Control%20of%20Microbial%20Growth

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Control of Microbial Growth
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• Introduction
• Early civilizations practiced salting, smoking,
  pickling, drying, and exposure of food and
  clothing to sunlight to control microbial growth.
• Use of spices in cooking was to mask taste of
  spoiled food. Some spices prevented spoilage.
• In mid 1800s Semmelweiss and Lister helped
  developed aseptic techniques to prevent
  contamination of surgical wounds.
• Before then
• Nosocomial infections caused death in 10 of
  surgeries.
• Up to 25 mothers delivering in hospitals died
  due to infection

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• Definitions
• Sterilization Killing or removing all forms of
  microbial life (including endospores) in a
  material or an object.
• Disinfection Reducing the number of pathogenic
  microorganisms to the point where they no longer
  cause diseases. Usually involves the removal of
  vegetative or non -endospore forming pathogens.

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• Sepsis Comes from Greek for decay or putrid.
  Indicates bacterial contamination.
• Asepsis Absence of significant contamination.
• Aseptic techniques are used to prevent
  contamination of surgical instruments, medical
  personnel, and the patient during surgery.
• Aseptic techniques are also used to prevent
  bacterial contamination in food industry.

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• Disinfectant Applied to inanimate objects.
• Antiseptic Applied to living tissue
  (antisepsis).
• Degerming Mechanical removal of most microbes in
  a limited area.
• Sanitization Use of chemical agent on
  food-handling equipment to meet public health
  standards and minimize chances of disease
  transmission.

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General Considerations in Sterilization
Disinfection

• 1) Population unknown.
• 2) Determination of level of kill.
• 3) Heterogeneity within population.
• 4) Troublesome forms.
• 5) Environmental factors.
• 6) No Universal Method.
• 7) Careless handling.
• 8) Effect on Bacteria as the principal criteria.
• 9) Critical, semi-critical, and non critical
  items.

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• Physical Methods of S/D
• Heat
• Kills microorganisms by denaturing their enzymes
  and other proteins.
• Heat resistance varies widely among microbes.
• Thermal Death Point (TDP) the temperature at
  which all of the test microorganisms in a 24 hour
  broth culture are killed in ten minutes.
• Thermal Death Time (TDT) the time required to
  kill all microorganisms of a known population at
  a given temperature under specific conditions.
• Decimal Reduction Time (DRT) Time in minutes at
  which 90 of bacteria at a given temperature will
  be killed.

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• Moist Heat
• Kills microorganisms by coagulating their
  proteins.
• In general, moist heat is much more effective
  than dry heat.
• Boiling Heating to 100oC at sea level. Kills
  vegetative forms of bacterial pathogens, almost
  all viruses, and fungi and their spores within 10
  minutes or less.
• - Hepatitis B virus Can survive up to 30
  minutes of
• boiling.
• - Endospores Can survive up to 20 hours or
  more of
• boiling.

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• Reliable sterilization with moist heat requires
  temperatures above that of boiling water.
• Steam at atmospheric pressure
• Steam at 100C has 540 calories of latent heat as
  compared to the 80 calories for boiling water at
  that temperature.
• - Used to disinfect pipes, vats, in diaries and
  breweries.

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• Steam under pressure( Autoclaving)
• At a temperature of 121C of moist heat for 15
  minutes spores are destroyed.
• In order to obtain this T saturated steam is
  placed under a pressure of 15 Psi (15 libra/inch2
  or 1.05 kg/cm2).
• Pressure (Psi) TC Time (minutes)
• 15 121 15
• 20 126 10
• 30 134 3

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Autoclave Closed Chamber with High Temperature
and Pressure
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• Pasteurization Developed by Louis Pasteur to
  prevent the spoilage of beverages. Used to
  reduce microbes responsible for spoilage of milk,
  juices, beer, wine, etc.
• Classic Method of Pasteurization exposure to a
  T of 65oC for 30 minutes.
• High Temperature Short Time Pasteurization
  (HTST) Used today. Exposure to a T of 72oC for
  15 seconds.
• Ultra High Temperature Pasteurization (UHT)
  Exposure to a T of 140oC for 3 seconds and then
  cooling very quickly in a vacuum chamber.
  Advantage can be stored at room temperature for
  several months.
• Fractional sterilization (Tyndalization )

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• Dry Heat Kills by oxidation effects.
• Direct Flaming Used to sterilize inoculating
  loops and needles. Heat metal until it has a red
  glow.
• Incineration Effective way to sterilize
  disposable items (paper cups, dressings) and
  biological waste.
• Hot Air Sterilization Place objects in an oven.
  Require 2 hours at 170oC for sterilization. Dry
  heat transfers heat less effectively to a cool
  body, than moist heat.

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• Low Temperature
• Effect depends on microbe and treatment applied.
• Refrigeration Temperatures from 2o to 8oC.
  Bacteriostatic effect. Reduces metabolic rate of
  most microbes so they cannot reproduce or produce
  toxins.
• Freezing Temperatures below 0oC.
• Flash Freezing Does not kill most microbes.
• Slow Freezing More harmful because ice crystals
  disrupt cell structure.
• Freeze- drying (lyophilization)
• Over a third of vegetative bacteria may survive
  1 year.
• Most parasites are killed by a few days of
  freezing.

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• Filtration
• Used to sterilize heat sensitive materials like
  vaccines, enzymes, antibiotics, and some culture
  media.
• Removal of microbes by passage of a liquid or gas
  through a screen like material with small pores.
• High Efficiency Particulate Air Filters (HEPA)
  Used in operating rooms and burn units to remove
  bacteria from air.
• Membrane Filters Uniform pore size. Used in
  industry and research. Different sizes
• 0.22 and 0.45um Pores Used to filter most
  bacteria. Dont retain spirochetes, mycoplasmas
  and viruses.
• 0.01 um Pores Retain all viruses and some large
  proteins.

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• Ultrasonication
• High frequency electrical energy ? transducers ?
  high frequency sound waves (mechanical)
• High frequency sound waves are above the human
  auditory level.
• - Cell disintegration
• - Sterilization
• - Cleansing purposes (ultrasonic cleaners) to
  remove soil and organic matter esp. from
  glassware.

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• Desiccation
• In the absence of water, microbes cannot grow or
  reproduce, but some may remain viable for years.
• Susceptibility to desiccation varies widely
• Neisseria gonnorrhea Only survives about one
  hour.
• Mycobacterium tuberculosis May survive several
  months.
• Viruses are fairly resistant to desiccation.
• Clostridium spp. and Bacillus spp. May survive
  decades.

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Radiation Forms of Radiation
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• Radiation
• Three types of radiation kill microbes
• Ionizing Radiation Gamma rays, X rays, electron
  beams, or higher energy rays.
• - Have short wavelengths (less than 1 nanometer).
  
• - Dislodge electrons from atoms and form ions.
• - Cause mutations in DNA and produce peroxides.
• - Used to sterilize pharmaceuticals and
  disposable medical supplies.
• - Food industry is interested in using ionizing
  radiation.

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• Their microbicidal action is due to the
  ionization of water.
• Ionized water interacts with nonionized water to
  produce hydroxyl radicals
• Radiation
• H2O ---------? H2O e
• H2O H2O ---? OH- H3O
• - Disadvantages Penetrates human tissues. May
  cause genetic mutations in humans.

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• Ultraviolet light (Nonionizing Radiation)
• Wavelength is longer than 1 nanometer.
• Damages DNA by producing thymine dimers, which
  cause mutations.
• Used to disinfect operating rooms, nurseries,
  cafeterias.
• Disadvantages Damages skin and eyes. Does not
  penetrate paper, glass, and cloth.

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• Microwave Radiation
• - Wavelength ranges from 1 millimeter to 1
  meter.
• Heat is absorbed by water molecules.
• May kill vegetative cells in moist foods.
• Bacterial endospores, which do not contain water,
  are not damaged by microwave radiation.
• Solid food is unevenly penetrated by microwaves.
• Trichinosis outbreaks have been associated with
  pork cooked in microwaves.

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Chemical Methods of S/D

• Qualities of a universal disinfectant
• Destroys all forms within a practical period of
  time
• Not irritant, allergenic or toxic
• Makes effective contact and not bypassed by
  dilution
• Readily soluble
• Chemically stable, no disagreeable odor, non
  discoloring and leaves no stains
• Not expensive

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• Important Factors
• Characteristics of microorganisms (chemical make
  up)
• Concentration of disinfectant
• PH of disinfectant (neutral to slightly acidic)
  affects degree of ionization
• Interfering matter
• 5) Time

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Types of Disinfectants

• Phenols and Phenolics
• Phenol (carbolic acid) was first used by Lister
  as a disinfectant.
• Rarely used today because it is a skin irritant
  and has strong odor.
• Used in some throat sprays and lozenges.
• Acts as local anesthetic.
• Phenolics are chemical derivatives of phenol
• Cresols Derived from coal tar (Lysol).
• Bisphenols (Hexachlorophene) Effective against
  gram-positive bacteria like staphylococci and
  streptococci. Used in nurseries. Excessive use
  in infants may cause neurological damage.
• Destroy plasma membranes and denature proteins.
• Advantages Stable, persist for long time after
  application, and remain active in the presence of
  organic compounds.

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• Alcohols
• Kill bacteria, fungi, but not endospores or naked
  viruses.
• Act by denaturing proteins and disrupting cell
  membranes.
• Evaporate, leaving no residue.
• Used to mechanically wipe microbes off skin
  before injections or blood drawing.
• Not good for open wounds, because it causes
  proteins to coagulate.
• Ethanol Optimum concentration is 70.
• Isopropanol Rubbing alcohol. Better
  disinfectant than ethanol. Also cheaper and less
  volatile.

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• Halogens
• Effective alone or in compounds.
• A. Iodine
• Tincture of iodine (alcohol solution) was one of
  first antiseptics used.
• - Combines with the amino acid tyrosine in
  proteins and denatures proteins.
• - Stains skin and clothes, somewhat irritating.
• Iodophors Compounds with iodine that are slow
  releasing, take several minutes to act. Used as
  skin antiseptic in surgery. Not effective
  against bacterial endospores.
• Betadine, Isodine, Wescodyne, Prepodyne.

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• B. Chlorine
• When mixed with water it forms hypochlorous acid
• Cl2 H2O ------gt H Cl- HOCl
• Used to disinfect drinking water, pools, and
  sewage.
• Chlorine is easily inactivated by organic
  materials.
• Sodium hypochlorite (NaOCl) Is the active
  ingredient of bleach.
• Chloramines Consist of chlorine and ammonia.
  Less effective as germicides.

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• Surfactants
• Property of concentrating at interfaces
• Posses hydrophobic and hydrophilic groups
• Lower surface tension and increase miscibility of
  molecules
• A. Soap
• Na or K salts of fatty acids
• degerm skin mechanically

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• B- Detergents
• Superior to soap because they do not form
  precipitates or deposits with water minerals
• Anionic (COO-, SO4-)
• Cationic (NH4, phosphonium, sulfonium)
• Quaternary ammonium ion compounds four organic
  groups covalently linked to nitrogen
• Interact with cell membrane of microorganisms.

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• Alkylating Agents
• Substitute alkyl groups for the hydrogen of
  reactive groups of enzymes, nucleic acids, and
  proteins
• Ethylene oxide
• - Chemosterilizer
• - Colorless, soluble in water and common
  organic solvents.
• - Explosive (mixed with CO2 or N2)
• - Toxic
• - Slow action influenced by concentration of
  gas, relative
• humidity, time of exposure, and
  temperature (4 hours at 50-
• 56C and 6-12 hours at RT).
• - Optimum humidity is 30.

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• Used to sterilize heat sensitive items.
• Gas must be dissipated before the item can be
  used (24 hours).
• Exposure time is reduced by 50 for each doubling
  of ETO concentration.
• likewise the activity of ETO doubles with each T
  increase of 10C.
• BetaPropiolactone Fumigant. (carcinogen)

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• Aldehydes
• Include some of the most effective
  antimicrobials.
• Inactivate proteins by forming covalent
  crosslinks with several functional groups.
• Formaldehyde gas
• Excellent disinfectant.
• Commonly used as formalin, a 37 aqueous
• solution.
• Formalin was used extensively to preserve
  biological
• specimens and inactivate viruses and
  bacteria in
• vaccines.
• Irritates mucous membranes, strong odor.

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• B. Glutaraldehyde
• Less irritating and more effective than
  formaldehyde.
• One of the few chemical disinfectants that is a
  sterilizing agent.
• A 2 solution of glutaraldehyde (Cidex) is
  Bactericidal, tuberculocidal, and viricidal in 10
  minutes but sporicidal in 3 to 10 hours.
• Commonly used to disinfect hospital instruments.

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• Heavy Metals
• Include copper, selenium, mercury, silver, and
  zinc.
• Oligodynamic action Very tiny amounts are
  effective.
• A. Silver
• 1 silver nitrate used to protect infants
  against gonorrheal eye infections (ophthalmia
  neonatorum) until recently.
• B. Mercury
• Organic mercury compounds like merthiolate and
  mercurochrome are used to disinfect skin wounds.

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• C. Copper
• Copper sulfate is used to kill algae in pools
  and fish tanks.
• D. Selenium
• Kills fungi and their spores. Used for fungal
  infections.
• Also used in dandruff shampoos.
• E. Zinc
• Zinc chloride is used in mouthwashes.
• Zinc oxide is used as antifungal agent in paints.

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• Oxidizing Agents
• Oxidize cellular components of treated microbes.
• Disrupt membranes and proteins.
• A. Ozone
• Highly reactive form of oxygen.
• Made by exposing oxygen to electricity or UV
  light.
• Used along with chlorine to disinfect water.
• Helps neutralize unpleasant tastes and odors.
• More effective killing agent than chlorine, but
  less stable and more expensive.

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• B. Hydrogen Peroxide
• Used as an antiseptic.
• Not good for open wounds because it is quickly
  broken down by catalase present in human cells.
• The active Oxidant is the free hydroxyl radical
• Effective in disinfection (3-6) of inanimate
  objects.
• Sporicidal (10-25) at higher temperatures.
• Used by food industry and to disinfect plastic
  implants, contact lenses, and surgical
  prostheses.
• Plasma Gas Vaporization of H2O2 with the
  generation of microwave or radiofrequency energy.
  It is a sterilant
• C. Benzoyl Peroxide
• Used in acne medications.

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• D. Peracetic Acid
• One of the most effective sporicidal liquid
  available.
• Generates acetic acid and Oxygen, both of which
  are nontoxic
• Sterilant
• Kills bacteria and fungi in less than 5 minutes.
• Kills endospores and viruses within 30 minutes.
• Used widely in disinfection of food and medical
  instruments because it does not leave toxic
  residues.

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• Chlorhexidine
• - Antiseptic at 4
• - Used for surgical scrub and hand washing.
• - Hibiclens, Hibitane.
• Parachlorometaxylenol (PCMX)
• - Active against gram positive bacteria.
• Triclosan
• - Active against bacteria. It is a common
  antiseptic in deodorant soap.

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• High-Level disinfectants
• Used for items involved with invasive procedure
  that cannot withstand sterilization procedures
  (certain types of endoscopes, surgical
  instruments with plastic or other components that
  cannot be autoclaved).
• Disinfection of these and other items is most
  effective if treatment is preceded by cleaning
  the surface to remove organic matter.
• Examples of high level disinfectants include
  treatment with moist heat, glutaraldehyde,
  hydrogen peroxide, peracetic acid, chlorine
  dioxide, and other chlorine compounds.

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• Intermediate Level disinfectants
• Alcohols, iodophors, and phenolics are used to
  clean surfaces or instruments in which
  contamination with bacterial spores and other
  highly resilient organisms is unlikely
  (semicritical items).
• Examples of items treated include flexible
  fiberoptic endoscopes, laryngoscopes, vaginal
  specula, anesthesia breathing circuits, and
  others.

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• Low-Level disinfectants
• Quaternary ammonium compounds are used to treat
  noncritical instrument and devices such as blood
  pressure cuffs, ECG electrodes, and stethoscopes.
  
• Although these items come into contact with
  patients, they do not penetrate through mucosal
  surfaces into sterile tissues.