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Destruction of Microorganisms for PS inter 2005

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					March 10, 2005   Destruction of Microorganisms   Slide No. 1
              Learning Objectives
  • Know the definitions of terms associated
       with sterilization and asepsis
  • Discuss the factors affecting microbial death
       rate
  • Know the conditions for sterilization by
       physical methods
  • Discuss the major classes of chemical
       disinfectants in terms of mode of killing and,
       where appropriate, major advantages or
       disadvantages Destruction of Microorganisms
March 10, 2005                                     Slide No. 2
                 Learning Objectives
  • To define the terms: sterilization,
    disinfection, decontamination, antisepsis and
    sanitization
  • To understand the principles and methods of
    sterilization and disinfection
  • To become aware of some agents used as
    disinfectants



March 10, 2005        Destruction of Microorganisms   Slide No. 3
                 Learning Objectives
  • compare and contrast the processes of
    disinfection, sanitization, antisepsis, and
    sterilization
  • compare the difficulties encountered when
    trying to kill endospores with those
    encountered when trying to kill vegetative
    cells
  • discuss the exponential pattern of microbial
    death
March 10, 2005        Destruction of Microorganisms   Slide No. 4
                                 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

March 10, 2005                        Destruction of Microorganisms            Slide No. 5
                              Definitions (1)
         ๏ Sterilization: Killing or removing all forms of
             microbial life (including endospores) in a
             material or an object.
                 • Heating is the most commonly used method of sterilization.


         ๏ Commercial Sterilization:       Heat treatment that
             kills endospores of Clostridium botulinum the
             causative agent of botulism, in canned food.
                 • Does not kill endospores of thermophiles, which are not
                   pathogens and may grow at temperatures above 45oC.



March 10, 2005                       Destruction of Microorganisms              Slide No. 6
                              Definitions (2)
         ๏ 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. May use physical or
             chemical methods.
                 • Disinfectant: Applied to inanimate objects.
                 • Antiseptic: Applied to living tissue (antisepsis).
                 • Degerming: Mechanical removal of most microbes in a limited
                   area. Example: Alcohol swab on skin.
                 • Sanitization: Use of chemical agent on food-handling
                   equipment to meet public health standards and minimize
                   chances of disease transmission. E.g: Hot soap & water
March 10, 2005                      Destruction of Microorganisms          Slide No. 7
                        Definitions (3)
         ๏ 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.

March 10, 2005               Destruction of Microorganisms   Slide No. 8
                              Definitions (4)
         ๏ Bacteriostatic Agent: An agent that inhibits
             the growth of bacteria, but does not necessarily
             kill them. Suffix stasis: To stop or steady.
         ๏ Germicide:   An agent that kills certain
             micoorganisms.
                 • Bactericide: An agent that kills bacteria. Most do not kill
                   endospores.
                 • Viricide: An agent that inactivates viruses.
                 • Fungicide: An agent that kills fungi.
                 • Sporocide: An agent that kills bacterial endospores of fungal
                   spores.


March 10, 2005                       Destruction of Microorganisms            Slide No. 9
                 Rate of Microbial Death (1)
             • When bacterial populations are heated or treated antimicrobial
               chemicals, they usually die at a constant rate.




March 10, 2005                     Destruction of Microorganisms           Slide No. 10
                 Rate of Microbial Death (2)
         ๏ Several factors influence the effectiveness of
             antimicrobial treatment.

                 1. Number of Microbes: The more microbes present, the more
                   time it takes to eliminate population.
                 2. Type of Microbes: Endospores are very difficult to destroy.
                   Vegetative pathogens vary widely in susceptibility to different
                   methods of microbial control.
                 3. Environmental influences: Presence of organic material
                   (blood, feces, saliva) tends to inhibit antimicrobials, pH,
                   temperature, biofilms etc.
                 4. Time of Exposure: Chemical antimicrobials and radiation
                   treatments are more effective at longer times. In heat treatments,
                   longer exposure compensates for lower temperatures.

March 10, 2005                        Destruction of Microorganisms            Slide No. 11
                 Rate of Microbial Death (3)




March 10, 2005            Destruction of Microorganisms   Slide No. 12
       Factors affecting microbial death
  • Nature of the organism-spore formers,
    Mycobacterium tuberculosis
  • Concentration of agent
  • Temperature
  • Effect of organic matter and pH
  • Concentration of cells
  • Time


March 10, 2005     Destruction of Microorganisms   Slide No. 13
           Sterilization-quality assurance
  • Process indicators
         ๏ Autoclave tape
         ๏ Temperature recorders
  • Biological indicators
         ๏ Spore containing ampules
         ๏ Spore bacterial test strips
         ๏ (Bacillus stearothermophilus is the industry
             index for heat killing)

March 10, 2005                Destruction of Microorganisms   Slide No. 14
 Actions of Microbial Control Agents
  • Alternation of membrane permeability
  • Damage to proteins
  • Damage to nucleic acids




March 10, 2005     Destruction of Microorganisms   Slide No. 15
        Resistance of Microorganisms to
            Disinfectants/Sterilants




March 10, 2005     Destruction of Microorganisms   Slide No. 16
     Decreasing Order of Resistance of
            Microorganisms to
         Disinfectants/Sterilants
                        Prions
                        Spores
                     Mycobacteria
                 Non-Enveloped Viruses
                         Fungi
                       Bacteria
                   Enveloped Viruses
March 10, 2005        Destruction of Microorganisms   Slide No. 17
                    Phsysical Methods (1)
      ๏ Heat: Kills microorganisms by denaturing their
           enzymes and other proteins. Heat resistance
           varies widely among microbes.

             • Thermal Death Point (TDP): Lowest temperature at which all of
               the microbes in a liquid suspension will be killed in ten minutes.
             • Thermal Death Time (TDT): Minimal length of time in which all
               bacteria will be killed at a given temperature.
             • Decimal Reduction Time (DRT): Time in minutes at which 90%
               of bacteria at a given temperature will be killed. Used in canning
               industry.




March 10, 2005                     Destruction of Microorganisms            Slide No. 18
         Decimal Reduction Time (DRT)




March 10, 2005     Destruction of Microorganisms   Slide No. 19
                    Phsysical Methods (2)
    ๏ Moist Heat: Kills microorganisms by coagulating
        their proteins.
           • In general, moist heat is much more effective than dry heat.
    ๏ Boiling: Heat to 100oC or more at sea level. Kills
        vegetative forms of bacterial pathogens, almost all
        viruses, and fungi and their spores within 10
        minutes or less. Endospores and some viruses are
        not destroyed this quickly. However brief boiling will
        kill most pathogens.
           • Hepatitis virus: Can survive up to 30 minutes of boiling.
           • Endospores: Can survive up to 20 hours or more of boiling.
March 10, 2005                    Destruction of Microorganisms             Slide No. 20
                       Phsysical Methods (3)
    ๏ Reliable sterilization with moist heat requires
        temperatures above that of boiling water.
    ๏ Autoclave: Chamber which is filled with hot steam
        under pressure. Preferred method of sterilization,
        unless material is damaged by heat, moisture, or
        high pressure.
           • Temperature of steam reaches 121oC at twice atmospheric pressure.
           • Most effective when organisms contact steam directly or are
             contained in a small volume of liquid.
                 – All organisms and endospores are killed within 15 minutes.
           • Require more time to reach center of solid or large volumes of liquid.

March 10, 2005                            Destruction of Microorganisms         Slide No. 21
                 Autoclave
                                                  • Closed
                                                    Chamber with
                                                    High
                                                    Temperature
                                                    and Pressure




March 10, 2005    Destruction of Microorganisms                Slide No. 22
                 COMMERCIAL CANNING




March 10, 2005         Destruction of Microorganisms   Slide No. 23
                              Pasteurization
         ๏ Developed by Louis Pasteur to prevent the
             spoilage of beverages. Used to reduce
             microbes responsible for spoilage of beer, milk,
             wine, juices, etc.
                 • Classic Method of Pasteurization: Milk was exposed to 65oC
                   for 30 minutes.
                 • High Temperature Short Time Pasteurization (HTST): Used
                   today. Milk is exposed to 72oC for 15 seconds.
                 • Ultra High Temperature Pasteurization (UHT): Milk is treated
                   at 140oC for 3 seconds and then cooled very quickly in a
                   vacuum chamber.
         ๏ Advantage:    Milk can be stored at room
             temperature for several months.
March 10, 2005                      Destruction of Microorganisms          Slide No. 24
                                    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 is transfers heat less
                   effectively to a cool body, than moist heat.
                                             (Hot Air Oven)



March 10, 2005                        Destruction of Microorganisms            Slide No. 25
                                        Filtration
    ๏ Removal of microbes by passage of a liquid or gas
        through a screen like material with small pores.
        Used to sterilize heat sensitive materials like
        vaccines, enzymes, antibiotics, and some culture
        media.
           • 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. Don’t retain spirochetes,
                   mycoplasmas and viruses.
                 – 0.01 um Pores: Retain all viruses and some large proteins.

March 10, 2005                            Destruction of Microorganisms                       Slide No. 26
                 Membrane filters




March 10, 2005       Destruction of Microorganisms   Slide No. 27
                 Membrane filtration
                                                      • Cellulose acetate, etc.
                                                      • Paper-thin
                                                      • Too small for passage
                                                        of microorganisms
                                                      • Large enough to allow
                                                        liquid through
                                                      • Various pore sizes
                                                      • Some smaller than
                                                        smallest known
                                                        viruses




March 10, 2005        Destruction of Microorganisms                      Slide No. 28
                 Membrane filtration




March 10, 2005        Destruction of Microorganisms   Slide No. 29
                 Filter Sterilization
                                                      ๏   Filtration: Used
                                                          for sterilization of
                                                          heat-sensitive
                                                          liquids or gases
                                                      ๏   Filter: device
                                                          with pores
                                                           • Too small for
                                                             passage of
                                                             microorganisms
                                                           • Large enough to
                                                             allow liquid through




March 10, 2005        Destruction of Microorganisms                        Slide No. 30
                 Air filtration
                                                   • High-efficiency particulate air
                                                     (hepa) filters
                                                   • Anything > 0.3 mm removed
                                                     from air
                                                   • Used for
                                                       – Specialized hospital rooms
                                                       – Laminar flow hoods




March 10, 2005     Destruction of Microorganisms                               Slide No. 31
                              Low Temperature
         ๏ Effect depends on microbe and treatment
             applied.

                 • Refrigeration: Temperatures from 0 to 7oC. 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.
                    –   Over a third of vegetative bacteria may survive 1 year.
                    –   Most parasites are killed by a few days of freezing.


March 10, 2005                              Destruction of Microorganisms                          Slide No. 32
                                  Dessication
         ๏ In the absence of water, microbes cannot grow
             or reproduce, but some may remain viable for
             years. After water becomes available, they start
             growing again.
         ๏ Susceptibility to dessication varies widely:
                 •   Neisseria gonnorrhea: Only survives about one hour.
                 •   Mycobacterium tuberculosis: May survive several months.
                 •   Viruses are fairly resistant to dessication.
                 •   Clostridium spp. and Bacillus spp.: May survive decades.



March 10, 2005                        Destruction of Microorganisms        Slide No. 33
                            Osmotic Pressure
         ๏ The use of high concentrations of salts and
             sugars in foods is used to increase the osmotic
             pressure and create a hypertonic environment.
         ๏ Plasmolysis: As water leaves the cell, plasma
             membrane shrinks away from cell wall. Cell
             may not die, but usually stops growing.

                 • Yeasts and molds: More resistant to high osmotic pressures.
                 • Staphylococci spp. that live on skin are fairly resistant to high
                   osmotic pressure.



March 10, 2005                        Destruction of Microorganisms             Slide No. 34
                                 Radiation (1)
         ๏ Three types of radiation kill microbes:
             1. 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.
         ๏ Disadvantages: Penetrates human tissues.
             May cause genetic mutations in humans.

March 10, 2005                        Destruction of Microorganisms              Slide No. 35
                 Forms of Radiation




March 10, 2005        Destruction of Microorganisms   Slide No. 36
                             Radiation (2)
        2. 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, eyes.                           Doesn’t
        penetrate paper, glass, and cloth.


March 10, 2005                     Destruction of Microorganisms         Slide No. 37
                                Radiation (3)
             3. 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 foods are unevenly penetrated by microwaves.
                 • Trichinosis outbreaks have been associated with pork cooked in
                   microwaves.




March 10, 2005                       Destruction of Microorganisms          Slide No. 38
                      Chemical Methods (1)
  • Selection of appropriate germicidal
    chemicals: Must consider
         ๏ 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

March 10, 2005                            Destruction of Microorganisms      Slide No. 39
                       Chemical Methods (2)


         ๏ Residue
                 • Toxic or corrosive residue
         ๏ Cost & availability
         ๏ Storage & stability
                 • Concentrated stocks, etc.
         ๏ Environmental risk
                 • Neutralization prior to disposal




March 10, 2005                        Destruction of Microorganisms   Slide No. 40
                        Chemical Methods (3)
         Classes of germicidal chemicals

                 •   Phenols and Phenolics
                 •   Halogens
                 •   Alcohols
                 •   Heavy Metals
                 •   Quaternary Ammonium Compounds (Quats)
                 •   Aldehydes
                 •   Gaseous Sterilizers
                 •   Peroxygens (Oxidizing Agents)




March 10, 2005                      Destruction of Microorganisms   Slide No. 41
                       Chemical Methods (4)
    1. 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).
                 – Biphenols (pHisoHex): Effective against gram-positive 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 times after applied, and remain
             active in the presence of organic compounds.
March 10, 2005                              Destruction of Microorganisms                        Slide No. 42
March 10, 2005   Destruction of Microorganisms   Slide No. 43
                 Disinfection: Measurement
  • Phenol coefficient (higher is better)
  • Mycobacterium tuberculosis - the industry
    index for chemical killing (look for
    "tuberculocidal")
  • Standard levels of activity:
                 Level Vegetative Fungi TB Bacillus               Viruses Spores
                 High     +        +      +                       +      +
                 Medium    +        +      +                      +       -
                 Low      +        +       -                      -       -



March 10, 2005                    Destruction of Microorganisms                    Slide No. 44
                       Chemical Methods (5)
         2. Halogens: Effective alone or in compounds.
         A. Iodine:
                 • Tincture of iodine (alcohol solution) was one of first
                   antiseptics used.
                     – Combines with 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


March 10, 2005                             Destruction of Microorganisms                       Slide No. 45
                       Chemical Methods (6)
      B. Chlorine:
      When mixed in water forms hypochlorous acid:
                 Cl2 + H2O ------>    H+       + Cl- + HOCl
                                                     Hypochlorous acid
           • Used to disinfect drinking water, pools, and sewage.
           • Chlorine is easily inactivated by organic materials.
    ๏ Sodium hypochlorite (NaOCl): Is active ingredient
        of bleach.
    ๏ Chloramines: Consist of chlorine and ammonia.
        Less effective as germicides.

March 10, 2005                       Destruction of Microorganisms       Slide No. 46
                       Chemical Methods (7)
         3. 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 cause proteins to
                   coagulate.
                     – Ethanol: Drinking alcohol. Optimum concentration is 70%.
                     – Isopropanol: Rubbing alcohol. Better disinfectant than ethanol. Also cheaper and
                       less volatile.


March 10, 2005                             Destruction of Microorganisms                        Slide No. 47
March 10, 2005   Destruction of Microorganisms   Slide No. 48
                       Chemical Methods (8)
         4. 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 until recently.
         B. Mercury
                 • Organic mercury compounds like merthiolate and
                   mercurochrome are used to disinfect skin wounds.
         C. Copper
                 • Copper sulfate is used to kill algae in pools and fish tanks.


March 10, 2005                        Destruction of Microorganisms                Slide No. 49
                       Chemical Methods (9)
         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.




March 10, 2005                        Destruction of Microorganisms            Slide No. 50
                  Chemical Methods (10)
    5. Quaternary Ammonium Compounds (Quats):
           • Widely used surface active agents.
           • Cationic (positively charge) detergents.
           • Effective against gram positive bacteria, less effective against
             gram-negative bacteria.
           • Also destroy fungi, amoebas, and enveloped viruses.
           • Zephiran, Cepacol, also found in our lab spray bottles.
           • Pseudomonas strains that are resistant and can grow in presence
             of Quats are a big concern in hospitals.

           Advantages: Strong antimicrobial action, colorless, odorless,
             tasteless, stable, and nontoxic.
           Diasadvantages: Form foam. Organic matter interferes with
             effectiveness. Neutralized by soaps and anionic detergents.

March 10, 2005                    Destruction of Microorganisms            Slide No. 51
                      Chemical Methods (11)
         6. Aldehydes:
                 • Include some of the most effective antimicrobials.
                 • Inactivate proteins by forming covalent crosslinks with several
                   functional groups (–NH2, –OH, –COOH, —SH).
         A. 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.
                 • Also used in mortuaries for embalming.




March 10, 2005                        Destruction of Microorganisms            Slide No. 52
                      Chemical Methods (12)
         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.
                     – Sporicidal in 3 to 10 hours.
                 • Commonly used to disinfect hospital instruments.
                 • Also used in mortuaries for embalming.




March 10, 2005                              Destruction of Microorganisms           Slide No. 53
                      Chemical Methods (13)
         7. Gaseous Sterilizers:
                 • Chemicals that sterilize in a chamber similar to an autoclave.
                 • Denature proteins, by replacing functional groups with alkyl
                   groups.
         A. Ethylene Oxide:
                 • Kills all microbes and endospores, but requires exposure of 4 to
                   18 hours.
                 • Toxic and explosive in pure form.
                 • Highly penetrating.
                 • Most hospitals have ethylene oxide chambers to sterilize
                   mattresses and large equipment.




March 10, 2005                        Destruction of Microorganisms            Slide No. 54
                      Chemical Methods (14)
         8. Peroxygens (Oxidizing Agents):
                 • Oxidize cellular components of treated microbes.
                 • Disrupt membranes and proteins.
         A. Ozone:
                 • 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.
                 • Highly reactive form of oxygen.
                 • Made by exposing oxygen to electricity or UV light.
         B. Benzoyl Peroxide:
                 • Used in acne medications.

March 10, 2005                        Destruction of Microorganisms             Slide No. 55
                       Chemical Methods (15)
         C. Hydrogen Peroxide:
                 • Used as an antiseptic.
                 • Not good for open wounds because quickly broken down by
                   catalase present in human cells.
                 • Effective in disinfection of inanimate objects.
                 • Sporicidal at higher temperatures.
                 • Used by food industry and to disinfect contact lenses.
         D. Peracetic Acid:
                 •   One of the most effective liquid sporicides available.
                 •   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.
March 10, 2005                         Destruction of Microorganisms           Slide No. 56
        CHEMICAL SITES OF ACTION




March 10, 2005   Destruction of Microorganisms   Slide No. 57
March 10, 2005   Destruction of Microorganisms   Slide No. 58
            Efficiency of Different
         Chemical Antimicrobial Agents




March 10, 2005     Destruction of Microorganisms   Slide No. 59
                        PRESERVATION (1)
         Preventing or slowing growth of microorganisms
         Extends shelf life of many products
                 • Foods, soaps, medicines, deodorants, cosmetics, etc.
         Approaches to preservation
                 1. Chemical preservatives
                 2. Low temperature storage
                 3. Reducing available water




March 10, 2005                       Destruction of Microorganisms        Slide No. 60
                          PRESERVATION (2)
  1. Chemical preservatives
         ๏ Germicidal chemicals used in non-food items
         ๏ Food preservatives must be non-toxic
                 • Weak organic acids
                     – Inhibit metabolism
                     – Sorbic acid, benzoic acid, calcium propionate
                     – Low pH effective
                 • Nitrate or nitrite
                     –   Prevent endospore germination
                     –   React with myoglobin ---> nice pink color
                     –   Other reactions ---> carcinogens
                     –   Risk vs. benefit
March 10, 2005                               Destruction of Microorganisms   Slide No. 61
                        PRESERVATION (3)
  2. 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




March 10, 2005                       Destruction of Microorganisms          Slide No. 62
                        PRESERVATION (4)
  3. Reducing available water
         ๏ Stops growth, doesn’t 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




March 10, 2005                       Destruction of Microorganisms       Slide No. 63
                 Definitions 2
  • Sterilization: Destruction of all living
    microbes.
  • Disinfection: Destruction of all harmful
    microbes – (not including spores).
  • Antisepsis: Destruction of all harmful
    microbes – (not incl. spores) on living
    tissue.
  • Sanitize: Reduction of microbes to an
    acceptable level
March 10, 2005      Destruction of Microorganisms   Slide No. 64
                 Disinfectants
  •    Alcohols
  •    Aldehydes
  •    Ampholytes
  •    Diguanides
  •    Halogens
  •    Phenols
  •    Quaternary Ammoniums (QUA’s)
  •    QUA’s + Diguanides
March 10, 2005      Destruction of Microorganisms   Slide No. 65
                 Sterilization by Heat
  •    121 OC for 15mins – moist heat
  •    - steam @ 15 lbs./sq inch.
  •    134 OC for 3.5 mins – moist heat
  •    - steam @ 30 lbs./sq inch.
  •    160 OC for 1 hr – dry heat (oven)
  •    Tyndallization – boiled for 1hr. X 3
  •    Pasteurization does NOT sterilize


March 10, 2005          Destruction of Microorganisms   Slide No. 66
                 Controlling Microorganism
  • General consideration in microbial control
    belongs to the general category of:
         ๏ Decontamination - destroys or removes
             contaminants
         ๏ Physical Agents
                 • heat or radiation
         ๏ Chemical           Agents
                 • disinfectants
                 • antiseptics
                 • drugs


March 10, 2005                         Destruction of Microorganisms   Slide No. 67
                 Conditions Influencing
                   Microbial Control
  •    Temperature
  •    Type of Microbe
  •    Physiological State Of The Microbe
  •    Environment




March 10, 2005          Destruction of Microorganisms   Slide No. 68
                        Actions of
                 Microbial Control Agents
  • Alteration of Membrane Permeability
  • Damage to Proteins And Nucleic Acids




March 10, 2005           Destruction of Microorganisms   Slide No. 69
                  Physical Methods
                 of Microbial Control
  •    Heat
  •    Moist Heat
  •    Pasteurization
  •    Dry Heat Sterilization
  •    Filtration
  •    Cold
  •    Desiccation


March 10, 2005         Destruction of Microorganisms   Slide No. 70
                  Chemical Methods
                 of Microbial Control
  • Principles of Effective Disinfection
  • Evaluating a Disinfectant
  • Filter Paper method




March 10, 2005         Destruction of Microorganisms   Slide No. 71
                 Types of Disinfectants
  •    Phenol and Phenolics
  •    Chlorhexidine
  •    Halogens
  •    Alcohols
  •    Heavy metals and their compounds
  •    Surface-Active Agents
  •    Quaternary Ammonium Compounds(Quats)


March 10, 2005          Destruction of Microorganisms   Slide No. 72
                 Types of Disinfectants
  •    Organic Acids and Derivatives
  •    Aldehydes
  •    Gaseuos Chemosterilizers
  •    Oxidizing Agents




March 10, 2005          Destruction of Microorganisms   Slide No. 73
                 Pattern of Microbial Death
  • Usually exponential (logarithmic)
  • the population is reduced by the same
    fraction at regular intervals
  • When the population has been greatly
    reduced, the rate of killing may slow due to
    the survival of a more resistant strain of the
    organism



March 10, 2005            Destruction of Microorganisms   Slide No. 74
                 Relative Resistance of
                   Microbial Forms
  • Highest resistance - Endospores
  • Moderate resistance - Protozoa cysts;
    Naked viruses; Sexual spores; Hepatitis B;
    TB; S. aureus; Pseudomonas
  • Least resistance - Trophs; fungal spores;
    yeast; enveloped viruses




March 10, 2005          Destruction of Microorganisms   Slide No. 75
                 Frequently Used Terms
  • Sterilization - all living cells, viable spores,
    viruses, viroids are either destroyed or
    removed

         ๏ When achieved by a chemical agent, the
             chemical is called a sterilant


  • Disinfectants - agents, usually chemical,
    used on inanimate objects
March 10, 2005                Destruction of Microorganisms   Slide No. 76
                 Frequently Used Terms
  • Disinfection is the killing, inhibition or
    removal of microorganisms

         ๏ A disinfectant does not necessarily sterilize an
             object because viable spores and a few
             microbes may remain




March 10, 2005              Destruction of Microorganisms   Slide No. 77
                 Frequently Used Terms
  • Sanitization- the microbial population is
    reduced to levels that are considered safe by
    public health standard
         ๏ inanimate objects are cleaned as well as
             partially disinfected
  • Antisepsis (prevention of infection) by using
    antiseptics- chemical agents applied to living
    tissue. Protect by killing or inhibiting
    pathogen growth
March 10, 2005                Destruction of Microorganisms   Slide No. 78
                 Frequently Used Terms
  • Common Suffixes

  • -cide (Latin cida, to kill)
         ๏ Germicide kills pathogens (germs), not
             necessarily endospores
         ๏ Bactericide kills bacteria
         ๏ Fungicide kills fungi
         ๏ Algicide kill Algae
         ๏ Viricide kills Viruses
March 10, 2005              Destruction of Microorganisms   Slide No. 79
                 Frequently Used Terms
  • Common Suffixes

  • -static or stasis (Greek statikos,causing to
    stand still or stopping)

         ๏ Bacteriostatic agents halt the growth of bacteria
         ๏ Fungistatic agents halt the growth of fungi

March 10, 2005             Destruction of Microorganisms   Slide No. 80
                 Sterilization
  • Heat-most efficient, reliable method




March 10, 2005      Destruction of Microorganisms   Slide No. 81
      Chemical Disinfectant Resistance
           (From most to least)
  •    Bacterial endospores
  •    Mycobacterium tuberculosis
  •    Small nonlipid viruses (hydrophilic)
  •    Poliovirus, rotavirus, Hep A
  •    Fungi
  •    Medium-sized lipid viruses (lipophilic)
  •    HIV, herpes, Hep B
  •    Vegetative bacteria
March 10, 2005          Destruction of Microorganisms   Slide No. 82
                 Chemical Class (Process)
  • STERILIZATION
         ๏ Sterilant/disinfectant (prolonged contact time)
  • HIGH-LEVEL DISINFECTION
         ๏ Sterilant/disinfectant (short contact time)
  • INTERMEDIATE-LEVEL DISINFECTION
         ๏ Hospital disinfectant (tuberculocidal activity)
  • LOW-LEVEL DISINFECTION
         ๏ Nontuberculocidal hospital disinfectant
March 10, 2005              Destruction of Microorganisms    Slide No. 83
                       Disinfection
  • Low-level
         ๏ Does not kill spores or M. tuberculosis
  • Intermediate-level
         ๏ Kills M. tuberculosis, not necessarily spores
  • High-level
         ๏ Kills M. tuberculosis, kills some spores


March 10, 2005             Destruction of Microorganisms   Slide No. 84
                 Surface Disinfectants
  •    CHLORINE COMPOUNDS
  •    PHENOLS
  •    IODOPHORS
  •    ALCOHOLS (poor cleaning agent)
  •    QUATERNARY AMMONIUM COMPOUNDS
       (Quats)
         ๏ Alcohol-free is not tuberculocidal
         ๏ Quat-alcohol is tuberculocidal
March 10, 2005             Destruction of Microorganisms   Slide No. 85
                  Ideal Disinfectant
  •    Broad spectrum                      •     Surface compatibility
  •    Fast acting                         •     Residual effect
  •    Non toxic                           •     Easy to use
  •    Hypoallergenic                      •     Odorless
  •    Not affected by                     •     Economical
       physical factors




March 10, 2005            Destruction of Microorganisms             Slide No. 86
                       Historical Perspective
                 • In order to eradicate these infections, a new industry was
                   developed—the disposable medical device industry.
                 • Nosocomial infections decreased significantly once this industry
                   became regulated and sterilization processes became
                   standardized.
                 • The new disposable products were created from a class of newly
                   developed low cost plastics that were produced and packaged to
                   maintain their sterile properties up to the time of use;
                 • Disposable plastic devices, such as syringes, blood transfusion
                   kits, and hospital gowns could not be subjected to the traditional
                   sterilization methods of dry heat or steam (autoclave) because
                   they would melt.
                 • New methods of low temperature sterilization had to be
                   developed in order to allow the use of these devices in a sterile
                   environment.


March 10, 2005                        Destruction of Microorganisms            Slide No. 87
   Sterilization or Disinfection of
 Medical Devices: General Principles
                 • In general, reusable medical devices or patient-care equipment
                   that enters normally sterile tissue or the vascular system or
                   through which blood flows should be sterilized before each use.

                 • Sterilization means the use of a physical or chemical procedure
                   to destroy all microbial life, including highly resistant bacterial
                   endospores.

                 • The major sterilizing agents used in hospitals are a) dry heat, b)
                   moist heat by steam autoclaving, c) ethylene oxide gas, and, d)
                   radiation.

                 • Disinfection means the use of a chemical procedure that
                   eliminates virtually all recognized pathogenic microorganisms
                   but not necessarily all microbial forms (e.g., bacterial
                   endospores) on inanimate objects.
March 10, 2005                         Destruction of Microorganisms             Slide No. 88
                        Sterilization Methods
                 • There is no ideal sterilization process but in general:

                 • For liquid products, where possible, utilize one of the variations
                   of steam sterilization. Small volume parenterals, however, also
                   might be compatible with radiation sterilization. Avoid aseptic
                   filtration / fill unless absolutely dictated by product compatibility.

                 • For non-liquid products, steam, dry heat, and radiation
                   sterilization are much preferred over EtO. The aforementioned
                   processes are relatively simple, are amenable to parametric
                   release, and do not leave toxic residues in the product.




March 10, 2005                          Destruction of Microorganisms               Slide No. 89
                                     Dry Heat
                 • Temperature: 140 -170oC
                   Exposure Time: 60 -180 minutes

                 • Dry heat sterilization is a relatively simple process that involves
                   exposure of the product to hot air in an appropriate sized
                   chamber.

                 • To assure temperature uniformity in the chamber, the air is
                   circulated via a fan/blower system.

                 • When glass vials or ampules are sterilized /depyrogenated prior
                   to the asceptic filling of pharmaceuticals, special equipment is
                   utilized that has particulate control systems to ensure that the
                   load is exposed to class 100 conditions or better during the
                   sterilization run.


March 10, 2005                         Destruction of Microorganisms             Slide No. 90
                        Dry Heat (continued)
                 • Typical products sterilized by dry heat, in addition to glass vials
                   and ampules, include heat stable dry powder pharmaceuticals,
                   oils, and products that are heat stable but either sensitive to
                   moisture or not penetrated by moist heat.

                 • The principal advantages of dry heat sterilization are its
                   simplicity, penetrating power, and lack of toxic residues.

                 • Its disadvantages are the relatively long processing time and the
                   high temperature, which limits the types of products and
                   packaging materials compatible with this process.




March 10, 2005                         Destruction of Microorganisms             Slide No. 91
                       Steam under Pressure
                 • Sterilization by steam under pressure also is a relatively simple
                   process which involves exposure of the product to steam at the
                   desired temperature and pressure.

                 • The process usually is carried out in a pressure vessel designed
                   to withstand the high temperature and pressure.

                 • To provide for uniform temperature distribution, it is important to
                   remove the air from the sterilization chamber; this may be
                   accompanied by gravity displacement or by a vacuum system.

                 • A vacuum system is generally preferred when compatible with
                   the product/package system to ensure efficient air removal and
                   optimum steam penetration.



March 10, 2005                         Destruction of Microorganisms             Slide No. 92
        Steam Sterilization: Autoclaving
                                                    • An autoclave is a self
                                                      locking machine that
                                                      sterilizes with steam under
                                                      pressure.

                                                    • Sterilization is achieved by
                                                      the high temperature that
                                                      steam under pressure can
                                                      reach.

                                                    • The high pressure also
                                                      ensures saturation of
                                                      wrapped surgical packs.

                                                    • Ideal for metal instruments.




March 10, 2005      Destruction of Microorganisms                            Slide No. 93
                 Operational Information
            Autoclave    Temperature                   Pressure   Time (min)
             Settings        (F)                         (PSI)

     General                 250                            20       30
     Wrapped Items

     Bottled solutions       250                            20       30


     “Flashing”              270                            20       4-7




March 10, 2005              Destruction of Microorganisms                  Slide No. 94
                 Preparation for Sterilization
                                                    • All instruments must be double
                                                      wrapped in linen or special
                                                      paper or placed in a special
                                                      metal box equipped with a filter
                                                      before sterilization.
                                                    • 'Flashing' is often used when a
                                                      critical instrument is dropped.
                                                    • The white stripes on the tape
                                                      change to black when the
                                                      appropriate conditions
                                                      (temperature) have been met.
                                                    • Indicators should be on the
                                                      inside and outside of equipment
                                                      pack.
                                                    • Expiration dates should be
                                                      printed on all equipment packs.



March 10, 2005             Destruction of Microorganisms                        Slide No. 95
                       Steam under Pressure
                 • The principal advantages of steam sterilization are its simplicity,
                   relatively short processing times, and lack of toxic residues;

                 • Parametric release, that is, the release of product for sale
                   without conducting microbiological sterility testing, generally is
                   easily validated;

                 • Its main disadvantage is the relatively high temperature
                   (generally lower than dry heat, however) making it unsuitable for
                   many plastic devices and lack of utility for products that are
                   moisture sensitive or moisture impermeable.




March 10, 2005                         Destruction of Microorganisms             Slide No. 96
      Steam under Pressure (continued)
                 • Products typically sterilized by steam under pressure include
                   small and large volume parenterals (SVPs, LVPs), surgical
                   dressings, water for injection, contact lenses, and so on.

                 • To be compatible with steam sterilization, a product must be
                   stable with respect to temperature and moisture, and the
                   product/package must be readily penetrated by steam.

                 • Without adequate steam penetration, sterilization can be
                   impeded or defeated entirely.




March 10, 2005                        Destruction of Microorganisms           Slide No. 97
                    Ethylene Oxide
                 Sterilization: ETO Gas
                                            • Colorless gas, very toxic and
                                              flammable;

                                            • Requires special equipment with
                                              special venting requirements;

                                            • Low temperature sterilization
                                              method of choice for heat sensitive
                                              instruments: plastics, suture
                                              material, lenses and finely
                                              sharpened instruments;

                                            • Materials must be well aerated after
                                              sterilization;

                                            • Materials/instruments must be dry.


March 10, 2005          Destruction of Microorganisms                      Slide No. 98
                               Ethylene Oxide
                 • Nonliquid products, contained in gas permeable packages not
                   compatible with the heat or moisture of dry heat or steam
                   sterilizaiton, and not compatible with radiation sterilization, are
                   candidates for sterilization with EtO gas.

                 • Because it is toxic and potentially carcinogenic, the use of EtO is
                   under ever increasing regulatory scrutiny and control.

                 • EtO is flammable and potentially explosive, so specialized
                   equipment and damage limiting facilities are required.

                 • EtO can be used undiluted in its pure form or with nitrogen as a
                   diluent.




March 10, 2005                         Destruction of Microorganisms              Slide No. 99
                              Ethylene Oxide
                 • The primary advantages associated with the use of EtO
                   sterilization are the low processing temperature and the wide
                   range of compatible materials.
                 • The disadvantages relate to the toxicity of the gas, only useful as
                   a surface sterilant unable to reach blocked-off surfaces, such as
                   those found in hypodermic plunger/barrel interfaces in
                   hypodermic needles, and residuals in the product and
                   manufacturing environment are present after treatment.
                 • The increasing cost of the gas and of the various engineering
                   and environmental controls required to assure safe low residual
                   products and low personnel exposure has raised and will
                   continue to escalate the cost of EtO sterilization.
                 • EtO is used for a wide range of products including blood
                   oxygenators, catheters, tracheostomy tubes, mechanical heart
                   valves, sutures, custom procedure kits, adhesive bandages,
                   tubing sets, and so on.

March 10, 2005                        Destruction of Microorganisms            Slide No. 100
             Radiation
(Co-60, Cs-137, accelerated electrons)
                 • Dose: 1.5-3.5 Mrad;
                 • Radiation sterilization, either by gamma rays from Co-60 or Cs-
                   137, radioisotopes, or accelerated electrons, offers a simple
                   sterilization alternative for moisture sensitive/thermolabile
                   nonliquid products;
                 • Inactivation of microorganisms occurs either through direct
                   ionization of a vital cellular molecule (DNA, key enzyme, etc.) or
                   indirectly through the reaction of the free radicals produced in
                   the cellular fluid;
                 • It also applies to small volume thermolabile liquid products that
                   are radiation compatible;
                 • Products to be sterilized are exposed to gamma rays from a Co-
                   60 or a Cs-137 source or to machine accelerated electrons until
                   the desired dose is received.



March 10, 2005                        Destruction of Microorganisms            Slide No. 101
                            Radiation
• No toxic agents are involved, and products may be released for sale on the
  basis of documentation that the desired dose was delivered; microbiological
  release testing generally is not required unless it is a local regulatory
  requirement.
• Gamma radiation is a penetrating sterilant.
• No area of the device or container is left with uncertain sterility. This
  includes prefilled containers.
• There is no need for specialized packaging.
• Since there is no requirement for pressure or vacuum, seals are not
  stressed.
• Gamma radiation is highly reliable due to its single variable to control—
  exposure time.
• Gamma processing has demonstrated lower overall costs. Both large and
  smallproduct volumes can be accommodated in a cost-effective manner.
• Many medical products are sterilized by radiation including sutures, gloves,
  gowns, face masks, dressing, syringes, surgical staplers, and so on.
March 10, 2005                Destruction of Microorganisms          Slide No. 102
                                  Drawbacks
                 • Gamma radiation sterilization is not without its drawbacks.

                 • Recently, tests have shown that the gamma radiation provides
                   an environment conducive to the oxidation of the UHMWPE
                   (Wright Medical Technology, 1995 and Naidu et al., 1997).

                 • Many researchers have concluded that this oxidation process
                   explains the diminished wear properties of the UHMWPE in the
                   human body by changing the percent crystallinity of the
                   UHMWPE (Naidu et al., 1997).




March 10, 2005                        Destruction of Microorganisms              Slide No. 103
                           Aseptic Processing
                 • Many liquid pharmaceutical and biological products cannot
                   withstand any form of thermal sterilization; so most of them are
                   relegated to aseptic filtration and then filled into presterilized
                   containers in a cleanroom environment.

                 • As mentioned above, a few thermolabile liquid products have
                   been demonstrated to be compatible with radiation sterilization.

                 • Aseptic filtration involves passing the solution through a sterile
                   0.1 to 0.22 mm microbiological filter and capturing the filtrate in a
                   presterilized bulk container.

                 • The liquid from the bulk container then must be aseptically
                   dispensed in presterilized containers such as bottles, vials,
                   ampules, or syringes.


March 10, 2005                         Destruction of Microorganisms             Slide No. 104
           Aseptic Processing (continued)
                 • Many parenteral and diagnostic products are asceptically filtered
                   and filled, including intravaneous drug solutions, ophthalmic drug
                   solutions, blood banking reagents, antibiotic solutions, and so
                   on.

                 • There is now increasing pressure in the United States not to
                   approve asceptic filtration / fill processes for products unless
                   terminal sterilization processes have been demonstrated to be
                   deleterious to the product.

                 • Once an asceptic filtration / fill facility has been established and
                   validated, it has been convenient to process subsequent
                   products by this method even though they might, for example,
                   be compatible with steam sterilization.



March 10, 2005                         Destruction of Microorganisms             Slide No. 105
                      Sterilization Principles
         ๏ Sterilization - destruction or removal of all
             forms of life (key to Infection Control program)
                 • Kills spores (most heat resistant microbe)
         ๏ Disinfection - inhibition or destruction of
             pathogens, not all organisms
                 • Spores are not destroyed
                 • Chemicals applied to inanimate surfaces
         ๏ Antiseptic - antimicrobial to living tissue
         ๏ Cleaning-removing debris, reducing total
         ๏ Do not disinfect when you can sterilize
March 10, 2005                        Destruction of Microorganisms   Slide No. 106
                                     Cleaning
  • Principles:
                 •   Removes organic material that:
                 •   protects microorganisms against sterilization and HLD
                 •   can inactivate disinfectants
                 •   Must be done for sterilization and HLD to be effective
                 •   Method of mechanically reducing the number of endospores

  • Practices:
                 • Wash with detergent and water.
                 • Scrub instruments until visibly clean.
                 • Thoroughly rinse with clean water.




March 10, 2005                        Destruction of Microorganisms        Slide No. 107
                                    Sterilization
  • Principles:
                 • Destroys all microorganisms including endospores
                 • Used for instruments, gloves, and other items that come in direct
                   contact with blood stream or tissue under the skin

  • Practices:
                 • Steam sterilization (autoclave):
                     – 121oC (250oF); 106 kPa (15 lbs/in2) pressure: 20 minutes for
                       unwrapped items, 30 minutes for wrapped items
                     – Allow all items to dry before removing
                 • Dry-heat (oven):
                     – 170oC (340oF) for 1 hour, or 160oC (320oF) for 2 hours
                 • Chemical sterilization:
                     – Soak items in glutaraldehyde for 8 -10 hours or formaldehyde for 24 hours
                     – Rinse with sterile water
March 10, 2005                            Destruction of Microorganisms                     Slide No. 108
                    High-Level Disinfection
  • Principles:
                 • Destroys all microorganisms including HBV and HIV; does not
                   reliably kill all bacterial endospores
                 • Only acceptable alternative when sterilization equipment is not
                   available

  • Practices:
                 • Boil instruments and other items for 20 minutes (sufficient up to
                   5500 meters/18,000 ft.).
                 • Always boil for 20 minutes in pot with lid.
                 • Start timing when water begins to boil.
                 • Do not add anything to pot after timing begins.
                 • Air dry before use or storage.


March 10, 2005                        Destruction of Microorganisms            Slide No. 109
     High-Level Disinfection Steaming
  • Practices:
                 • Steam instruments, gloves and other items for 20 minutes.
                 • Always steam for 20 minutes.
                 • Be sure there is enough water in bottom pan for entire steam
                   cycle.
                 • Bring water to rolling boil.
                 • Start timing when steam begins to come out from under lid.
                 • Do not add anything to pan after timing starts.
                 • Air dry and store in covered steamer pans.




March 10, 2005                       Destruction of Microorganisms          Slide No. 110
                    Steamer Used for
                 High-Level Disinfection




March 10, 2005          Destruction of Microorganisms   Slide No. 111
     Chemical High-Level Disinfection
  • Practices:
                 •   Cover all items completely with disinfectant.
                 •   Soak for 20 minutes.
                 •   Rinse with boiled water.
                 •   Air dry before use and storage.




March 10, 2005                          Destruction of Microorganisms   Slide No. 112
                      Preparing
 a High-Level Disinfected Container
   ๏ Boil (if small), or
   ๏ Fill a clean container with 0.5% chlorine solution.
   ๏ Soak for 20 minutes.
   ๏ Pour out solution. (The chlorine solution can
             then be transferred to a plastic container and
             reused.)
         ๏ Rinse thoroughly with boiled water.
         ๏ Air dry and use for storage of HLD items.

March 10, 2005               Destruction of Microorganisms   Slide No. 113
                              Waste Disposal
  • Principles:
                 • Prevents spread of infection to clinic personnel who handle
                   waste
                 • Prevents spread of infection to local community
                 • Protects those who handle wastes from accidental injury
                 • Practices:
                 • Wearing utility gloves, place contaminated items (gauze or
                   cotton) in leak-proof container (with a lid) or plastic bag.
                 • Dispose by incineration or burial.




March 10, 2005                        Destruction of Microorganisms           Slide No. 114
     Traffic Flow and Activity Patterns
  • Goal: To eliminate level of microbial
    contamination in areas where “clean
    activities” take place:
                 • procedure rooms
                 • surgical areas
                 • areas for final processing and instrument storage

  • Number of microorganisms in area is related
    to number of people present and their
    activity


March 10, 2005                       Destruction of Microorganisms     Slide No. 115
  Human blood transmissible viruses




March 10, 2005   Destruction of Microorganisms   Slide No. 116
                 Newly Emerging Viruses
๏   GB virus type C (Flavivirus)
๏   TT virus (Circovirus)
๏   West Nile Virus (Flavivirus)
๏   SARS CoV (Coronavirus)
๏   Nipah Virus (Paramyxovirus)
๏   Monkey pox virus (Orthopoxvirus)
๏   Avian Influenza Virus
๏   …and next year ???




March 10, 2005              Destruction of Microorganisms   Slide No. 117
                         Viruses
  • Klein-Deforest
  • Lipid Enveloped Viruses
         ๏ HIV, HBV, HSV, HCV
  • Small N/E Viruses
         ๏ Picorna, HAV, HEV
  • Larger N/E Viruses
    Adenovirus


March 10, 2005          Destruction of Microorganisms   Slide No. 118
       Current Viral Clearance Methods
  Clearance = Inactivation (die-off) or Removal

                   Inactivation                                               Removal
         ๏   Heating:                                             ๏      Partitioning during
                 • Heating in solution                                   purification
                 • Terminal dry heat                                      • Ethanol fractionation
                 • Vapor heating                                          • Other precipitation steps
         ๏   Chemical:                                                    • Chromatography
                 • Solvent / detergent                            ๏      Nanofiltration
                 • Ethanol / methanol                                     • 15-100 nm
                 • Low pH                                                 • Adsorption



March 10, 2005                           Destruction of Microorganisms                            Slide No. 119
                 Current Safety and Concerns
  • Plasma derivatives enjoy excellent safety
    profiles, so why seek improved methods?
                 • Current viral clearance methods are marginally effective for non-
                   enveloped viruses
                 • Uncertainty about characteristics of new/emerging infectious
                   agents
                 • Lack of robustness of some viral clearance methods
                 • Desire to improve existing safety margins
                 • Increasingly stringent standard guidelines in many countries




March 10, 2005                        Destruction of Microorganisms           Slide No. 120
                      Some Alternative
                 Viral Inactivation Methods
                  • Irradiation               • Ionizing (gamma)
                                              • Ultraviolet (UVC)
                                              • Visible

                  • Chemical                  •
                                              •
                                                  Inactine
                                                  Beta-propiolactone
                                              •   Aldehydes/amines
                                              •   S-303 ("FRALE")
                                              •   Iodine

                  • Photochemical             •
                                              •
                                                  Types 1 and 2
                                                  Psoralens (UVA)
                                              •   Riboflavin (visible
                                              •   Phenothiazines (620-670 nm)
                                              •   Phthalocyanines (>570 nm)

                  • Physical                  • Supercritical Fluids
                                              • Pressure
                                              • Severe heat or microwavw
March 10, 2005                 Destruction of Microorganisms                    Slide No. 121
            Adverse Effects
    on Product After Viral Inactivation
         Thermal Denaturation
                 • Heat, irradiation
         Chemical Modification
                 • Possible with most methods
         Chemical Contamination
                 • Toxicity/mutagenicity of chemicals
                 • Validated removal or conversion
         ROS & Free Radical Oxidation
                 • Irradiation and photochemical inactivation




March 10, 2005                         Destruction of Microorganisms   Slide No. 122
Methods of viral inactivation (die-off)
  • Chemical: organic solvents; pH extremes;
    solvent detergent; alcohol
  • Physical: Heat treatment (dry heat or
    pasteurization)
  • pH, HEAT treatment, ultraviolet radiation,
    IONIZING RADIATION; DESICCATION;
    ANTISEPTICS; DISINFECTANTS; organic
    solvents, and DETERGENTS.

March 10, 2005     Destruction of Microorganisms   Slide No. 123
                 General Considerations
         ๏ Manufacturing processes for blood derived
             products should contain two effective steps for
             removal/inactivation of viruses
         ๏ At least one step should be effective against
             non-enveloped viruses
         ๏ At least one stage in a production process must
             inactivate rather than remove viruses
         ๏ A single step having a large effect gives more
             assurance of viral safety than several steps
             having the same overall effect
March 10, 2005               Destruction of Microorganisms   Slide No. 124
                 QUESTIONS




March 10, 2005    Destruction of Microorganisms   Slide No. 125
                   Internet Resources
     ๏ Laboratory biosafety manual (WHO 2004).
          Available at
          http://www.who.int/entity/csr/resources/publication
          s/biosafety/en/Biosafety7.pdf
     ๏ Inactivation (disinfection) process. Available at
          http://www.who.int/entity/water_sanitation_health/
          dwq/en/watreatpath3.pdf
     ๏ Ultraviolet Radiation. Available at
          http://www.epa.gov/safewater/mdbp/pdf/alter/cha
          pt_8.pdf
March 10, 2005             Destruction of Microorganisms   Slide No. 126

				
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