Lecture PowerPoint to accompany

Foundations in
Microbiology
Eighth Edition

Talaro
Chapter 11
Physical and Chemical
Agents for Microbial
Control
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 Controlling Microorganisms
• Physical, chemical, and mechanical methods to
destroy or reduce undesirable microbes in a given
area
• Primary targets are microorganisms capable of
causing infection or spoilage:
– vegetative bacterial cells and endospores
– fungal hyphae and spores, yeast
– protozoan trophozoites and cysts
– worms
– viruses
– prions

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 Relative Resistance of Microbes
• Highest resistance
– prions
– bacterial endospores (Bacillus/Clostridium)
• Moderate resistance
– Pseudomonas, Acinetobacter
– Staphylococcus
– Mycobacterium
– protozoan cysts/fungal zygospores/naked virus
• Least resistance
– most bacterial vegetative cells
– fungal spores (other than zygospores) and hyphae, yeast
– enveloped viruses
– protozoan trophozoites 4
 Terminology and Methods of Control
• Sterilization – a process that destroys all viable microbes,
including viruses and endospores; microbicidal (tiệt/diệt
trùng)
• Disinfection – a process to destroy vegetative pathogens,
not endospores; inanimate objects (khử trùng)
• Antiseptic – disinfectants applied directly to exposed body
surfaces (sát trùng)
• Sanitization – any cleansing technique that mechanically
removes microbes (vệ sinh)
• Degermation – reduces the number of microbes
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Sepsis: the growth of microorganisms in the
blood and/or other tissues
Asepsis: any practice that blocks the entry of
infectious agents into sterile tissues and thus
prevents infection
Aseptic techniques: commonly practiced sterile
methods that exclude all microbes to antisepsis

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 Microbial death
• Permanent loss of reproductive capability, even
under optimum growth conditions

Cell that is “alive” and able to reproduce:

Viable cell
#
CFU

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 Factors That Affect Death Rate
The effectiveness of a particular agent is governed by
several factors:
• Number of microbes
• Nature of microbes in the population
• Temperature and pH of environment
• Concentration or dosage of agent
• Mode of action of the agent
• Presence of solvents, interfering organic matter,
or inhibitors
• Duration 10
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Practical Concerns in Microbial Control
Selection of method of control depends on circumstances:
• Does the application require sterilization?
• Is the item to be reused?
• Can the item withstand heat, pressure, radiation, or
chemicals?
• Is the method suitable?
• Will the agent penetrate to the necessary extent?
• Is the method cost- and labor-efficient and is it safe?

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Antimicrobial Agents’ Modes of Action
Cellular targets of physical and chemical agents:
1. The cell wall – cell wall becomes fragile and cell
lyses: some antimicrobial drugs, detergents, and
alcohol
2. The cell membrane - loses integrity: detergent
surfactants
3. Cellular synthetic processes (DNA, RNA) –
prevention of replication, transcription: some
antimicrobial drugs, radiation, formaldehyde,
ethylene oxide
4. Proteins – interfere at ribosomes to prevent
translation, disrupt or denature proteins: alcohols,
phenols, acids, heat
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 Methods of Physical Control

1. Heat – moist and dry

2. Cold temperatures
3. Desiccation
4. Radiation
5. Filtration

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 Mode of Action and Relative
Effectiveness of Heat
• Moist heat – lower temperatures and
shorter exposure time than using Dry heat;
coagulation and denaturation of proteins

• Dry heat – moderate to high temperatures;

dehydration, alters protein structure;
incineration (tro hóa)

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 Heat Resistance and Thermal
Death
• Bacterial endospores most resistant –
usually require temperatures above boiling

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 Thermal Death Measurements
• Thermal death time (TDT) – shortest length
of time required to kill all test microbes at a
specified temperature
• Thermal death point (TDP) – lowest
temperature required to kill all microbes in a
sample in 10 minutes

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 Moist Heat Methods
• Steam under pressure – sterilization
• Autoclave 15 psi (1.055 kg/cm2)/121oC/10-40min
• Steam must reach surface of item being sterilized
• Item must not be heat or moisture sensitive
• Mode of action
denaturation of proteins
destruction of membranes and DNA

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 Nonpressurized Steam
• Tyndallization – intermittent (gián đoạn) sterilization
for substances that cannot withstand autoclaving
• Items exposed to free-flowing steam for 30 – 60 minutes,
incubated for 23-24 hours and then subjected to steam
again
• Repeat cycle for 3 days.
• Used for some canned foods and laboratory media
• Disinfectant

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 Boiling Water
• Boiling at 100oC for 30 minutes to destroy non-
spore-forming pathogens
• Disinfection

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 Pasteurization
• Pasteurization – heat is applied to kill potential
agents of infection and spoilage without
destroying the food flavor or value
• 65°C for 30 minutes (batch method) (LTLT)
• 70°C for 15 seconds (flash method) (HTST)
• Not sterilization - kills non-spore-forming
pathogens and lowers overall microbe count;
does not kill endospores or many nonpathogenic
microbes
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 Dry Heat
Dry heat using higher temperatures than
moist heat
• Incineration – flame or electric heating coil
– ignites and reduces microbes and other
substances
• Dry ovens – 150-180oC- coagulate proteins

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 Cold
• Microbiostatic  slows biological activities of
the cell  the growth of microbes

• Refrigeration 0-15oC and freezing <0oC

• Used to preserve food, media and cultures

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 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
• Lyophilization – freeze drying; preservation

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 Radiation
• Ionizing radiation – deep penetrating power
that has sufficient energy to cause electrons to
leave their orbit, breaks DNA,
– gamma rays, X-rays, cathode rays
– used to sterilize medical supplies and food
products

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 Radiation
• Nonionizing radiation – little penetrating
power – must be directly exposed
• UV light (240-280 nm) creates Thymine
dimers, which interfere with replication.

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 Filtration
• Physical removal of microbes based on
their sizes
• By passing a gas or liquid through filter
• Used to sterilize heat sensitive liquids and
air in hospital isolation units and industrial
clean rooms

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 Chemical Agents in Microbial
Control
• Disinfectants, antiseptics, sterilants, degermers, and
preservatives
• Desirable qualities of chemicals:
– rapid action in low concentration
– solubility in water or alcohol, stable
– broad spectrum, low toxicity
– penetrating
– noncorrosive and nonstaining
– affordable and readily available
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 Levels of Chemical Decontamination
• High-level germicides – kill endospores; may be sterilants
– devices that are not heat sterilizable and intended to be used
in sterile environments (body tissue)
• Intermediate-level – kill fungal spores (not endospores),
tubercle bacillus, and viruses
– used to disinfect devices that will come in contact with
mucous membranes but are not invasive
• Low-level – eliminate only vegetative bacteria, vegetative
fungal cells, and some viruses
– clean surfaces that touch skin but not mucous membranes

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 Factors that Affect Germicidal
Activity of Chemicals
• Nature of the material being treated
• Degree of contamination
• Time of exposure
• Strength and chemical action of the
germicide

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 Germicidal Categories
1. Halogens
2. Phenolics
3. Chlorhexidine
4. Alcohols
5. Hydrogen peroxide
6. Detergents & soaps
7. Heavy metals
8. Aldehydes
9. Gases
10. Dyes
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 Halogens
• Chlorine - Cl2, hypochlorites (chlorine bleach), chloramines
– denature proteins by disrupting disulfide bonds
– damage the structure of DNA, RNA, and fatty acids
– intermediate level
– unstable in sunlight, inactivated by organic matter
– water, sewage, wastewater, inanimate objects

• Iodine - I2, iodophors (betadine)

– rapidly penetrate the cell and denature proteins
– intermediate level
– milder medical & dental degerming agents, disinfectants,
ointments

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 Phenolics
• Disrupt cell walls and membranes and
precipitate proteins
• Low to intermediate level - bactericidal,
fungicidal, virucidal, not sporicidal
– Benzalkonium chloride (Lysol)
– Triclosan- antibacterial additive to soaps

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 Chlorhexidine
• A surfactant and protein denaturant with
broad microbicidal properties
• Low to intermediate level
• Hibiclens, Hibitane
• Used as skin degerming agents for
preoperative scrubs, skin cleaning and burns

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 Alcohols
• Ethyl, isopropyl in solutions of 50-95%
• Act as surfactants dissolving membrane
lipids and coagulating proteins of vegetative
bacterial cells and fungi
• Intermediate level

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 Hydrogen Peroxide
• Weak (3%) to strong (25%)
• Produce highly reactive hydroxyl-free radicals
that damage protein and DNA while also
decomposing to O2 gas – toxic to anaerobes
• Antiseptic at low concentrations; strong
solutions are sporicidal

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 Detergents and Soaps
• Quaternary ammonia compounds (quats) act as
surfactants that alter membrane permeability of
some bacteria and fungi.
• Soaps - mechanically remove soil and grease
containing microbes
• Very low level

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 Heavy Metals
• Solutions of silver and mercury kill vegetative
cells in low concentrations by inactivating proteins
• Oligodynamic action, Low level
• Silver nitrate, Silver sulfadiazine
• Merbromin/Mercurochrome

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 Aldehydes
• Glutaraldehyde and formaldehyde kill by
alkylating protein and DNA.
• Glutaraldehyde in 2% solution (Cidex) used as
sterilant for heat sensitive instruments
• Formaldehyde - disinfectant, preservative, toxicity
limits use
– formalin – 37% aqueous solution
• Intermediate to high level

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 Gases and Aerosols
• Ethylene oxide, propylene oxide
• Strong alkylating agents
• High level
• Sterilize and disinfect plastics and prepackaged
devices, foods

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 Dyes
• Crystal/Gentian violet and
Malachite green are most
active against gram-positive
bacteria
• Methylene blue: active
against both Gram+/- bacteria,
fungi, virus
• Insert into nucleic acids and
cause mutations
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