Tortora Chapter 7

1
Tortora Chapter 7

• Control of Microbial Growth
• Consult Tables 7.1, 7.5, 7.8

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Terminology

• Sterilization-destruction of ALL forms of
  microbial life
• Commercial sterilization-apply just enough heat
  in food preparations to destroy endospores of
  Clostridium botulinum
• Disinfection-destruction of vegetative pathogens
  on inert surfaces
• Antisepsis-destruction of vegetative pathogens on
  living tissue

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Terms continued

• Degerming-mechanical removal of microbes in a
  limited area (alcohol swab before a shot)
• Sanitization-lowers microbial counts to safe
  levels (washing then dipping in chemical
  disinfectant in a bar)
• icide-to kill
• stasis-to stop or to steady
• Aseptic-object or area is free of pathogens
• Asepsis-absence of significant contamination
• Sepsis-bacterial contamination

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Rate of microbial death

• Populations die at a specific rate when heated or
  treated with antimicrobial chemicals
• 1 million treated for 1 minute results in a death
  rate of 90 (leaves 100,000)
• Treat again, leaves 10,000
• Treat again, leaves 1,000
• Plot the curve logarithmically (see figure 7.1)

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Factors contributing to effectiveness of
antimicrobial treatments

• 1. number of microbes (more takes longer)
• 2. environmental influences (organic matter like
  vomit or blood may harbor organisms)
• 3. Time of exposure (follow directions on
  bottles for maximum effectiveness)
• 4. microbial characteristics (some are just
  harder to kill due to virulence factors)

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Good web sites

• http//www.cellsalive.com/ecoli.htm

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Actions of microbial control agents

• 1. Alteration of membrane permeability-damage to
  lipids or proteins can cause leakage
• 2. Damage to proteins-heat can denature proteins
  by breaking hydrogen bonds, disulfide bridges
• 3. nucleic acids-DNA or RNA can be damaged by
  heat, radiation, or chemicals
• 4. Cell wall can be damaged (often the
  peptidoglycan layer is damaged)

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Physical methods of control

• Heat resistance varies among microorganisms
• TDP (thermal death point) is the lowest
  TEMPERATURE required to kill bacteria in a liquid
  suspension at a given time (10 minutes)
• TDT (thermal death time) is the lowest TIME
  required to kill bacteria in a liquid suspension
  at a set temperature
• DRT-decimal reduction time is the time in minutes
  in which 90 of population at a given temp. is
  killed

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Heat

• 1. Dry heat-(denature enzymes) oven (170 Degrees
  C for 2 hours
• and incineration (direct flame is an example)
• 2. moist heat-(coagulation of proteins)
• Boiling (20 minutes usually)
• Autoclave-steam under pressure (121 C-15 psi-15
  minutes)
• Cant use aluminum foil to steam!

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Pasteurization

• Set times and temps used to eliminate or reduce
  pathogens
• Classic pasteurization of milk-63 C 30 minutes
• Modern pasteurization-72 C 15 seconds (HTST)
• UHT allows milk to be sterilized 140 C for 3
  seconds (used in non-refrigerated situations)

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Filtration

• Passage of liquid or gas through screen like
  filters
• Used to sterilize heat sensitive materials
• HEPA-high efficiency particulate air filters are
  used in operating rooms and remove bacteria
  larger than 0.3 microns
• Membrane filters are made from cellulose esters
  or plastic polymers can filter liquids (see
  figure 7.4)

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Low temperature

• Refrigeration usually reduces metabolic rate and
  is bacteriostatic
• Freezing is more harmful because it causes ice
  crystals to form and disrupts the cellular and
  molecular structure

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High pressure

• Alters molecular structure of proteins and
  carbohydrates
• Endospores are often resistant to high pressure,
  but are susceptible if this is combined with
  something else (like steam)

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Other methods

• Desiccation-removing water by lyophilization
  (freeze drying)
• Osmotic pressure-high concentration of salt or
  sugar (molds and yeasts can still grow)
• Microwaves-not very good, must have moisture,
  heat kills the vegetative pathogens
• Uneven distribution
• Be careful!
• http//iowalakes.edu/faculty/klepper/Microbiology/
  Lecture/07Control20of20Microbial20Growth.pdf

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Radiation-2 types that kill

• Ionizing radiation-gamma rays, x-rays, high
  energy electron beams, have short wavelengths
• This causes ionization of water and releases
  hydroxyl radicals which react with cellular
  components including DNA
• Nonionizing radiation-UV light, longer
  wavelength, forms thymine dimers which causes
  mutations in DNA (doesnt penetrate glass or
  plastic)

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Chemical methods

• Evaluation of disinfectant is done by either
• Use dilution test uses Salmonella,
  Staphylococcus, and/or Pseudomonas dipped into a
  solution of the disinfectant for 10 minutes at 20
  C, then growth is checked.
• Disk-diffusion test uses a disc soaked in the
  disinfectant then placed on a petri dish with the
  bacteria, incubate, then look at zone of
  inhibition (see figure 7.6)

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Phenolics and Phenol

• Phenol is the standard and was used by Lister
  (Phenol coefficient is 1)
• Phenol is caustic and nasty and is rarely used
  anymore
• Phenolics are derivatives of phenol that injure
  the plasma membrane
• These remain active in organic material, are
  stable, and persist for long periods

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Phenolics continued

• One example is derived from coal tar and is a
  group called cresols
• One cresol is O-phenylphenol and is the main
  ingredient in Lysol.

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Bisphenols

• are derivatives of phenol with 2 phenolic groups.
• One example is hexachlorophene which is used in
  pHisoHex (not used to bathe infants any longer,
  found nerve damage in rats)
• Another example is Triclosan which is in soaps,
  toothpaste, and cutting board plastic (some
  resistance has been reported)
• Triclosan inhbits an enzyme needed to synthesize
  fatty acids and affects the plasma membrane

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Biguanides

• Chlorhexidine is example
• Broad spectrum, but endospores and Mycobacteria
  are resistant
• Used on skin and mucous membranes, can damage
  eyes so use caution!
• Used as scrub and preoperative skin preparation
• Avagard combines chlorhexidine with ethanol
• Blocks lipid synthesis in plasma membrane

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Halogen Group

• Iodine is one of the oldest antiseptics
• Impairs protein synthesis and is very effective
• Tincture is iodine mixed with alcohol
• Iodophor is combination of iodine with organic
  molecule (released slowly)
• Iodophors are less staining and less irritating
  (Betadine is example)
• Can use iodine to disinfect water in emergency

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Halogens continued

• Chlorine is a gas or may be mixed with water or
  other chemicals
• Calcium hypochlorite is used to disinfect dairy
  equipment (used by Semmelweis to control
  infections during childbirth)
• Liquid chlorine gas is used to disinfect drinking
  water
• Sodium hypochlorite is household bleach
  (Clorox)light sensitive
• Can disinfect water supply with 2 drops bleach
  per liter of water and set 30 minutes.

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Chloramines

• Chloramines (chlorine and ammonia) disinfect
  glassware in restaurants
• Adding ammonia to chlorine will cut taste and
  odor problems caused by reaction of chlorine with
  nitrogenous waste in water
• Must add a lot of chlorine because these are less
  effective
• These are toxic to aquarium fish, so must add
  neutralizer.

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Alcohols

• Dont kill endospores or nonenveloped viruses
• May disrupt lipid in plasma membrane or protein
  denaturated
• Leave no residue
• Used to degerm skin before injection
• Do not use on wounds, will cause coagulation of
  protein in skin allowing bacteria to grow under
  that.

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Types of alcohol

• 1. ethanol-70 is recommended concentration
  (denaturation requires water)
• 2. Isopropanol (rubbing alcohol) is slightly
  better as antiseptic and disinfectant and is
  cheaper!
• Often used to enhance other chemicals (tinctures)
• 3. Methanol-TOXIC!

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Heavy Metals

• Oligodynamic action-metal ions combine with
  sulfhydryl groups on cellular proteins,
  denaturing them
• 1. Silver is used as an antiseptic in the form of
  silver nitrate (1)-once used to treat newborns
  eyes to guard against gonorrheal opthalmia
  neonatorium
• Silver-sulfadiazine dressings and creams used for
  burns
• Surfacine is new antimicrobial for living or
  non-living surfaces, lasts 13 days!

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Heavy Metals continued

• 2. Mercury compounds including mercuric chloride
  is bacteriostatic (merthiolate was tincture,
  mercurochrome wasnt)
• Use is limited due to toxicity, and
  ineffectiveness on organic matter
• Older generation still swears by this stuff
• Now used to control mildew in paint
• 3. copper sulfate-used in swimming pools to
  destroy algae
• 4. Zinc chloride is common in mouthwash, zinc
  oxide is antifungal agent in paint

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Surface Active Agents

• Also called surfactants and reduce the surface
  tension http//www.exploratorium.edu/ronh/bubbles/
  sticky_water.html
• 1. soaps and detergents-good in degerming
  (mechanical removal)
• 2. acid-anionic sanitizer-clean dairy utensils

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More surfactants

• 3. quaternary ammonium compounds or QUATS
• very good against gram positive and less so
  against gram negatives and mycobacteria
• Also fungicidal, ameobicidal, virucidal against
  enveloped viruses
• Brand names Zephiran and Cepacol (foam when
  shaken up)
• Neutralized by soaps and anionic detergents
• Pseudomonas loves to grow in this stuff! See p.
  203 box

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Chemical food preservatives

• Sulfur dioxide used in wine making
• Sodium benzoate, sorbic acid, calcium propionate
  are common organic acids
• Often act as antifungals and inhibit mold growth
  on bread, etc.
• Sodium nitrate and sodium nitrite are meat
  preservatives that help prevent botulism
• May cause nitrosamines that are carcinogens

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Aldehydes

• Among the most effective antimicrobials
• 1. formaldehyde gas-more commonly used as
  formalin (37)
• Has a residue that may cause allergic reactions
• 2. glutaraldehyde-relative of formaldehyde that
  is used as a 2 solution (Cidex)
• Kills viruses, spores, and bacteria quickly
• Can be considered a true sterilizing agent
• Light sensitive

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Gaseous chemosterilizers

• Pumped into chambers
• Ethylene oxide-kills all microbes and endospores
  after long exposure time
• Toxic and explosive in pure form so mix with
  carbon dioxide or nitrogen
• Used to clean space craft (NASA)
• Also used to clean mattresses and other large
  items in hospitals

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Peroxygens (oxidizing agents)

• Ozone-used to supplement chlorine in water
  because it neutralizes taste and odor
• Hydrogen peroxide-antiseptic in the brown bottle,
  not good on open wounds and slows healing (never
  use on punctures either)
• Benzoyl peroxide-used in treating wounds infected
  by anaerobic bacteria and as an anti-acne
  medicine
• Peracetic acid is one of the most effective
  sporicids and is a sterilant (no toxic residue)