Microbiology: A Systems

Approach, 2nd ed.

Chapter 11: Physical and Chemical
Control of Microbes
 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
Figure 11.1
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
• Contaminants that need to be controlled
– Bacterial vegetative cells and endospores (so resistant, the
goal is sterilization)
– Fungal hyphae and spores
– Yeasts
– Protozoan trophozoites and cysts
– Worms
– Viruses
– Prions
Terminology and Methods of Microbial
Control
• Sterilization
– Removes all viable microorganisms including
viruses
– Material is said to be sterile
– Usually reserved for inanimate objects
– Mostly performed with heat
– Sometimes chemicals called sterilants are used
 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
 Antisepsis
• Antiseptics applied directly to exposed body
surfaces 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
 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
– Bacteristatic: prevent the growth of bacteria
– Fungistatic: inhibit fungal growth
– Microbistatic: materials used to control microorganisms in the
body, for example
 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
• If a cell can no longer reproduce under ideal
environmental conditions
• Death begins when a certain threshold of
microbicidal agent is met, and continues in a
logarithmic manner
Figure 11.2
 Factors that Influence the Action of
Antimicrobial Agents
• 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
– Block its synthesis
– Digest it
– Break down its surface
– The cell becomes fragile and is lysed easily
• The Cell Membrane
Figure 11.3
Protein and Nucleic Acid Synthesis
• Binding to ribosomes to stop translation
• Bind irreversibly to DNA preventing
transcription and translation
• Mutagenic agents
 Protein Function

Figure 11.4
 11.2 Methods of Physical Control
• Heat as an Agent of Microbial Control
– Generally, elevated temperatures are microbicidal
and lower temperatures are microbistatic
– Can use moist heat or dry heat
Heat Resistance and Thermal Death of
Spores and Vegetative Cells
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
– Pressure raises the temperature of steam
– Autoclave is used
– Most efficient pressure-temperature combination
for sterilization: 15 psi which yields 121°C
Figure 11.5
 Nonpressurized 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
 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
 Boiling Water
• For disinfection and not sterilization
• Expose materials to boiling water for 30
minutes
Dry Heat: Hot Air and Incineration
• Incineration
– Ignites and reduces microbes to ashes and gas
– Common practice in microbiology lab- incineration
on inoculating loops and needles using a Bunsen
burner
– Can also use tabletop infrared incinerators
Figure 11.6
 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
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
Radiation as a Microbial Control Agent
• Radiation: energy emitted from atomic
activities and dispersed at high velocity
through matter or space
• For microbial control:
– Gamma rays
– X rays
– Ultraviolet radiation
 Modes of Action of Ionizing Versus
Nonionizing Radiation
• Ionizing radiation: if the radiation ejects
orbital electrons from an atom causing ions to
form
• Nonionizing radiation: excites atoms by
raising them to a higher energy state but does
not ionize them
Figure 11.7
 Ionizing Radiation: Gamma Rays, X
Rays, and Cathode Rays
• Cold sterilization
• Dosage of radiation- measured in Grays
• Exposure ranges from 5 to 50 kiloGrays
• Gamma rays, most penetrating; X rays,
intermediate; cathode rays, least penetrating
 Applications of Ionizing Radiation
• Food products
• Medical products
 Nonionizing Radiation: Ultraviolet
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
Figure 11.9
 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
Figure 11.10
 Decontamination by Filtration:
Techniques for Removing Microbes
• Effective for removing microbes from air and
liquids
• Fluid strained through a filter with openings
large enough for fluid but too small for
microorganisms
• Filters are usually thin membranes of cellulose
acetate, polycarbonate, and a variety of plastic
materials
• Pore size can be controlled and standardizes
Figure 11.11
 Applications of Filtration
• Prepare liquids that can’t withstand heat
• Can decontaminate beverages without
altering their flavor
• Water purification
• Removing airborne contaminants (HEPA
filters)
 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
• Tinctures: solutions dissolved in pure alcohol
or water-alcohol mixtures
 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: liquid and gaseous chlorine,
hypochlorites, chloramines
• Kills bacteria and endospores
• Also kills fungi and viruses
• Example: Household bleach
– Iodine compounds: free iodine and iodophors
• Topical antiseptic
• Disinfectant
 Phenol and its Derivatives
• Phenol coefficient: compares a chemical’s
antimicrobic properties to those of phenol
• High concentrations: cellular poisons
• Lower concentrations: inactivate certain
critical enzyme systems
 Chlorhexidine
• 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 gram-
negative bacteria but inactive against spores
• Mild, low toxicity, rapid action
 Alcohols as Antimicrobial Agents
• Only ethyl and isopropyl alcohols are suitable for
microbial control
• Mechanism of action depends in part upon its
concentration
– 50% and higher dissolve membrane lipids, disrupt cell
surface tension, and compromise membrane integrity
– 50% to 90% denatures proteins through coagulation; but
higher concentration does not increase microbicidal activity
– 100% (absolute alcohol) dehydrates cells and inhibits their
growth
• 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
• Bactericidal, virucidal, and fungicidal
• In higher concentrations is sporicidal
 Chemicals with Surface Action:
Detergents
• Act as surfactants
• Anionic detergents have limited microbicidal
power
• 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
Figure 11.14
Figure 11.15
 Heavy Metal Compounds
• Hg, Ag, Au, Cu, As, and Zn have been used
• Only Hg and Ag still have significance as germicides
• Oligodynamic action: having antimicrobial effects in
exceedingly small amounts
• Bind onto functional groups of proteins and inactivating
them
• 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
Figure 11.16
 Aldehydes as Germicides
• –CHO functional group on the terminal carbon
• Glutaraldehyde and formaldehyde (formalin-
aqueous solution)- most often used in
microbial control
Figure 11.17
 Gaseous Sterilants and Disinfectants
• Ethylene oxide (ETO)
• Propylene oxide
• Chlorine dioxide
 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
• 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