CHAPTER 9: CONTROLLING MICROBIAL GROWTH IN THE
ENVIRONMENT
Basic Principles of Microbial Control
Terminology of Microbial Control
- Sterilization
• refers to the removal of all microbes in or on an object
• indicates only the eradication of harmful microbes (ex commercial sterilization
does not kill hyperthermophilic microbes, whom does not thrive in ambient
temperatures)
• typically achieved by steam under pressure, incineration, or ethylene oxide gas
- Aseptic
• Describes an environment free of contamination by pathogens
• ex preparation of surgical field, handwashing, flame sterilization of lab equipment
- Disinfection
• Refers to the use of chemical/physical disinfectants (such as ultraviolet light,
heat, alcohol, bleach) to destroy microorganisms, especially pathogens
• Does not guarantee that all pathogens are eliminated
• Used only in reference to treatment of inanimate objects
• When chemical is used on skin or other tissue, process is referred to as
antisepsis, where the chemical is called antiseptic (ex iodine, alcohol)
• Disinfectants can be left on a surface for longer periods of time compared to
antiseptics
• ex phenolics, alcohols, aldehydes, soaps
- Degerming
• Removal of microbes form a surface by scrubbing with the assistive use of
chemicals (soap, alcohol)
• ex handwashing, preparing a specific area of the body for injection
- Sanitization
• Process of disinfecting places and utensils used by the public to reduce number
of pathogenic microbes to meet accepted public health standards
• Difference between disinfection is where the objects are used (private vs. public)
• ex the use of steam, high-pressure hot water, and scrubbing to sanitize utensils
and dishes, the use of chemicals to sanitize public toilets
- Pasteurization
• Use of heat to kill pathogens and reduce number of spoilage microorganisms in
food and beverages
➢ IMPORTANT SUFFIXES TO REMEMBER
• -cide & -cidal: indicates destruction of a type of microbe (ex ethylene oxide,
propylene oxide, aldehydes)
• -static & -stasis: indicates inhibition but not complete destruction of a type of
microbe (ex refrigeration, freezing, use of some chemicals)
Microbial Death Rates
- Defined as the permanent loss of reproductive ability under ideal environmental
conditions
- Used as a technique to evaluate efficacy of antimicrobial agent
- Plots the constant killing percentage of cells over time by microbicidal agents
Action of Antimicrobial Agents
1. Alteration of Cell Walls & Membranes
• Physical/chemical agents disrupt the cell wall, causing cell to burst as water
moves into it by osmosis
• Physical/chemical agents damages the phospholipids or and/or proteins
within the cytoplasmic membrane, causing cellular contents to leak out and if
not immediately repaired, can cause death to the cell
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 • Physical/chemical agents damage the envelope covering certain viruses,
which fatally interrupts viral replication (note that non-enveloped viruses have
greater tolerance of harsh environmental conditions, including antimicrobial
agents)
2. Damage to Proteins & Nucleic Acids
• Extreme heat or the presence of certain chemicals break bonds that maintain
a protein’s shape and consequent function (denaturation)
• Chemicals, radiation, and heat also alters and destroys nucleic acids that
produces fatal mutations to the cell’s or virus’s genes
• Physical/chemical agents can also interfere with nucleic acids and their
ability to catalyze ribozymes, thus stopping protein synthesis
Selection of Microbial Control Methods
Factors Affecting Efficacy of Antimicrobial Methods
➢ Site to be Treated
- Choice of antimicrobial method depends of nature of site to be treated, in
such that the method used will not compromise the site exposed (ex
equipment used on the outer defenses of the body may carry greater
potential for infection, hence the need for sterilization)
➢ Relative Susceptibility of Microorganisms
- Antimicrobial methods are chosen based on the hardiest microbe present,
assuming that such treatment will eliminate the more fragile microbes as well
- The most resistant microbes are the following:
❖ Bacterial endospores: can survive environmental extremes of
temperature, acidity, & dryness, and can withstand many chemical
disinfectants
❖ Species of Mycobacterium: cell walls contain a large amount of
waxy lipid, allowing these bacteria to survive drying and protects
them from water-based chemical (strong disinfectants or heat are
then used to treat such microbes)
❖ Cysts of protozoa: its wall prevents entry of most disinfectants,
drying, and shields against radiation and heat
➢ Environmental Conditions
- Temperature & pH affect microbial death rates and efficacy of
antimicrobial agents (chemicals react faster at higher temperatures, while
acidic conditions enhance antimicrobial effect of heat)
- Some chemical disinfectants are more effective at a low pH (ex chlorine
bleach)
- Organic materials (ex feces, vomit, biofilms, blood, fat) interfere with the
penetration of heat, chemicals, and some forms of radiation, and can
sometimes inactivate chemical disinfectants
Biosafety Levels
- Four safety levels have been established by the Centers for Disease Control
and Prevention (CDC) for microbiological labs dealing with pathogens
1. Biosafety Level 1
• Suitable for handling microbes (such as E. coli) that are not known to
cause disease in healthy humans
• Precautions are minimal and include handwashing with antimicrobial
soap and washing surfaces with disinfectants
2. Biosafety Level 2
• Designed for handling more hazardous agents (such as hepatitis
and influenza viruses)
• Access is limited when work is being conducted, and extreme
precautions are taken with contaminated sharp objects
• Procedures that might produce aerosols are conducted within safety
cabinets
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 3. Biosafety Level 3
• Requires all manipulations to be done within safety cabinets
containing high-efficiency particulate air filters
• Specifies special design features for lab, including double sets of
doors and ventilation systems that only allow air movement through
an open door
• Designed for experimentation on microbes (such as bacteria of TB or
viruses of yellow fever)
4. Biosafety Level 4
• Designated for working with dangerous or exotic microbes that cause
severe or fatal diseases in humans (such as Ebola and smallpox)
• Either separate buildings or completely isolated from all other areas,
with points of entry and exit strictly controlled through electronically
sealed airlocks
• Safety measures include a vacuum room, multiple showers, an
ultraviolet light room, and other safety precautions designed to
destroy all traces of the biohazard
• All air and water entering and leaving facility are filtered to prevent
accidental release
• Suits and the lab itself are pressurized so that microbes are swept
away from workers
Physical Methods of Microbial Control
Heat-Related Methods
- High temperatures can denature proteins, interfere with integrity of
cytoplasmic membranes and cell walls, and disrupt function and structure of
nucleic acids
- Thermal death point refers to the lowest temperature that kills all
microbes in a broth in 10 minutes
- Thermal death time refers to the time it takes to completely sterilize a
particular volume of liquid at a set temperature
- decimal reduction time refers to the time required to destroy 90% of the
microbes in a sample
- Moist Heat is commonly used to disinfect, sterilize, and pasteurize, and is
more effective because water is a better conductor of heat than air
➢ Boiling: kills the vegetative cells of bacteria and fungi, the
trophozoites of protozoa, and most viruses within 10 minutes at sea
level at 100℃ (remember: water in higher elevations boil at lower
temperatures due to lowered atmospheric pressure); boiling is not
recommended when true sterilization is required
➢ Autoclaving: applies pressure to boiling water to prevent heat escape
through steam to achieve true sterilization at 121℃ in 15 minutes;
used to sterilize chemicals and objects that can tolerate moist heat;
solid objects must be wrapped in porous cloth/paper, while
containers with liquids must be loosely shut to allow steam to
circulate freely; sterility indicators (in the form of endospores of
Bacillus stearothermophilus) are used (absence of color change
indicates sterility)
➢ Pasteurization: (note: pasteurization is not sterilization); combination of
time and temperature required for effective pasteurization depends
on product; milk can be pasteurized 3 ways- batch method (30
minutes at 63℃), flash pasteurization (15 seconds at 72℃), &
ultra-high-temperature pasteurization (1 second at 135℃)
➢ Ultra-High Temperature Sterilization: involves passing liquid through
superheated steam at about 140℃ for 1-3 seconds then cooling it
rapidly; treated liquids can be stored indefinitely at room temperature
without microbial spoilage
- Dry Heat is used for substances such as powders and oils that cannot be
sterilized by boiling or steam or for materials that can be damaged by
repeated exposure to steam; requires higher temperatures for longer
times (160℃ for 2 hours or 171℃ for an hour); complete incineration (1
second in 1000℃) is ultimate means of sterilization
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Refrigeration & Freezing
- the most convenient method of microbial control
(refrigeration between 0-7℃ & freezing at temperatures below 0℃)
- Decrease microbial metabolism, growth, and reproduction as chemical
reactions occur more slowly in lower temperatures
- Refrigeration halts growth of most pathogens, which are predominantly
mesophiles
- Slow freezing involves the formation of ice crystals to puncture cell
membranes to inhibit microbial metabolism
Desiccation & Lyophilization
- Desiccation is a means of preservation by drying foods; inhibits most
pathogens as microbial metabolism requires liquid water
- Lyophilization is a technique of freezing and drying to preserve microbes
and other cells for many years; uses liquid nitrogen or frozen carbon dioxide
to freeze a culture then subject it to a vacuum that removes water via
sublimation (-196℃ for a few minutes while drying)
Filtration
- Passage of fluid (liquid or gas) through a sieve designed to trap particles
(cells or viruses) and separate them from the fluid
- Typically uses circular membrane filters made of mitrocellulose or plastic that
contain specific pore sizes
- High-efficiency particulate air (HEPA) filters are crucial parts of biological
safety cabinets, where they are mounted in air ducts of rooms that may
house airborne viruses or bacteria
Osmotic Pressure
- The use of high concentration of salt or sugar to inhibit microbial growth
(exposure of cell to a hypertonic solution)
- recall: Osmosis is the net movement of water across an area of higher
concentration to an area of lower concentration
- removal of water inhibits cellular metabolism because enzymes cannot
function in non-aqueous environments
Radiation
- two types:
1. Particulate radiation- consists of high-speed subatomic particles that
have been freed from their atoms
2. Electromagnetic radiation- defined as energy without mass traveling in
waves at the speed of light released from atoms that have undergone
internal changes (the shorter of the wavelength of an electromagnetic
wave, the more energy it carries)
- Can be described as either ionizing or nonionizing
➢ Ionizing Radiation
• Wavelengths that are shorter than 1nm
• Have sufficient energy to eject electrons from atoms when they
strike molecules, thus creating ions
• Released ions disrupt hydrogen bonding, oxidize covalent
bonds, and create highly reactive hydroxyl radicals that denature
other molecules (particularly DNA), causing fatal mutations and
death
• ex electron beams produced by cathode ray machines (has the
ability to kill microbes in just a few seconds, but cannot sterilize
thick objects or objects coated with large amounts of organic
matter); gamma rays emitted by radioactive elements that can
penetrate farther than electron beams but require hours to kill
microbes; x-rays travel the farthest through matter but have less
energy than gamma rays and require a prohibitive amount of
time to be made practical for microbial control
➢ Nononizing Radiation
• Wavelengths greater than 1nm
• Do not have sufficient energy to force electrons out of orbit, but
contains enough energy to excite electrons and cause them to
make new covalent bonds which then affects 3-D protein
structures and nucleic acids
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 • exultraviolet light has sufficient energy to be a practical
antimicrobial agent; has a wavelength of 260 nm, causes
nucleotide bases in DNA to bond covalently with each other
rather than thru hydrogen bonding with the complementary
bases
Chemical Methods of Microbial Control
- effect of chemicals varies with pH, concentration, and freshness of the
chemical used
- adversely affects cell membranes, cell walls, cytoplasmic membranes, 3-D
Halogens
structures of proteins, and nucleic acid
Phenols & Phenolics
- the efficacy of phenol serves as the one standard to which the actions of
antimicrobial agents can be compared
- these denature proteins and disrupt cell membranes in a wide variety of
pathogens
- considered to have intermediate to low level antiseptics
- effective even in the presence of contaminating organic material (such as
saliva, urine, feces, etc) and remain active for a prolonged period of time
- Phenolics are compounds derived from phenol molecules that have been
chemically modified with the addition of halogens or organic functional
groups
- Bisphenolics are composed of two covalently linked phenolics
- negative aspects include disagreeable odor and possible side effects ( ex skin
irritation)
Alcohols
- are bactericidal, fungicidal, and virucidal against enveloped viruses, but are
not effective against bacterial endospores or fungal spores
- considered intermediate-level disinfectants
- denatures proteins and disrupts cytoplasmic membranes
- commonly used alcohols are rubbing alcohol (isopropanol) and drinking
alcohol (ethanol)
- Isopropanol is slightly superior to ethanol as a disinfectant
- Tinctures are solutions of other antimicrobial chemicals in alcohol, and are
more often effective than the same chemicals dissolved in water
- Pure alcohol is not an effective antimicrobial agent because protein
denaturation requires water (hence the use of 70-90% alcohol to control
microbes)
- Evaporates rapidly that it leaves no residue but may not be in contact with
microbes long enough to be effective
- Composed of the four highly reactive, non-metallic chemical elements:
iodine, chlorine, bromine, & fluorine
- Are considered intermediate-level antimicrobial chemicals that are effective
against vegetative bacterial and fungal cells, fungal spores, some bacterial
endospores and protozoan cysts, and many viruses
- Used both alone or combined with other elements in organic and inorganic
compounds
- Exert antimicrobial effect by denaturing essential proteins, including enzymes
➢ Iodine is a well-known antiseptic that is either used as a tincture or as an
iodophor (iodine-containing organic compound that releases iodine)
➢ Chlorine in its elemental form is used to treat water, while compounds
containing chlorine are also effective disinfectants
➢ Bromine is an effective disinfectant in hot tubs as it evaporates slower
that chlorine at high temperatures, and it is also used as an alternative to
chlorine for disinfecting water containers
➢ Fluorine (in the form of fluoride) is an antibacterial in drinking water that
can reduce incidence of dental cavities, and works in part by disrupting
the metabolism in the biofilm of dental plaque
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Oxidizing Agents
- Kills microbes by oxidizing enzymes, thereby preventing metabolism
- Are high-level disinfectants and antiseptics that release oxygen radicals
which are particularly effective against anaerobic microorganisms
- Hydrogen peroxide can disinfect and sterilize surfaces of inanimate objects
but if often mistakenly used to treat open wounds (this is so because
catalase released from damaged cells neutralize hydrogen peroxide into
water and oxygen gas)
- Ozone is a reactive form of oxygen that is generated when molecular O 2 is
subjected to electrical discharge; more effective than chlorine, but is more
expensive and is difficult to maintain at an effective concentration
- Peracetic acid is an extremely effective sporicide that can sterilize surfaces
and leave no toxic residue
Surfactants
- “surface active” chemicals
- One of its actions include reducing the surface tension of solvents by
decreasing the attraction among molecules
- Considered as low level detergents and antiseptics
- Two common surfactants are soaps and detergents
- Soap molecules have one end that is composed of fatty acids (and thus
hydrophobic) which are effective at breaking down oily deposits into tiny
droplets, while the other side is hydrophilic (negatively charged), thus
attracting water molecules
- Synthetic detergents are positively charged organic surfactants that are
more soluble than soaps in water
- Quaternary ammonium compounds (commonly known as quats) are the
most popular detergents for microbial control as they are composed of NH 4+
in which the hydrogen atom is replaced by other functional groups or
hydrocarbon chains
➢ are colorless, tasteless, and harmless to humans (except at high
concentrations) and functions by disrupting cellular membranes so
that affected cells lose essential internal ions
➢ are bactericidal (particularly against Gram-positive bacteria),
fungicidal, and virucidal against enveloped viruses, but are not
effective against nonenveloped viruses, mycobacteria, or
endospores
Heavy Metals
- exzinc, arsenic, mercury, silver, and copper
- combines with sulfur atoms in molecules of cysteine (and amino acid) to
denature a protein, thus inhibiting or eliminating its function
- low-level bacteriostatic and fungistatic agents
- used as a means to preserve vaccines, serves as an antimicrobial agent in
surgical dressings, burn creams, and catheters, and controls algal growth
and mildew
Aldehydes
- compounds containing terminal –CHO groups
- functions by cross-linking amino, hydroxyl, sulfhydryl, and carboxyl organic
functional groups to denature proteins and inactivate nucleic acids
- known as a high level disinfectant
- two common aldehydes used in the medical field are glutaraldehyde and
formaldehyde
➢ Glutaraldehyde is used by hospital personnel and scientists at 2%
to kill bacteria, viruses and fungi (10 minutes effectively disinfects,
10 hours sterilizes); less irritating and more effective than
formaldehyde, but is also more expensive
➢ Formaldehyde is used by morticians and health care workers to
make a 37% dissolved solution called formalin, which is used to
disinfect hospital rooms, instruments, and machines, and to embalm;
must be treated with caution as it can irritate mucous membranes
and is carcinogenic (cancer-causing)
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Gaseous Agents
- Highly reactive microbicidal and sporicidal gases
- Rapidly penetrates paper and plastic wraps and diffuses into every crack
- Denatures proteins and DNA by cross-linking organic functional groups,
thereby killing everything they contact without harming the inanimate object
over time (usually 4-18 hours)
- Can be extremely hazardous to people using them (must be combined with
10-20% nitrogen gas or carbon dioxide because they are highly explosive)
- Are also extremely poisonous and beta-carcinogenic
- ex ethylene oxide, propylene oxide, beta-propiolactone
➢ Ethylene oxide is frequently used as a sterilizing agent in hospitals,
and is used by NASA to sterilize spacecrafts deigned to land on
other worlds should they accidentally export unearthly microbes
Enzymes
- Considered high-level antimicrobial enzymes towards target substrates
- Produced by organisms to inhibit or destroy bacteria, viruses, and fungi
- Scientists are currently researching ways to use naturally and chemically
modified antimicrobial enzymes to control microbes in the environment
- It was recently discovered that prioenzymes can remove target prions
Antimicrobials
- Considered as intermediate to low level disinfectants
- Include antibiotics, semisynthetics, and synthetics
- Act against cell walls, cell membranes, protein synthesis, and DNA
transcription and replication
- Antibiotics are naturally produced by microogranisms
- Semisynthetic refers to antibiotics modified by scientists
- Synthetic refers to wholly developed antimicrobial drugs
- Typically used for treatment of diseases and not for the environmental control
of microbes
Methods for Evaluating Disinfectants & Antiseptics
➢ Phenol Coefficient
- can be evaluated by calculating the ratio that compares a given agent’s
ability to control microbes to that of phenol under standardized conditions
- phenol coefficients greater than 1.0 indicates that an agent is more effective
than phenol (the larger the ratio, the greater the effectiveness)
➢ Use-Dilution Test
- Researcher dip several metal cylinders into broth cultures of bacteria and
briefly dries them at 37℃ then immerses each contaminated cylinder into
different dilutions of disinfectants being evaluated
- Each cylinder is then removed after 10 minutes, rinsed with water and placed
into a sterile medium for 48 hours of incubation
- The most effective agent is the one that entirely prevents microbial growth at
the highest dilution
➢ Kelsey-Sykes Test
- standard alternative assessment to determine capacity of a given chemical to
inhibit bacterial growth
- researchers add a suspension of bacterium to a suitable concentration of the
chemical being tested, then at predetermined times will move the samples of
the mixture into growth medium containing a disinfectant inhibitor
- after incubating for 48 hours, turbidity will indicate the bacteria’s survival of
the treatment (the lack of turbidity indicates the effectiveness of the chemical
being tested)
➢ In-Use Test
- a more realistic but time-consuming method
- swabs are taken from actual objects before and after application of a
disinfectant or antiseptic, then are inoculated into appropriate growth media
and evaluated for microbial growth after incubation
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