNA
while also decomposing to O2 gas
toxic to anaerobes
Strong solutions are sporicidal

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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 (higher conc. available)
Esp. effective against anaerobic
bacteria (e.g.:
Effervescent action, may be
useful for wound cleansing
through removal of tissue
debris

6. Detergents & soaps

Quaternary ammonia cpds act as
surfactants that alter membrane
permeability of some bacteria &
fungi
Not sporicidal

Soaps- mechanically remove soil and

grease containing microbes

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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, noncorrosive, and fast acting. Laundry soap,
dairy industry.
Cationic detergents Quarternary
ammonium compounds (Quats). Strongly
against wide range, but
Soap bactericidal against Degerming
esp. Gram+ bacteriaSanitizing
Acid-anionic detergents
Quarternary ammonium
compounds
(cationic detergents)

Strongly bactericidal,
denature proteins, disrupt
plasma membrane

7. Heavy metals
Solutions of silver & mercury kill
vegetative cells in low concentrations
by inactivating proteins
Oligodynamic action
Not sporicidal

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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

8. Aldehydes
Glutaraldehyde & formaldehyde kill
by alkylating protein & DNA
glutaraldehyde in 2% solution (Cidex)
used as sterilant for heat sensitive
instruments
formaldehyde - disinfectant,
preservative, toxicity limits use

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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 sensive material

Denature proteins
Ethylene oxide

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)

Plasma
Luminous gas with free radicals that
destroy microbes
Use: Tubular instruments, hands, etc.

Microbial
Characteristics and
Microbial Control

Fig 7.11

the end

Gases & aerosols

Ethylene oxide, propylene oxide,
betapropiolactone & chlorine dioxide
Strong alkylating agents, sporicidal

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