Disinfection

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Disinfection

• Disinfection is the elimination of pathogens,
  except spores, from inanimate objects
• Disinfectants are chemical solutions used to
  clean inanimate objects
• (physical processes, e.g., UV radiation, may
  also be employed to effect disinfection)
• Germicides are chemicals that can be applied to
  both animate (living) and inanimate objects for
  the purpose of eliminating pathogens
• Antiseptics are formulated for application to
  living tissue

Destruction/Removal of Harmful Microorganisms
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The Ideal Disinfectant

• Resistant to inactviation
• Broadly active (killing pathogens)
• Not poisonous (or otherwise harmful)
• Penetrating (to pathogens)
• Not damaging to non-living materials
• Stable
• Easy to work with
• Otherwise not unpleasant

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Disinfectant Performance

• Is dependent on Disinfectant concentrations
• Is dependent on length (time) of administration
• Is dependent on temperature during administration
• (usual chemical reaction 2x increase in rate
  with each 10C increase in temperature)
• Microbe type (e.g., mycobacteria, spores, and
  certain viruses can be very resistant to
  disinfectionin general vegetative cells in log
  phase are easiest to kill)
• Substrate effects (e.g., high organic content
  interferes with disinfectionstainless steel
  bench easier to disinfect than turd)
• It is easier (and faster) to kill fewer microbes
  than many microbes

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Cleansing

• Cleansing is the removal of soil or organic
  material from instruments and equipment may be
  done, clinically, in four steps
• Rinsing the object under cold water
• Applying detergent and scrubbing object
• Rinsing the object under warm water
• Drying the object prior to sterilization or
  disinfection

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Sterilization

• Sterilization is the total elimination of all
  microorganisms including spores
• Typically the last things to die are the highly
  heat- and chemical-resistant bacterial endospores
• Instruments used for invasive procedures must be
  sterilized prior to use
• Moist heat or steam, radiation, chemicals (e.g.,
  glutaraldehyde), and ethylene oxide (a gas) are
  employed for sterilization
• Sterilization by autoclaving, which uses moist
  heat, is used in most hospital and microbiology
  laboratory settings

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Other Terms

• Sanitization Lowering of microbial counts to
  prevent transmission in public setting (e.g.,
  restaurants public rest rooms)
• Degerming Mechanical removal of microbes, e.g.,
  from hands with washing
• Sepsis Bacterial contamination
• Antisepsis Reduction of or Inhibition of
  microbes found on living tissue
• Germincides, Fungicides, Virucides
• Physical versus Chemical disinfectants
• Static (stasis) versus Cidal (e.g.,
  bacteriostatic versus bacteriocidal)

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Exponential Death
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Different Kinds of Bacteria Death
1. Bacteriostatic
2. Bacteriocidal
3. Bacteriolytic
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Resistance to Killing

• Gram-negative bacteria (with their outer
  membrane) are generally more resistant than
  gram-positive bacteria to disinfectants and
  antiseptics
• Stationary-phase (I.e., non-growing) bacteria
  generally are more resistant than log-phase
  (I.e., growing) bacteria
• Mycobacteria, endospores, and protozoan cysts and
  oocysts are very resistant to disinfectants and
  antiseptics
• Nonenveloped viruses are generally more resistant
  than enveloped viruses to disinfectants and
  antiseptics
• Organic matter (such as vomit and feces)
  frequently affects the actions of chemical
  control agent
• Disinfectant activity is inhibited by cold
  temperatures
• Longer application times are preferable to
  shorter
• Higher concentrations, though, are not always
  preferable to lower concentration (e.g., alcohols)

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Chemical Antimicrobials
Agent Mechanisms of Action Comments
Surfactants Membrane Disruption increased penetration Soaps detergents
Quats (cationic detergent) Denature proteins Disrupts lipids Antiseptic - benzalconium chloride, Cepacol Disinfectant
Organic acids and bases High/low pH Mold and Fungi inhibitors e.g., benzoate of soda
Heavy Metals Denature protein Antiseptic Disinfectant Silver Nitrate
Halogens Oxidizing agent Disrupts cell membrane Antiseptic - Iodine (Betadine) Disinfectant - Chlorine (Chlorox)
Alcohols Denatures proteins Disrupts lipids Antiseptic Disinfectant Ethanol and isopropyl
Phenolics Disrupts cell membrane Disinfectant Irritating odor
Aldehydes Denature proteins  Gluteraldehyde - disinfectant (Cidex) Formaldehyde - disinfectant
Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize
Oxidizing agents Denature proteins Hydrogen peroxide antiseptic Hydrogen peroxide disinfectan Benzoyl peroxide antiseptic
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Surfactants
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Soap Detergents

• Soaps are sodium or potassium salts of fatty
  acids, a natural product
• Detergents, instead, are artificial surfactants
• While soaps are always negatively charged, some
  detergents are negatively charged while others
  are positively charged
• One example of a positively charged detergent are
  quaternary ammonium compounds (a.k.a., quats)

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Quats

• Quats are cationic detergents that act by
  disrupting lipid bilayers
• Quats are bactericidal, fungicidal, viricidal
  (enveloped), and amoebicidal
• Quats are most effective against Gram-positive
  bacteria
• Quats do not kill endospores, Mycobacteria spp.,
  nor non-enveloped viruses
• Quats are rapidly inactivated by organics
  including cotton and soap
• Zephiran ? Benzalkonium chloride
• Cepacol ?Cetylpyridinium chloride

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Heavy Metals

• Ag, Cu, Hg, Ni, Zn, Ag(NO3)2, CuSO4, ZnCl2, HgCl2
• These metals (and metal ions) react with
  sulfhydral (SH) groups of proteins, denaturing
  proteins
• Silver nitrate is used to treat Ophthalmia
  neonatorum in newborns as caused by Neisseria
  gonorrhoeae
• Oligodynamic action the ability of very small
  amounts of heavy metals (especially silver and
  copper) to exert antimicrobial activity

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Halogens

• Halogens are the seventh (VII) column of the
  periodic table of elements
• Two halogens are regularly employed as
  antimicrobials Iodine and Chloride
• Iodine commonly used as an antiseptic against
  all microbes, fungi, and viruses
• Iodine It inhibits protein synthesis and
  oxidizes SH groups of amino acids
• Chlorine Used as a disinfectant (10 bleach)
• Chlorine Hypochlorous acid (HOCl) is a product,
  formed in water, that is the active form of the
  disinfectant
• Chlorine Applied in treatment of drinking water,
  swimming pool, and sewage

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Chlorination

• 1744 discovered in Sweden
• 1810 identified as an element
• 1835 first used to control odors
• 1890s started to be used as a disinfectant
• 1896 earliest recorded use in experiments on
  water supplies
• 1897 used in England to sterilize water mains
  following typhoid outbreak
• 1902 first continuous use in water supplies in
  Belgium
• 1909 liquid chlorine (compressed gas) became
  commercially available
• Subsequent rapid spread in use of chlorine
  throughout the world
• WWI Chlorine gas used as chemical warfare agent

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Chlorination

• Hypochlorite may either be added directly (i.e.,
  in the form of bleach) or created within water by
  bubbling chlorine gas through the water
• Chlorine gas - preferred for medium to large
  disinfection systems
• Sodium Hypochlorite (liquid) - typically used for
  small disinfection systems and large swimming
  pools
• Calcium Hypochlorite (powder, tablet) - typically
  used for private swimming pools
• For water purification, do not use scented bleach
• Bromine sometimes used as a less-smelly
  alternative
• Hypochlorite is less effective in the presence of
  significant organic compounds

What is known as modern chemical warfare began
during World War I. The first chemical agent to
be used was large amounts of chlorine gas, about
one hundred sixty tons, which was released from
6,000 pressurized cylinders into the wind by the
Germans against the Allies. The chlorine floated
in a huge clouds toward the Allies until it
reached the Allied lines causing men to die from
the effects of the chlorine gas. Because of the
large amounts of gas released the chlorine caused
large amounts of yellowish fluid to form in the
lungs of its victim, also causing eye, nose, and
throat burning before causing death by
choking. http//www.geocities.com/CapeCanaveral/
Lab/4239/chemweapons/history.html
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Alcohols

• Aqueous ethanol (60-95) and isopropanol are used
  as disinfectants
• Effectively kill bacteria and fungi but not
  endospores nor nonenveloped viruses
• Fast acting, no residue (evaporate away), no
  staining
• But not very penetrating and no residual activity
  (once gone gone)
• Exert their action by denaturing proteins and
  dissolving lipids
• In tinctures, they enhance the effectiveness of
  other antimicrobial chemicals
• Flammable also may damage rubber, plastic, etc.

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Tincture

• Formulae Fresh juice of Organic Habanero
  peppers, New Mexico Jalapeno, African Bird
  peppers and Hatch Chili peppers.
• Dosage Five to thirty drops, three times daily. 
  CAUTION EXTREMELY HOT!!
• Therapeutic Action Cayenne is the greatest
  herbal aid to circulation and should be used on a
  regular basis.  The extract is very concentrated
  and gets into the bloodstream quickly and makes
  it a perfect first aid remedy for heart attacks,
  stroke, fainting, shock, dizziness, hemorrhage,
  internal and external bleeding.  Use a few drops
  to 10 droppers full.  It has saved many lives.

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Tincture II

• Formulae Fresh Garlic Juice, Goldenseal root,
  Usnea lichen, Myrrh gum, Pine resin, Echinacea
  root juice, Tea Tree oil, Kelp, Black Walnut
  inner hulls, Oak galls and Cayenne pepper in 80
  grain alcohol.
• Dosage Generally for external use but can be
  used in the oral cavity.  Soak a cotton swab in
  the tincture and scrub into the infected area,
  let air dry.  It has a burning sensation. If the
  wound is tender, just flush it with multiple
  droppers full of the tincture but no need to
  scrub it in.
• Therapeutic Action There has never been an
  infected occur when this formula has been used. 
  It's excellent for treating any cut or wound and
  it is anti-bacterial, anti-viral and
  anti-fungal.  The tree resins in this formula
  leave an invisible protective, anti-bacterial
  coating over the wound.  This formula was use on
  a man in England who had the top of his knee torn
  off in an automobile accident.  In 24 hours it
  literally glued his knee back together.  A nurse
  from Ireland on the scene said in all the years
  in the hospital, she had never seen such a severe
  wound close right up and heal, and with no
  infections.

A tincture is a nonvolatile substance (medicine)
presented as an alcohol solution, e.g., (for fun
with numerous sic)
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Iodine Iodophores
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Phenol, Carbolic Acid, Phenolics

• Phenol (carbolic acid) and derivatives
• Affect plasma membrane, inactivates enzymes, and
  denature proteins
• Stable, persistant, and especially effective when
  dealing with disinfecting materials contaminated
  with organics
• but leave residual films, can irritate skin,
  dont kill endospores, and are corrosive to
  rubber and plastics
• Some phenolics are mild enough for use as
  antiseptics while others are too harsh or
  otherwise dangerous to be employed on living
  tissue
• Hexachlorophene, Triclosan, Lysol, soap

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Oxidizing Agents

• HOOH, hydrogen peroxide, is most common
• HOOH is not a terribly effective disinfectant or
  anticeptic
• This is because bacteria and body tissues contain
  enzymes (catalase) that inactivate hydrogen
  peroxide
• However, the oxygen released upon inactivation
  can help oxygenate deep wounds and thus kill
  strict-anaerobe contaminants, e.g., Clostridium
  tetani
• Ozone and peracetic acid are also oxidizing
  antimicrobial agents
• They exert their effect by oxidizing cell
  macromolecules (e.g., proteins, DNA, etc.)

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Glutaraldehyde
Glutaraldehyde is capable of effectiving
sterilizationat room temperature, even against
endospores, and even in the presence of
organics, but achieving sterilization requries
many hours of exposure and it is nasty stuff to
work with!
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Gaseous Chemosterilizers

• Propylene oxide (C3H6O)
• Chlorine gas (Cl2)
• Chlorine dioxide (ClO2)
• Ozone (O3)
• Ethylene oxide (C2H4O)
• is used to sterilize heat- or moisture-sensitive
  items
• is used for items damaged by heat or moisture
• is not corrosive, not damaging to delicate
  instruments, microscopes, disposable plastic
  instruments and materials
• permeates porous materials
• dissipates rapidly from material
• but is costly, toxic, carcinogenic, explosive,
  and relatively lengthy process

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Physical Antimicrobials
Agent Mechanisms of Action Comments
Moist Heat, boiling Denatures proteins Kills vegetative bacterial cells and viruses Endospores survive
Moist Heat, Autoclaving Denatures proteins 121C at 15 p.s.i. for 30 min kills everything
Moist Heat, Pasteurization Denatures proteins Kills pathogens in food products
Dry Heat, Flaming Incineration of contaminants Used for inoculating loop
Dry Heat, Hot air oven Oxidation Denatures proteins 170C for 2 hours Used for glassware instrument sterilization
Filtration Separation of bacteria from liquid (HEPA from air) Used for heat sensitive liquids
Cold, Lyophilization (also desiccation) Desiccation and low temperature Used for food drug preservation Does not necessarily kill so used for Long-term storage of bacterial cultures
Cold, Refrigeration Decreased chemical reaction rate Bacteriostatic
Osmotic Pressure, Addition of salt or sugar Plasmolysis of contaminants Used in food preservation (less effective against fungi)
Radiation, UV DNA damage (thymine dimers) Limited penetration
Radiation, X-rays DNA damage Used for sterilizing medical supplies
Strong vis. Light Line-drying laundry
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Application of Heat

• Heat is frequently used to kill microorganisms
• Thermal death point (TDP) is the lowest
  temperature at which all bacteria in a liquid
  culture will be killed in 10 minutes
• Thermal death time (TDT) is the length of time
  required to kill all bacteria in a liquid culture
  at a given temperature
• Decimal reduction time (DRT) is the length of
  time in which 90 of a bacterial population will
  be killed at a given temperature (especially
  useful in canning industry)
• Dry heat kills by oxidation (slow, uneven
  penetration)
• Moist heat kills by protein coagulation
  (denaturation) so requires lower temperatures or
  shorter times, but the moisture must penetrate to
  pathogens to be effective (grease oil can block)

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Moist Heat

• Moist heat kills microbes by denaturing enzymes
  (coagulation of proteins)
• Boiling (at 100C, I.e., at sea level) kills many
  vegetative cells and viruses within 10 minutes
• Autoclaving steam applied under pressure (121C
  for 15 min) is the most effective method of moist
  heat sterilizationthe steam must directly
  contact the material to be sterilized
• Pasteurization destroys pathogens (Mycobacterium
  tuberculosis, Salmonella typhi, etc.) without
  altering the flavor of the fooddoes not
  sterilize (63C for 30 seconds)
• Higher temperature short time pasteurization
  applies higher heat for a much shorter time (72C
  for 15 seconds)
• An ultra-high-temperature, very short duration
  treatment (140C for 3 sec.) is used to sterilize
  dairy products

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Sterilization Times

• 171o C, 60 minutes, dry heat
• 160o C, 120 minutes, dry heat
• 149o C, 150 minutes, dry heat
• 141o C, 180 minutes, dry heat
• 121o C, 12 hours, dry heat
• 121o C, 15 minutes, moist heat (but dont start
  the clock until entire item is up to tempe.g.,
  large volumes fluid)

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Filtration Air Fluids
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Evaluating Disinfectants

• Phenol Coefficient
• compares efficacy to that of phenol, with
  greater efficacy indicated with coefficient gt1
• Salmonella typhi and Staphylococcus aureus
  commonly used to determine coefficients
• Filter-Paper/Disk Diffusion method
• placement of disinfectant impregnated filter
  paper on well-inocated agar
• Use-Dilution test
• drying bacteria to surface followed by exposure
  to disinfectant and subsequent washing and
  inoculation of sterile broth

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Disk Diffusion Method
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Hands Spread Disease
WASH 'EM !!!
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Link to Next Presentation
35
Acknowledgements
http//www.sw.vccs.edu/rth/RTH_132/decont.ppt http
//www.nt.gov.au/powerwater/html/pdf/tennant_creek
_chlorination.ppt http//www.bugcruncher.com/ws/do
cs/lekcii/DS_INQUAL_eng.ppt (nice overview of
specific antimicrobials) http//www.delmarhealthca
re.com/olcs/white/ppts/chapter2021.ppt (nice
overview of microbilogy in nursing) http//www.cou
ntyofkings.com/health/ehs/forms/FDHNDLR.ppt http/
/bioeng.ttu.edu.tw/chunyi/microbes/Chapter7.ppt h
ttp//microbiology.okstate.edu/courses/micro2124/b
abus/Chpt7_files/chapter7.ppt
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Chemical Antimicrobials
Type of Disinfectant H High level I
Intermediate level L Low level