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Chapter 05
Control of Microbial
Growth
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1
A Glimpse of History
 British Medical Journal stated British physician
Joseph Lister (1827–1912)
“saved more lives by the introduction of his system
than all the wars of the 19th century together had
sacrificed.”
• Lister revolutionized surgery: introduced methods to
prevent infection of wounds
• Impressed with Pasteur’s work, he wondered if
‘minute organisms’ might be responsible for infections
• Applied carbolic acid (phenol) directly onto damaged
tissues, where it prevented infections
• Improved methods further by sterilizing instruments and
maintaining clean operating environment
A Glimpse of History
 Until late 19th century, patients undergoing even
minor surgeries were at great risk of developing
fatal infections
• Physicians did not know their hands could pass
diseases from one patient to the next
• Did not understand airborne microbes could infect
open wounds
 Modern hospitals use strict
procedures to avoid
microbial contamination
• Most surgeries performed
with relative safety
5.1. Approaches to Control
 Principles of Control
• Sterilization: removal of all microorganisms
• Sterile item is free of microbes including endospores
and viruses (but does not consider prions)
• Disinfection: elimination of most or all pathogens
• Some viable microbes may remain
• Disinfectants used on inanimate objects
– May be called biocides, germicides, bactericides
• Antiseptics used on living tissues
• Pasteurization: brief heating to reduce number of
spoilage organisms, destroy pathogens
• Foods, inanimate objects
5.1. Approaches to Control
 Principles of Control (continued…)
• Decontamination
:
: reduce pathogens to levels
considered safe to handle
• Sanitized: substantially reduced microbial population
that meets accepted health standards
• Not a specific level of control
• Preservation : process of delaying spoilage of foods
and other perishable products
• Adjust conditions
• Add bacteriostatic (growth-inhibiting) preservatives
5.1. Approaches to Control
 Situational
considerations:
Microbial control
methods depend
upon situation and
level of control
required
5.1. Approaches to Control
 Daily Life
• Washing and scrubbing with soaps and detergents
achieves routing control
• Soap aids in mechanical removal of organisms
• Beneficial skin microbiota reside deeper on underlying
layers of skin, hair follicles
– Not adversely affected by regular use
• Hand washing with soap and water most important step
in stopping spread of many infectious diseases
5.1. Approaches to Control
 Hospitals
• Minimizing microbial population very important
•
•
•
•
Danger of healthcare-associated infections
Patients more susceptible to infection
May undergo invasive procedures (e.g., surgery)
Pathogens more likely found in hospital setting
– Feces, urine, respiratory droplets, bodily secretions
• Instruments must be sterilized to avoid introducing
infection to deep tissues
• Prions relatively new concern; difficult to destroy
5.1. Approaches to Control
 Microbiology Laboratories
• Routinely work with microbial cultures
• Use rigorous methods of control
• Must eliminate microbial contamination to both
experimental samples and environment
• Careful treatment both before (sterile media) and after
(sterilize cultures, waste)
• Aseptic techniques used to prevent contamination of
samples, self, laboratory
• CDC guidelines for labs working with microbes
• Biosafety levels range from BSL-1 (microbes not known
to cause disease) to BSL-4 (lethal pathogens for which
no vaccine or treatment exists)
5.1. Approaches to Control
 Food and Food Production Facilities
• Perishables retain quality longer when contaminating
microbes destroyed, removed, inhibited
• Heat treatment most common and reliable mechanism
– Can alter flavor, appearance of products
• Irradiation approved to treat certain foods
• Chemical additives can prevent spoilage
• FDA regulates because of risk of toxicity
• Facilities must keep surfaces clean and relatively free
of microbes
5.1. Approaches to Control
 Water Treatment Facilities
• Ensure drinking water free of pathogens
• Chlorine traditionally used to disinfect water
• Can react with naturally occurring chemicals
– Form disinfection by-products (DBPs)
– Some DBPs linked to long-term health risks
• Some organisms resistant to chemical disinfectants
– Cryptosporidium parvum (causes diarrhea)
• Regulations require facilities to minimize DBPs and
C. parvum in treated water
5.2. Selection of an Antimicrobial Procedure
 Selection of effective procedure is complicated
• Ideal method does not exist
• Each has drawbacks and procedural parameters
 Choice depends on numerous factors
•
•
•
•
Type and number of microbes
Environmental conditions
Risk of infection
Composition of infected item
5.2. Selection of an Antimicrobial Procedure
 Type of Microorganism
• Multiple highly resistant microbes
• Bacterial endospores: only extreme heat or chemicals
completely destroys
• Protozoan cysts and oocysts: resistant to disinfectants;
excreted in feces; causes diarrheal disease if ingested
• Mycobacterium species: waxy cell walls makes
resistant to many chemical treatments
• Pseudomonas species: resistant to and can actually
grow in some disinfectants
• Naked viruses: lack lipid envelope; more resistant to
disinfectants
5.2. Selection of an Antimicrobial Procedure
 Number of Microorganisms
• Time for heat, chemicals to kill affected by population size
• Fraction of population dies during given time interval
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• Decimal reduction time
(D value) gauges
commercial effectiveness
• Time required to kill 90% of
population under specific
conditions
108
Log decrease of 1
Number of surviving cells (logarithmic scale)
• Large population = more time
• Removing organisms by
washing reduces time required
107
106
Logarithmic
killing
105
104
103
Log
decrease
of 1
102
101
“D”
“D”
1
0
30
60
90
Time (min)
120
150
5.2. Selection of an Antimicrobial Procedure
 Environmental Conditions
• Dirt, grease, body fluids can interfere with heat
penetration, action of chemicals
• Important to thoroughly clean
• pH, temperature can influence effectiveness
• E.g., sodium hypochlorite (household bleach) solution can
kill suspension of M. tuberculosis at 55°C in half the time
as at 50°C
• Even more effective at low pH
5.2. Selection of an Antimicrobial Procedure
 Risk for Infection
• Medical instruments categorized according to risk for
transmitting infectious agents
• Critical items come in contact with body tissues
• Must be sterile
• Include needles and scalpels
• Semicritical instruments contact mucous membranes
but do not penetrate body tissues
• Must be free of viruses and vegetative bacteria
• Few endospores blocked by mucous membranes
• Includes endoscopes and endotracheal tubes
• Non-critical instruments contact unbroken skin only
• Low risk of transmission
• Countertops, stethoscopes, blood pressure cuffs
5.2. Selection of an Antimicrobial Procedure
 Composition of Item
• Some sterilization and disinfection methods
inappropriate for certain items
• Heat inappropriate for plastics and other sensitive items
• Irradiation provides alternative, but damages some types
of plastic
• Moist heat, liquid chemical disinfectants cannot be used
to treat moisture-sensitive material
5.3. Using Heat to Destroy Microorganisms
and Viruses
 Heat treatment useful for microbial control
• Reliable, safe, relatively fast, inexpensive, non-toxic
• Can be used to sterilize or disinfect
• Methods include moist heat, dry heat
 Moist heat: irreversibly denatures proteins
• Boiling destroys most microorganisms and viruses
• Does not sterilize: endospores can survive
• Pasteurization destroys pathogens, spoilage organisms
• High-temperature–short-time (HTST): most products
– Milk: 72°C for 15 s; ice cream: 82°C for 20 s
• Ultra-high-temperature (UHT): shelf-stable boxed juice
and milk; known as “ultra-pasteurization”
– Milk: 140°C for a few seconds, then rapidly cooled
5.3. Using Heat to Destroy Microorganisms
and Viruses
 Sterilization Using Pressurized Steam
• Autoclave used to sterilize using pressurized steam
• Increased pressure raises temperature; kills endospores
• Sterilization typically at 121°C and 15 psi in 15 minutes
– Longer for larger volumes
• Flash sterilization at higher
temperature can be used
• Prions thought destroyed
at 132°C for 1 hour
Exhaust valve to
remove steam
after sterilization
Valve to
control steam
to chamber
Pressure gauge
Safety
valve
Door
Steam
Air
Jacket
Thermometer
Trap
Pressure
regulator
Steam supply
5.3. Using Heat to Destroy Microorganisms
and Viruses
 Commercial Canning Process
• Uses industrial-sized autoclave called retort
• Designed to destroy Clostridium botulinum endospores
• Reduce 1012 endospores to only 1 (a 12 D process)
• Virtually impossible to have so many endospores
• Critical because otherwise endospores can germinate in
canned foods; cells grow in low-acid anaerobic
conditions and produce botulinum toxin
• Canned food commercially sterile
• Endospores of some thermophiles may survive
• Usually not a concern; only grow at temperatures well
above normal storage
5.3. Using Heat to Destroy Microorganisms
and Viruses
 Dry heat
• Less effective than moist heat; longer times, higher
temperatures necessary
• 200°C for 90 minutes vs. 121°C for 15 minutes
• Hot air ovens oxidize cell components, denature proteins
• Incineration a method of dry heat sterilization
• Oxidizes cell to ashes
• Used to destroy medical waste and animal carcasses
• Laboratory inoculation loop sterilized by flaming
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
 Some materials cannot withstand heat treatment
 Filtration retains bacteria
• Filtration of fluids used extensively
• Membrane filters
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– Small pore size (0.2 µm)
– Thin
• Depth filters
Filter
– Thick porous filtration
material (e.g., cellulose)
Flask
– Larger pores
– Electrical charges trap cells
• Filtration of air
Sterilized
fluid
• High-efficiency particulate air (HEPA) filters remove
nearly all microbes from air
Vacuum
pump
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
 Radiation
• Electromagnetic radiation: radio waves, microwaves,
visible and ultraviolet light, X rays, and gamma rays
• Energy travels in waves; no mass
• Wavelength inversely proportional to frequency
• High frequency has more energy than low frequency
200
Wavelength (nm)
400
500
300
Ultraviolet (UV) light
600
700
Visible light
Ionizing radiation
Gamma
rays
10–5
10–3
X rays
UV
1
Infrared
Microwaves Radio waves
103
106
Wavelength (nm)
109
Increasing energy
Crest
Increasing wavelength
1012
One wavelength
Trough
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
 Radiation (continued…)
• Ionizing radiation can remove electrons from atoms
•
•
•
•
Destroys DNA
Damages cytoplasmic membranes
Reacts with O2 to produce reactive oxygen species
Gamma rays and X rays important forms
– Used to sterilize heat-sensitive materials
– Generally used after packing
– Approved for use on foods, although consumer
resistance has limited use
– FDA has approved for fruits, vegetables, and grains
(for insect control), pork (for parasite control), and
poultry, beef, lamb, and pork (for bacterial control)
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
 Radiation (continued…)
• Ultraviolet radiation destroys microbes directly
• Damages DNA
• Used to destroy microbes in air, water, and on surfaces
• Poor penetrating power
– Thin films or coverings can limit effect
– Cannot kill microbes in solids or turbid liquids
– Most glass and plastic block
• Must be carefully used since damaging to skin, eyes
• Microwaves kill by generated heat, not directly
• Microwave ovens heat food unevenly, so cells can survive
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
 High Pressure
• Used in pasteurization of commercial foods
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•
•
•
E.g., guacamole
Avoids problems with high temperature pasteurization
Employs high pressure up to 130,000 psi
Destroys microbes by denaturing proteins and altering cell
permeability
• Products maintain color, flavor associated with fresh food
5.4. Using Other Physical Methods to Remove
or Destroy Microbes
5.5. Using Chemicals to Destroy Microorganisms
and Viruses
 Potency of Germicidal Chemical Formulations
• Sterilants destroy all microorganisms
• Used on heat-sensitive critical instruments
• High-level disinfectants destroy viruses, vegetative cells
• Do not reliably kill endospores
• Semi-critical instruments
• Intermediate-level disinfectants destroy vegetative
bacteria, mycobacteria, fungi, and most viruses
• Disinfect non-critical instruments
• Low-level disinfectants destroy fungi, vegetative bacteria
except mycobacteria, and enveloped viruses
• Do not kill endospores, naked viruses
• Disinfect furniture, floors, walls
5.5. Using Chemicals to Destroy Microorganisms
and Viruses
 Selecting the Appropriate Germicidal Chemical
• Toxicity: benefits must be weighed against risk of use
• Activity in presence of organic material
• Many germicides inactivated
• Compatibility with material being treated
• Liquids cannot be used on electrical equipment
• Residues: can be toxic or corrosive
• Cost and availability
• Storage and stability
• Concentrated stock decreases storage space
• Environmental risk
• Agent may need to be neutralized before disposal
Classes of Germicidal Chemicals
 Alcohols
• 60–80% aqueous solutions of ethyl or isopropyl alcohol
• Kills vegetative bacteria and fungi
• Not reliable against endospores, some naked viruses
• Coagulates essential proteins (e.g., enzymes)
• More soluble in water; pure alcohol less effective
• Damage to lipid membranes
• Commonly used as antiseptic and disinfectant
• Limitations
• Evaporates quickly, limiting contact time
• Can damage rubber, some plastics, and others
Classes of Germicidal Chemicals
 Aldehydes
• Glutaraldehyde, formaldehyde, and orthophthalaldehyde
• Inactivates proteins and nucleic acids
• 2% alkaline glutaraldehyde common sterilant
• Immersion for 10–12 hours kills all microbial life
• Formaldehyde used as gas or as formalin (37% solution)
• Effective germicide that kills most microbes quickly
• Used to kill bacteria and inactivate viruses for vaccines
• Used to preserve specimens
Classes of Germicidal Chemicals
 Biguanides
• Chlorhexidine most effective
•
•
•
•
•
Extensive in antiseptics
Stays on skin, mucous membranes
Relatively low toxicity
Destroys vegetative bacteria, fungi, some enveloped viruses
Common in many products: skin cream, mouthwash
Classes of Germicidal Chemicals
 Ethylene oxide
• Useful gaseous sterilant
• Destroys microbes including endospores and viruses
• Reacts with proteins
• Penetrates fabrics, equipment, implantable devices
• Pacemakers, artificial hips
• Useful in sterilizing heat- or moisture-sensitive items
• Many disposable laboratory items
• Petri dishes, pipettes
• Applied in special chamber resembling autoclave
• Limitations: mutagenic and potentially carcinogenic
Classes of Germicidal Chemicals
 Halogens: oxidize proteins, cellular components
 Chlorine: Destroys all microorganisms and viruses
•
•
•
•
Used as disinfectant
Caustic to skin and mucous membranes
1:100 dilution of household bleach effective
Very low levels disinfect drinking water
• Cryptosporidium oocysts, Giardia cysts survive
• Presence of organic compounds a problem
• Chlorine dioxide used as disinfectant and sterilant
 Iodine: Kills vegetative cells, unreliable on endospores
• Commonly used as iodophore
• Iodine slowly released from carrier molecule
• Some Pseudomonas species can survive in stock solution
Classes of Germicidal Chemicals
 Metal Compounds
• Combine with sulfhydryl groups of enzymes, proteins
• High concentrations too toxic to be used medically
• Silver still used as disinfectant: creams, bandages
• Silver nitrate eyedrops were required to prevent Neisseria
gonorrhoeae infections acquired during birth
– Antibiotics have largely replaced
• Compounds of mercury, tin, copper, and others once
widely used as preservatives
•
•
•
•
In industrial products
To prevent microbial growth in recirculating cooling water
Extensive use led to environmental pollution
Now strictly regulated
Classes of Germicidal Chemicals
 Ozone
•
•
•
•
O3: unstable form of oxygen
Decomposes quickly, so generated on-site
Powerful oxidizing agent
Used as alternative to chlorine
• Disinfectant for drinking and wastewater
Classes of Germicidal Chemicals
 Peroxygens: powerful oxidizers used as sterilants
• Readily biodegradable, no residue
• Less toxic than ethylene oxide, glutaraldehyde
• Hydrogen peroxide: effectiveness depends on surface
• Aerobic cells produce enzyme catalase
– Breaks down H2O2 to O2, H2O
• More effective on inanimate object
• Doesn’t damage most materials
• Hot solutions used in food industry
• Vapor-phase can be used as sterilant
• Peracetic acid: more potent than H2O2
• Effective on organic material
• Useful on wide range of material
Classes of Germicidal Chemicals
 Phenolic Compounds (Phenolics)
• Phenol one of earliest disinfectants
• Has unpleasant odor, irritates skin
• Phenolics kill most vegetative bacteria
• Mycobacterium at high concentrations
• Not reliable on all virus groups
• Destroy cytoplasmic membranes, denature proteins
• Wide activity range, reasonable cost, remain effective in
presence of detergents and organic contaminants
• Leave antimicrobial residue
• Some sufficiently non-toxic; used in soaps, lotions
• Triclosan, hexachlorophene
Classes of Germicidal Chemicals
 Quaternary Ammonium Compounds (Quats)
• Cationic (positively charged) detergents
• Nontoxic, used to disinfect food preparation surfaces
• Charged hydrophilic and uncharged hydrophobic regions
• Reduces surface tension of liquids
• Aids in removal of dirt, organic matter, organisms
• Most household soaps, detergents are anionic
• But positive charge of quats attracts them to negative
charge of cell surface
•
•
•
•
Reacts with membrane
Destroys vegetative bacteria and enveloped viruses
Not effective on endospores, mycobacteria, naked viruses
Pseudomonas resists, can grow in solutions
Classes of Germicidal Chemicals
5.6. Preservation of Perishable Products
 Chemical preservatives
• Food preservatives must be non-toxic for safe ingestion
 Weak organic acids (benzoic, sorbic, propionic)
• Inhibit metabolism, alter cell membrane function
• Control molds and bacteria in foods and cosmetics
 Nitrate and nitrite used in processed meats
• Inhibit endospore germination and vegetative cell growth
• Stops growth of Clostridium botulinum
• Higher concentrations give meats pink color
• Shown to be carcinogenic—form nitrosamines
5.6. Preservation of Perishable Products
 Low-Temperature Storage
• Refrigeration inhibits growth of pathogens and spoilage
organisms by slowing or stopping enzyme reactions
• Psychrotrophs, psychrophilic organisms can still grow
• Freezing preserves by stopping all microbial growth
• Some microbial cells killed by ice crystal formation, but
many survive and can grow once thawed
5.6. Preservation of Perishable Products
 Reducing Available Water
• Accomplished by salting, adding sugar, or drying food
• Addition of salt, sugar increases environmental solutes
• Causes cellular plasmolysis (water exits bacterial cells)
• Some bacteria grow in high salt environments
• Staphylococcus aureus
• Drying often supplemented by salting
• Lyophilization (freeze drying) foods
• Coffee, milk, meats, fruits, vegetables
• Drying stops microbial growth but does
not reliably kill
• Numerous cases of salmonellosis from
dried eggs
Contamination of an Operating Room
 Contamination occurs readily
 Cleaning afterwards critical