Chapter 7 Control of Microorganisms

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Chapter 7Control of Microorganisms

• Definitions
• Conditions Influencing Antimicrobial Activity
• Physical Methods
• Chemical Agents
• Preservation of Microbial Cultures

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Definitions

• Sterilization A treatment that kills or removes
  all living cells, including viruses and spores,
  from a substance or object
• Disinfection A treatment that reduces the total
  number of microbes on an object or surface, but
  does not necessarily remove or kill all of the
  microbes
• Antiseptic A mild disinfectant agent suitable
  for use on skin surfaces

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Definitions

• Sanitation Reduction of the microbial population
  to levels considered safe by public health
  standards
• -cidal A suffix meaning that the agent kills.
  For example, a bacteriocidal agent kills bacteria
• -static A suffix that means the agent inhibits
  growth. For example, a fungistatic agent
  inhibits the growth of fungi, but doesnt
  necessarily kill it.

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Conditions Influencing Antimicrobial Activity

• Under most circumstances, a microbial population
  is not killed instantly by an agent but instead
  over a period of time
• The death of the population over time is
  exponential, similar to the growth during log
  phase

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Conditions Influencing Antimicrobial Activity

• Several critical factors play key roles in
  determining the effectiveness of an antimicrobial
  agent, including
• Population size
• Types of organisms
• Concentration of the antimicrobial agent
• Duration of exposure
• Temperature
• pH
• Organic matter
• Biofilm formation

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Physical Methods

• Moist Heat
• Dry Heat
• Low Temperatures
• Filtration
• Radiation

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Physical Methods Moist Heat

• Mechanism of killing is a combinantion of
  protein/nucleic acid denaturation and membrane
  disruption
• Bacterial spores much more difficult to kill than
  vegetative cells

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Physical Methods Moist Heat

• Methods of Moist Heat
• Boiling at 100C
• Effective against most vegetative cells
  ineffective against spores unsuitable for heat
  sensitive chemicals many foods
• Autoclaving/pressure canning
• Temperatures above 100C achieved by steam
  pressure
• Most procedures use 121.1C, achieved at approx.
  15 psi pressure, with 15 - 30 min autoclave time
  to ensure sterilization
• Sterilization in autoclave in biomedical or
  clinical laboratory must be periodically
  validated by testing with spores of Clostridium
  or Bacillus stearothermophilus

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Physical Methods Moist Heat

• Methods of Moist Heat
• Pasteurization
• Used to reduce microbial numbers in milk and
  other beverages while retaining flavor and food
  quality of the beverage
• Retards spoilage but does not sterilize
• Traditional treatment of milk, 63C for 30 min
• Flash pasteurization (high-temperature short term
  pasteurization) quick heating to about 72C for
  15 sec, then rapid cooling

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Physical Methods Moist Heat

• Methods of Moist Heat
• Ultrahigh-temperature (UHT) sterilization
• Milk and similar products heated to 140 - 150C
  for 1 - 3 sec
• Very quickly sterilizes the milk while keeping
  its flavor quality
• Used to produce the packaged shelf milk that
  does not require refrigeration

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Physical Methods Dry Heat

• Incineration
• Burner flames
• Electric loop incinerators
• Air incinerators used with fermenters generally
  operated at 500C
• Oven sterilization
• Used for dry glassware heat-resistant metal
  equipment
• Typically 2 hr at 160C is required to kill
  bacterial spores by dry heat this does not
  include the time for the glass to reach the
  required temp (penetration time) nor does it
  include the cooling time

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Physical MethodsLow Temperatures

• Refrigerator
• around 4C
• inhibits growth of mesophiles or thermophiles
  psychrophiles will grow
• Freezer
• ordinary freezer around -10 to -20C
• ultracold laboratory freezer typically -80C
• Generally inhibits all growth many bacteria and
  other microbes may survive freezing temperatures

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Physical Methods Filtration

• Used for physically removing microbes and dust
  particles from solutions and gasses often used
  to sterilize heat-sensitive solutions or to
  provide a sterilized air flow
• Depth filters eg. Diatomaceous earth, unglazed
  porcelean
• Membrane filters eg. Nitrocellulose, nylon,
  polyvinylidene difluoride
• HEPA filters High efficiency particulate air
  filters used in laminar flow biological safety
  cabinets

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Physical Methods Radiation

• Ionizing Radiation
• Gamma radiation produced by Cobalt-60 source
• Powerful sterilizing agent penetrates and
  damages both DNA and protein effective against
  both vegetative cells and spores
• Often used for sterilizing disposable plastic
  labware, e.g. petri dishes as well as
  antibiotics, hormones, sutures, and other
  heat-sensitive materials
• Also can be used for sterilization of food has
  been approved but has not been widely adopted by
  the food industry

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Physical Methods Radiation

• Non-Ionizing Radiation
• Ultraviolet Radiation
• DNA absorbs ultraviolet radiation at 260 nm
  wavelength
• This causes damage to DNA in the form of thymine
  dimer mutations
• Useful for continuous disinfection of work
  surfaces, e.g. in biological safety cabinets

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Chemical Agents

• Phenolics
• Alcohols
• Halogens
• Heavy metals
• Quaternary Ammonium Compounds
• Aldehydes
• Sterilizing Gases
• Evaluating Effectiveness of Chemical Agents

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Chemical Agents Phenolics

• Aromatic organic compounds with attached -OH
• Denature protein disrupt membranes
• Phenol, orthocresol, orthophenylphenol,
  hexachlorophene
• Commonly used as disinfectants (e.g. Lysol)
  effective in presence of organic matter, remain
  on surfaces long after application
• Disagreeable odor skin irritation
  hexachlorophene once used as an antiseptic but
  its use is limited as it causes brain damage

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Chemical Agents Alcohols

• Ethanol isopropanol used at concentrations
  between 70 95
• Denature proteins disrupt membranes
• Kills vegetative cells of bacteria fungi but
  not spores
• Used in disinfecting surfaces thermometers
  ethanol-flaming technique used to sterilize
  glass plate spreaders or dissecting instruments
  at the lab bench

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Chemical Agents Halogens

• Act as oxidizing agents oxidize proteins other
  cellular components
• Chlorine compounds
• Used in disinfecting municiple water supplies (as
  sodium hypochlorite, calcium hypochlorite, or
  chlorine gas)
• Sodium Hypochlorite (Chlorine Bleach) used at 10
  - 20 dilution as benchtop disinfectant
• Halazone tablets (parasulfone dichloroamidobenzoic
  acid) used by campers to disinfect water for
  drinking

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Chemical Agents Halogens

• Iodine Compounds
• Tincture of iodine (iodine solution in alcohol)
• Potassium iodide in aqueous solution
• Iodophors Iodine complexed to an organic
  carrier e.g. Wescodyne, Betadyne
• Used as antiseptics for cleansing skin surfaces
  and wounds

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Chemical Agents Heavy Metals

• Mercury, silver, zinc, arsenic, copper ions
• Form precipitates with cell proteins
• At one time were frequently used medically as
  antiseptics but much of their use has been
  replaced by less toxic alternatives
• Examples 1 silver nitrate was used as opthalmic
  drops in newborn infants to prevent gonorrhea
  has been replaced by erythromycin or other
  antibiotics copper sulfate used as algicide in
  swimming pools

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Chemical Agents QuaternaryAmmonium Compounds

• Quaternary ammonium compounds are cationic
  detergents
• Amphipathic molecules that act as emulsifying
  agents
• Denature proteins and disrupt membranes
• Used as disinfectants and skin antiseptics
• Examples cetylpyridinium chloride, benzalkonium
  chloride

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Chemical Agents Aldehydes

• Formaldehyde and gluteraldehyde
• React chemically with nucleic acid and protein,
  inactivating them
• Aqueous solutions can be used as disinfectants

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Chemical Agents Sterilizing Gases

• Ethylene oxide (EtO)
• Used to sterilize heat-sensitive equipment and
  plasticware
• Explosive supplied as a 10 20 mixture with
  either CO2 or dichlorofluoromethane
• Its use requires a special EtO sterilizer to
  carefully control sterilization conditions as
  well as extensive ventilation after sterilization
  because of toxicity of EtO
• Much of the commercial use of EtO (for example,
  plastic petri dishes) has in recent years been
  replaced by gamma irradiation

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Chemical Agents Sterilizing Gases

• Betapropiolactone (BPL)
• In its liquid form has been used to sterilize
  vaccines and sera
• Decomposes after several hours and is not as
  difficult to eliminate as EtO, but it doesnt
  penetrate as well as EtO and may also be
  carcinogenic
• Has not been used as extensively as EtO
• Vapor-phase hydrogen peroxide
• Has been used recently to decontaminate
  biological safety cabinets

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Chemical AgentsEvaluating the Effectiveness

• Phenol Coefficient Test
• A series of dilutions of phenol and the
  experimental disinfectant are inoculated with
  Salmonella typhi and Staphylococcus aureus and
  incubated at either 20C or 37C
• Samples are removed at 5 min intervals and
  inoculated into fresh broth
• The cultures are incubated at 37C for 2 days
• The highest dilution that kills the bacteria
  after a 10 min exposure, but not after 5 min, is
  used to calculate the phenol coefficient

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Chemical AgentsEvaluating the Effectiveness

• Phenol Coefficient Test (cont.)
• The reciprocal of the maximum effective dilution
  for the test disinfectant is divided by the
  reciprocal of the maximum effective dilution for
  phenol to get the phenol coefficient
• For exampleSuppose that, on the test with
  Salmonella typhiThe maximum effective dilution
  for phenol is 1/90The maximum effective dilution
  for Disinfectant X is 1/450The phenol
  coefficient for Disinfectant X with S. typhi
  450/90 5

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Chemical AgentsEvaluating the Effectiveness

• Phenol Coefficient Test (cont.)
• Phenol coefficients are useful as an initial
  screening and comparison, but can be misleading
  because they only compare two pure strains under
  specific controlled conditions
• Use dilution tests and simulated in-use tests
• Are tests designed to more closely approximate
  actual normal in-use conditions of a disinfectant

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Preservation of Microbial Cultures

• Periodic Transfer and Refrigeration
• Mineral Oil Slant
• Freezing in Growth Medium
• Drying
• Lyophilization
• Ultracold Freezing

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Preservation of Microbial CulturesPeriodic
Transfer and Refrigeration

• Stock cultures are aseptically transferred at
  appropriate intervals to fresh medium and
  incubated, then stored at 4C until they are
  transferred again
• Many labs use agar slants care has to be taken
  to avoid contamination
• Major problem with possible genetic changes in
  strains most labs need a way to keep long term
  storage of original genetic stocks

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Preservation of Microbial CulturesMineral Oil
Slant

• Sterile mineral oil placed over growth on agar
  slants to preserve cultures for longer period of
  time in the refrigerator
• Contamination problems messy many organisms are
  sensitive to this generally it is a poor
  technique and doesnt work well

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Preservation of Microbial CulturesFreezing in
Growth Medium

• Used as a long term storage strategy
• Broth cultures of the organisms are frozen at
  -20C
• Often, sterile glycerin (glycerol) is added at a
  25 50 final concentration this helps to
  prevent ice crystal formation and increases
  viability of many organisms

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Preservation of Microbial CulturesDrying

• Suitable for some bacterial species
• Samples are grown on sterile paper disks
  saturated with nutrient, then the disks are
  allowed to air dry and stored aseptically
• Reconstituted by dropping disk into nutrient
  broth medium

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Preservation of Microbial CulturesLyophilization

• Suitable for many bacterial species as well as
  fungi and viruses
• Broth cultures are placed in special ampules and
  attached to a vacuum pump the vacuum removes all
  of the water from the cells leaving a
  freeze-dried powder
• The culture is reconstituted by adding broth to
  the lyophilized powder and incubating it
• Considered the best method of long-term storage
  for most bacterial species

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Preservation of Microbial CulturesUltracold
Freezing

• Similar to freezing, but at very cold temperature
• At about -70 to -80C, in liquid nitrogen or in
  an ultracold freezer unit