HOW TO KILL A NASTY LITTLE BUG

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CONTROL OF
MICROBIAL GROWTH

. . . OR . . .
HOW TO KILL A NASTY LITTLE BUG
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CONTROL OF MICROBES

• The undesired growth of microorganisms is
  problematic in many ways
• Infection during surgery
• Food production
• Food storage
• Etc.
• It is important to control the growth of microbes

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APPROACHES TO CONTROL

• Physical methods
• Heat
• Irradiation
• Filtration
• Mechanical removal
• Etc.
• Chemical methods
• Various chemicals

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STERILIZATION

• Removal or destruction of all microorganisms
• There is no such thing as somewhat sterile
• Accomplished by various means
• Endospores destroyed
• Prions not destroyed by standard treatment

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DISINFECTION

• Elimination of most or all disease-causing
  microbes
• Some microbes may persist
• Generally involves use of antimicrobial chemicals
• Disinfectants
• Biocides / germicides / antiseptics
• May involve processing
• Pasteurization
• Brief heat treatment
• Kills disease-causing organisms
• Reduces numbers of spoilage organisms

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DISINFECTION

• Decontamination
• Numbers of disease-causing organisms reduced
• Safe to handle
• Sanitization
• Reduced microbial population
• Generally meets some health standard
• Preservation
• Growth-inhibiting ingredient added
• Spoilage delayed

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MICROBIAL CONTROL

• Methods of microbial control differ greatly
• Dependent on situation
• Different requirements for
• Daily life
• Hospitals
• Microbiology laboratories
• Foods food production facilities
• Other industries

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MICROBIAL CONTROL

• Daily life
• Washing scrubbing
• Routine control of undesirable microbes
• Plain soap does not kill microbes
• Aids in mechanical removal of transient microbes
• Normal skin flora deeper in skin, not adversely
  impacted
• Antibacterial soap
• Cooking refrigeration of foods
• Cleaning of surfaces

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MICROBIAL CONTROL

• Hospitals
• Danger of hospital-acquired (noscomial)
  infections
• Disease-causing organisms more plentiful
• Numerous patients with infectious diseases
• Infectious particles shed
• Patients often more susceptible to infection
• Weakened immune system
• Invasive procedures penetrate skin
• Disinfection sterilization

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MICROBIAL CONTROL

• Microbiology laboratories
• Routinely work with microbial cultures
• Various methods of control
• Media sterilized prior to inoculation
• Aseptic technique
• Treatment of material prior to disposal

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MICROBIAL CONTROL

• Foods production facilities
• Foods last longer when growth of contaminating
  microbes is prevented or reduced
• Remove or destroy microbes
• e.g., Sterilization
• Inhibit growth
• e.g., Pasteurization, irradiation, chemical
  additives
• Production facilities must keep surfaces clean

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MICROBIAL CONTROL

• Other industries
• Specialized concerns in diverse industries
• e.g., cosmetics, deodorant, etc.

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ANTIMICROBIAL PROCEDURES

• Selection of a procedure
• No universal best procedure
• Many factors to consider
• Type of microorganism
• Numbers of microbes initially present
• Environmental conditions
• Potential risk of infection

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ANTIMICROBIAL PROCEDURES

• Selection of a procedure
• Type of microorganism
• Endospores require extreme treatment
• Waxy cell walls render Mycobacterium species
  resistant to many chemicals
• Pseudomonas resistant to many chemicals
• Naked viruses more resistant to disinfectants

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ANTIMICROBIAL PROCEDURES

• Selection of a procedure
• Numbers of microbes initially present
• It takes longer to kill a large population than
  to kill a small one
• e.g., If three minutes is sufficient to kill 90
  of the microbes, an additional three minutes will
  kill 90 of those remaining, and so on

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ANTIMICROBIAL PROCEDURES

• Selection of a procedure
• Environmental conditions
• Death rate strongly influenced by temperature,
  pH, and presence of fats and other materials
• e.g., ? Temperature ? ? death rate
• e.g., ? pH ? ? death rate (bleach)
• Dirt, etc. hides bacteria from antimicrobial
  agent, ? death rate
• Cleaning important prior to sterilization

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ANTIMICROBIAL PROCEDURES

• Selection of a procedure
• Potential risk of infection
• More rigorous sterilization procedures required
  for items more likely to transmit infectious
  agents
• Critical items
• Direct contact with body tissue
• e.g., Needles, scalpels, etc.
• Semicritical items
• Contact with mucous membranes
• e.g., Endoscopes, specula, etc.
• Noncritical items
• Contact with unbroken skin
• e.g., Stethoscopes, b.p. cuffs, etc.

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USE OF HEAT

• Effectiveness known prior to discovery of
  microbes
• Alexander the Greats army boiled water to
  prevent disease
• Aristotle was a good teacher
• Still very useful
• Fast, reliable, inexpensive
• Can be used to sterilize
• e.g., Autoclaving
• Can be used to disinfect
• e.g., Pasteurization

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USE OF HEAT

• Moist heat
• e.g., boiling water, pressurized steam
• Irreversibly denatures proteins
• Endospores resistant

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USE OF HEAT

• Moist heat Pasteurization
• Developed by Louis Pasteur
• Initially developed to avoid spoilage of wine
• Originally 62oC for 30 minutes (milk)
• Now 72oC for 15 seconds (milk)
• High-temperature-short-time (HTST) method
• 82oC for 20 seconds (ice cream)
• Richer in fats than milk
• Also used for juices, wine, vinegar, etc.
• Preferentially eliminates spoilage microorganisms
• Does not sterilize substances
• Taste not significantly altered

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USE OF HEAT

• Moist heat pressurized steam
• Pressure heat
• ? pressure ? ? boiling point
• Steam temperatures gt 100oC
• e.g., pressure cooker
• e.g., autoclave
• Sterilize at i5 psi, 121oC, 15 minutes
• Verify process in various ways
• e.g., heat-sensitive indicator, etc.

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AUTOCLAVE
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USE OF HEAT

• Moist heat commercial canning
• Industrial-sized autoclave
• Retort
• Process designed to destroy endospores
• Esp. Clostridium botulinum
• Destroys all endospores except those of some
  thermophilic bacteria
• Usually of no concern
• Incapable of growth at storage temperatures

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COMMERCIAL CANNING
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USE OF HEAT

• Moist heat commercial canning
• Clostridium botulinum
• Endospore-forming
• Obligate anaerobe
• Germinates in canned goods
• Produces potent toxin
• Among most potent known toxins
• Ingestion of minute amounts is lethal
• Must destroy in canning process

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USE OF HEAT

• Dry heat
• Burns destroys cells
• Takes longer than wet heat
• 1.5 hrs _at_ 200oc vs. 15 min _at_ 121oc
• Used for
• Bacterial culture loops
• Glassware
• Etc.

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CHEMICAL METHODS

• Germicidal chemicals
• Used to disinfect
• Sometimes used to sterilize
• Most specifically target a cellular component
• e.g., ribosomes, an enzyme, etc.
• Less reliable than heat, but
• Can treat large surfaces
• Can treat heat-sensitive items

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GERMICIDAL CHEMICAL SITES OF ACTION
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CHEMICAL METHODS

• Germicidal chemicals
• Sterilants
• High-level disinfectants
• Intermediate-level disinfectants
• Low-level disinfectants

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CHEMICAL METHODS

• Selection of appropriate germicidal chemicals
• Must consider
• Toxicity
• Activity in presence of organic matter
• Compatibility with material being treated
• Residue
• Cost availability
• Storage stability
• Environmental risk

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CHEMICAL METHODS

• Selection of appropriate germicidal chemicals
• Toxicity
• Most germicides are somewhat toxic to humans
  the environment
• Risk vs. Benefit analysis
• Activity in presence of organic matter
• Some germicidal chemicals inactivated by organic
  matter
• Compatibility with material being treated
• Some equipment cannot tolerate some germicides
• E.G., Electrical equipment liquid germicides

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CHEMICAL METHODS

• Selection of appropriate germicidal chemicals
• Residue
• Toxic or corrosive residue
• Cost availability
• Storage stability
• Concentrated stocks, etc.
• Environmental risk
• Neutralization prior to disposal

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CHEMICAL METHODS

• Classes of germicidal chemicals
• Alcohols
• Aldehydes
• Biguanides
• Ethylene oxide
• Halogens
• Metal compounds
• Ozone
• Peroxygens
• Phenolics
• Quaternary ammonium compounds

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GERMICIDAL CHEMICALS
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FILTRATION

• Some solutions are heat-sensitive
• e.g., sugar solutions
• Microorganisms can be removed by filtration
• Two types of filters
• Depth filters
• Membrane filters

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FILTRATION
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FILTRATION

• Depth filters
• Used for over a century
• Microbes retained by torturous passageway
• Passageway often larger than microorganisms
• Electrical charges assist in retention of
  microorganisms
• Fluid passes through these passageways

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FILTRATION

• Membrane filters
• Developed recently
• Cellulose acetate, etc.
• Paper-thin
• Fluid readily passes through
• Various pore sizes
• Some smaller than smallest known viruses

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FILTRATION

• Air filtration
• High-efficiency particulate air (hepa) filters
• Anything gt 0.3 mm removed from air
• Used for
• Specialized hospital rooms
• Laminar flow hoods

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RADIATION

• Various types of waves
• Gamma rays
• X-rays
• Ultraviolet (uv) light waves
• Microwaves
• Etc.

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RADIATION

• Gamma irradiation
• Ionizing radiation
• Produces reactive molecules
• e.g., Superoxide (O2-), OH radicals
• Gram-negative bacteria highly susceptible
• Endospores highly resistant
• Useful for heat-sensitive materials
• Useful for foods
• Sometimes sterilization
• Usually pasteurization

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RADIATION

• Ultraviolet irradiation
• Damages DNA ? kills microorganisms
• Forms thymine dimers (T-T)
• More effective vs. actively multiplying organisms
• Same reaction responsible for skin cancer
• Penetrates poorly
• Treatments sensitive to extraneous materials

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RADIATION

• Microwaves
• Do not kill directly
• Kill microbes by heating material
• Many microbes survive uneven heating during
  cooking

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PRESERVATION

• Preventing or slowing growth of microorganisms
• Extends shelf life of many products
• Foods, soaps, medicines, deodorants, cosmetics,
  etc.
• Approaches to preservation
• Chemical preservatives
• Low temperature storage
• Reducing available water

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PRESERVATION

• Chemical preservatives
• Germicidal chemicals used in non-food items
• Food preservatives must be non-toxic
• Weak organic acids
• Low pH effective
• Nitrate or nitrite
• Prevent endospore germination
• React with myoglobin ? nice pink color
• Other reactions ? carcinogens
• Risk vs. benefit

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PRESERVATION

• Low-temperature storage
• Temperature affects microbial growth
• Refrigeration
• Reactions are slow at low temperatures
• Growth of most microorganisms is slow
• Freezing
• Water becomes unavailable
• Microbial growth stopped
• Ice crystals kill some, remainder can grow when
  thawed

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PRESERVATION

• Reducing available water
• Stops growth, doesnt reliably kill
• Drying directly removes water
• Desiccation
• Salting removes water through plasmolysis
• High concentration of salt or sugar
• Water exits cells via osmosis
• Some microbes can tolerate high salt
  concentrations