Physical and Chemical
Control of Microbes
Chapter 11
Adapted from McGraw Hill
by Dr. G Cornwall
11.1 Controlling Microorganisms
• General Considerations in Microbial Control
• Sterilization: the destruction of all microbial
life
• Disinfection: destroys most microbial life,
reducing contamination on inanimate surfaces
• Antisepsis: destroys most microbial life,
reducing contamination on a living surface
• Decontamination: the mechanical removal of
most microbes from an animate or inanimate
surface
Microbial Control Methods
Relative Resistance of Microbial
Forms
Primary targets of microbial control: microorganisms that
can cause infection or spoilage that are constantly present
in the external environment
• Highest resistance
• Bacterial endospores; prions
• Moderate resistance
• Protozoan cysts, fungal sexual spores, naked viruses,
resistant vegetative bacteria
• Least resistance
• Most bacterial vegetative cells, fungal spores (nonsexual), enveloped viruses, yeasts, protozoan
trophozoites
Comparison of Resistance
Terminology and Methods of Microbial
Control
• Sterilization
• Removes all viable microorganisms including
viruses and bacterial endospores
• Material is said to be sterile
• Usually reserved for inanimate objects
• Mostly performed with heat
• Sometimes chemicals called sterilants are
used
• Generally reserved for inanimate objects
• Disinfection
• The use of a physical process or chemical
agent (disinfectant) to destroy vegetative
pathogens
• Does not destroy bacterial endospores
• Usually used only on inanimate objects
• Also removes toxins
• 5% bleach solution, boiling water, iodine
solutions
Antisepsis
• Antiseptics: applied directly to exposed body
surfaces (and mucous membranes) to destroy or
inhibit vegetative pathogens
• Sepsis: the growth of microorganisms in the
blood and other tissues
• Asepsis: any practice that prevents the entry of
infectious agents into sterile tissues
• Examples: skin preparations with iodine for
surgery, hydrogen peroxide in root canal, hand
washing with germicidal soap.
The Agents Versus the Processes
• –cide: to kill
• Bactericide: chemical that destroys bacteria (not
endospores)
• Fungicide: a chemical that can kill fungal spores, hyphae,
and yeasts
• Virucide: a chemical that inactivates viruses
• Sporicide: can destroy bacterial endospores
• Germicide and microbicide: chemical agents that kill
microorganisms
• Stasis and static: to stand still, prevent
multiplication
• Bacteristatic, Fungistatic
• Microbiostatic: materials used to control microorganisms in
the body
Decontamination
• Used when actual sterilization isn’t needed but
need to decrease the risk of infection or spoilage
(ex. food industry)
• Sanitization: any cleansing technique that
mechanically removes microorganisms to reduce
contamination to safe levels
• Sanitizer: compound such as soap or
detergent that sanitizes
• Sanitary: may not be free from microbes but
are safe for normal use
• Degermation: reduces the numbers of microbes
on the human skin (ex. alcohol wipes)
Practical Concerns in Microbial
Control
• Does the application require sterilization, or is
disinfection adequate?
• Is the item to be reused or permanently discarded?
• If it will be reused, can it withstand heat, pressure,
radiation, or chemicals?
• Is the control method suitable for a given
application?
• Will the agent penetrate to the necessary extent?
• Is the method cost- and labor-efficient, and is it
safe?
What is Microbial Death?
• When various cell
structures become
dysfunctional and the
entire cell sustains
irreversible damage
• The permanent loss of
reproductive capability,
even under optimal
growth conditions, is the
accepted microbiological
definition of death
Factors that Affect Death Rate
• The number of
microorganisms
• The nature of the
microorganisms in the
population
• The temperature and pH of
the environment
• The concentration of the
agent
• The mode of action of the
agent
• The presence of solvents,
interfering organic matter, and
inhibitors
How Antimicrobial Agents Work:
Their Modes of Action
• The Cell Wall
• The Cell Membrane
• Protein and Nucleic Acid Synthesis
• Protein Function
Antimicrobial Effects on Cell Well
• Block its synthesis
• Digest it
• Break down its surface
• The cell becomes fragile and is lysed easily.
Cell Membrane
• All microorganisms
have cell membrane
• Many viruses as well
• Disrupt cell membrane
= cell loss of selective
permeability
• Detergents
(surfactants) disrupt
membrane
Protein and Nucleic Acid Synthesis
• Any level can be affected: Replication,
Transcription, and/or Translation
• Some agents bind to ribosomes to stop
translation
• Some agents bind irreversibly to DNA preventing
transcription and translation
• Mutagenic agents
Protein Function
• Proteins must be in
native state
• Disrupt the native
state (denature)
• Break the bonds of
secondary or tertiary
structures
• Coagulation: heat,
alcohol, acids,
phenolics
Concept Check
What type of compound is isopropanol when the
nurse wipes your arm with it before giving you an
injection?
A. Antibiotic
B. Disinfectant
C. Mutagen
D. Antiseptic
11.2 Methods of Physical Control
• Heat as an Agent of Microbial Control
• Generally, elevated temperatures are microbicidal and lower
temperatures are microbiostatic
• Can use moist heat or dry heat
• Moist heat is more effective
• Mode of Action
• Moist- coagulation and denaturation
• Dry –remove water from organisms, incineration
Heat Resistance and Thermal Death of
Spores and Vegetative Cells
• Bacterial endospores exhibit the greatest resistance
• Spore destruction requires temperatures above
boiling
• Vegetative states of fungi and bacteria are least resistant
to both moist and dry heat
• also vary in their heat sensitivity to both dry and
moist heat
• Viruses surprisingly resistant to heat
• For practical purposes, all non-heat resistant forms of
bacteria, yeasts, molds protozoa, worms, and viruses
are destroyed by exposure to 80∘C for 20 minutes
Practical Concerns in the Use of Heat:
Thermal Death Measurements
• Temperature and length of exposure must be
considered
• Higher temperatures generally allow shorter
exposure times; lower temperatures generally require
longer exposure times
• Thermal death time (TDT):
• the shortest length of time required to kill all test
microbes at a specified temperature
• Thermal death point (TDP):
• the lowest temperature required to kill all microbes
in a sample in 10 minutes
Common Methods of Moist Heat
Control
• Steam under pressure
• Non-pressurized steam
• Pasteurization
• Boiling water
Steam Under Pressure
• Pressure raises the
temperature of steam
• Autoclave is used
• Most efficient pressuretemperature combination for
sterilization: 30 psi which
yields 121°C (15 psi above
our atmospheric pressure)
• Kills all endospores
• Home pressure cookerssame principle
Non-pressurized Steam
• Intermittent sterilization or tyndallization
• Expose to free-flowing steam for 30-60 minutes,
incubate for 23-24 hours, treat again; repeat for 3
days in a row
• A single exposure is not sufficient to kill all spores
• Used some for canned foods and certain media
preparations
Pasteurization & Boiling
Pasteurization
• Used to disinfect beverages
• Heat is applied to liquids to kill potential agents of infection and spoilage,
while retaining the liquid’s flavor and food value
• Special heat exchangers
• Flash method: expose to 71.6°C for 15 seconds
• Batch method: expose to 63°C to 66°C for 30 minutes
• Does not kill endospores or thermoduric microbes
• Pasteurized milk is NOT sterile
Boiling
• For disinfection and not sterilization (used in home and clinics)
• Expose materials to boiling water for 30 minutes
• will kill most non-spore forming pathogens, including resistant
species (staphylococci)
• Also for unsafe drinking water
Dry Heat: Hot Air and Incineration
• Incineration
• Ignites and reduces
microbes to ashes and
gas
• Common practice in
microbiology labincineration on
inoculating loops and
needles using a
Bunsen burner
• Can also use tabletop
infrared incinerators
Dry Oven
• Usually an electric oven
• Coils radiate heat within an enclosed
compartment
• Exposure to 150°C to 180°C for 2 to 4 hours
• Used for heat-resistant items that do not sterilize
well with moist heat
• Glassware, metallic instruments, powders & oils
that steam does not penetrate well
Concept Check
What temperature does an autoclave achieve at 15
p.s.i.?
A. 100°C
B. 121°C
C. 132°C
D. 250°C
The Effects of Cold and Desiccation
• To slow growth of cultures and microbes in food
during processing and storage
• Cold does not kill most microbes; freezing can
actually preserve cultures
• Desiccation:
• dehydration of vegetative cells when directly
exposed to normal room air
• Lyophilization:
• a combination of freezing and drying; used to
preserve microorganisms and other cells in a
viable state for many years
Modes of Action of Ionizing Versus
Non-ionizing Radiation
• Radiation: energy
emitted from atomic
activities, and
dispersed at high
velocity through matter
or space
• Ionizing: ejects
electron, causing ions
to form
• Non-ionizing: excites
atoms but does not
ionize them
Ionizing Radiation: Gamma Rays, X
Rays, and Cathode Rays
• Cold sterilization – no heat
• Dosage of radiation- measured in Grays
• Exposure ranges from 5 to 50 kiloGrays (kGray = 1000 grays)
• Gamma rays, most penetrating; X rays, intermediate; cathode
rays, least penetrating
Applications
• Food products
• Medical products
• Main advantages:
• Speed
• Penetrating power
• No heat
Non-ionizing Radiation: UV Rays
• Wavelength approximately 100
nm to 400 nm
• Germicidal lamp: 254 nm
• Not as penetrating as ionizing
radiation
• Powerful tool for destroying
fungal cells and spores, bacterial
vegetative cells, protozoa, and
viruses
• Wavelength: 200-300 nm
• Poor penetrating power
• Pyrimidine dimers
• Mutations induced
Applications of Ultraviolet Radiation
• Usually disinfection
rather than sterilization
• Hospital rooms,
operating rooms,
schools, food prep
areas, dental offices
• Treat drinking water or
purify liquids
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Filtration
Liquid
• Pass liquid or gas
through a filter with
sufficiently small pore
size
Filter
• HEPA – high-efficiency
particulate air filters
• Advantages: No thermal
damage
Pore
Filter
Sterilized
fluid
Vacuum
Pump suction
(a)
• Disadvantages: viruses
not eliminated and must
be either liquid or gas
(b)
b: © Fred Hossler/Visuals Unlimited
Osmotic Pressure
• Adding large amount of salt or sugar to food
• creates hypertonic environment for bacteria
• causes plasmolysis
• impossible for cells to multiply
• Curing meat
• high sugar in preserves
Concept Check
Which of these is least likely to result in
sterilization?
A. Incineration
B. Baking at 170°C
C. Boiling
D. Autoclaving
11.3 Chemical Agents in Microbial
Control
• Approximately 10,000 different antimicrobial
chemical agents are manufactured
• Approximately 1,000 used routinely in health care
and the home
• Occur in liquid, gaseous, or solid state
• Aqueous: dissolved in pure water
• Tinctures: solutions dissolved in pure alcohol or
water-alcohol mixtures
Chemical Agents in Health Care
Choosing a Microbicidal Chemical
• Rapid action even in low concentrations
• Solubility in water or alcohol and long-term stability
• Broad-spectrum microbicidal action without being toxic to
human and animal tissues
• Penetration of inanimate surfaces to sustain a cumulative
or persistent action
• Resistance to becoming inactivated by organic matter
• Noncorrosive or nonstaining properties
• Sanitizing and deodorizing properties
• Affordability and ready availability
Factors that Affect the Germicidal
Activity of Chemicals
• Nature of microorganisms
being treated
• Nature of the material
being treated
• Degree of contamination
• Time of exposure
• Strength and chemical
action of the germicide
Germicidal Categories According to
Chemical Group
• Halogen Antimicrobial Chemicals
• Fluorine, bromine, chlorine, and iodine
• Microbicidal and sporicidal with longer exposure
• Chlorine compounds
• Kills bacteria and endospores
• Also kills fungi and viruses
• Example: Household bleach
• Iodine compounds
• Topical antiseptic
• Disinfectant
• All classes of microorganisms are killed by iodine (proper
concentration & exposure time)
• Iodophores are complexes of iodine & alcohol (Betadine,
Isodine) used in medical antisepsis (less irritating)
Phenol and its Derivatives
• Phenol coefficient: compares a
chemical’s anti-microbic
properties to those of phenol
• High concentrations: cellular
poisons
• Lower concentrations: inactivate
certain critical enzyme systems
• Strongly microbicidal, but not
reliably sporicidal
• Bisphenols - a component of lysol
• Triclosan - most widely used,
found in soaps, kitty litter,
sponges, laundry aides.
Chlorhexidine
• Brands: Hibiclens, Hibitane, Peridex
• Complex organic base containing chlorine
and two phenolic rings
• Targets cell membranes and protein structure
• At moderate to high concentrations, it is
bactericidal for both gram-positive and gramnegative bacteria but inactive against spores
• Mild, low toxicity, rapid action
• Hand scrubbing, Preparing skin for surgery,
obstetric antiseptic, preservative in eye
solutions, irrigant, mouthwashes etc.
Alcohols as Antimicrobial Agents
• Only ethyl and isopropyl alcohols
are suitable for microbial control
• Mechanism of action depends in
part upon its concentration
• > 50% dissolves membrane lipids
• 50-95% denatures proteins
• Does not destroy bacterial spores at
room temperature but can destroy
resistant vegetative forms
• More effective in inactivating
enveloped viruses than nonenveloped viruses
Hydrogen Peroxide and Related
Germicides
• Germicidal effects are due to the direct and indirect actions
of oxygen
• Oxygen forms hydroxyl free radicals which are highly toxic
and reactive to cells
• Can be harmful to tissue
• Bactericidal, virucidal, and fungicidal
• In higher concentrations is sporicidal
Chemicals with Surface Action:
Detergents
• Act as surfactants
• Cationic detergents are
more effective because
the positively charged end
binds well with the
predominantly negatively
charged bacterial surface
proteins
• Soaps are weak
microbicides but gain
germicidal value when
mixed with agents such as
chlorhexidine or iodine
Heavy Metal Compounds
• Hg, Ag, Au, Cu, As, and Zn have been used
• Oligodynamic action: having antimicrobial
effects in exceedingly small amounts
• Bind onto functional groups of proteins and
inactivating them
• destroys most microbes (not endospores)
• Drawbacks to using metals in microbial
control:
• Can be very toxic to humans
• Often cause allergic reactions
• Large quantities of biological fluids and
wastes neutralize their actions
• Microbes can develop resistance to
them
Concept Check
What kind of property do surfactants have?
A. Hydrophilic
B. Hydrophobic
C. Amphipathic
D. Hypotonic
Aldehydes as Germicides
• –CHO functional group on the
terminal carbon
• Glutaraldehyde and
formaldehyde (formalinaqueous solution) - most
often used in microbial control
• cross-linking proteins on
cell surface
• Glutaraldehyde is more
commonly used, often in
conjunction with autoclaving
Gaseous Sterilants and
Disinfectants
• Ethylene oxide (ETO) - a sporicide
• Sterilization of pre-packaged medical devices,
plastic materials, disposable petri dishes etc.
• highly explosive, carcinogenic
• Propylene oxide - less toxic
• sterilization of foods (nuts, powders, spices)
• Chlorine dioxide - also a sporicide
• used for treatment of drinking water, waste
water, and food processing equipment
• Block DNA replication, protein function
Dyes as Antimicrobial Agents
• Primary source of certain drugs used in
chemotherapy
• Aniline dyes (crystal violet and malachite green)
are very active against gram-positive species of
bacteria and various fungi
• treatment of ringworm
• Yellow acridine dyes (acriflavine and proflavine)
sometimes used for antisepsis and wound
treatment
• Limited applications because they stain and have
a narrow spectrum of activity
Acids and Alkalis
• Very low or high pH can destroy or inhibit
microbial cells
• Limited in applications due to their corrosive,
caustic, and hazardous nature
• aqueous ammonium hydroxide is a component of
many detergents, deodorizers, and cleansers
• Organic acids are used in food preparation
• Acetic acid as a pickling agent
• propionic acid, lactic acid, benzoic acid, sorbic
acids
Main Ingredients Antimicrobial
Products
Concept Check
Which of these would be the least likely to be
removed by passage through a HEPA filter?
A. Virus
B. Yeast
C. Bacteria
D. Protist