Foundations in Microbiology

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					Microbial
  Control
  Chapter 11




               1
• We share our world with many microbes;
  – eliminating them is impossible and completely
    unwarranted

  – controlling their growth is essential
     • We clean, cook, refrigerate to prevent spoilage

     • Hand washing, sterilization, disinfection procedures in medical
       settings target pathogens to prevent infection


• Consider living in a time BEFORE there was a
  basic knowledge of microbes……
  – Seasonal foods (in various degrees of spoilage), kill it and
    eat it, parasites and all, or starve
     • Spices were precious
  – Shortened life spans, fear and suffering                             2
• Microbes capable of causing spoilage or
  infection include:
  –   vegetative bacterial cells and endospores
  –   fungal hyphae, spores, yeast
  –   protozoan trophozoites and cysts
  –   worms
  –   viruses and prions




• Mechanisms of control involve:
  – physical, chemical, and mechanical methods to destroy
    or reduce undesirable microbes in a given environment
                                                        3
4
        Decontamination Procedures

•   Any process used to control or destroy
    contaminants
    –   Contaminants are defined as microbes growing in a
        place or at a time deemed undesirable


•   Method of control based on many overlapping
    considerations:
    –   Level of destruction necessary or desired
    –   System to be decontaminated
    –   Relative resistance of microbes
    –   Practical concerns, cost effectiveness
                                                            5
                  Level of Control
• Disinfection
  – Destruction or removal of vegetative pathogens, but
    not endospores
• Sterilization
  – The complete destruction of all viable microorganisms
     • Includes viruses and endospores
     • Essential for invasive equipment
• Sanitation
  – Any cleansing technique that mechanically removes
    microbes
     • Reduces the numbers of microbes (does not necessarily
       destroy them)
     • Removes material harboring or supporting microbes       6
             More Control Issues
• Sepsis is growth in/on tissues, antisepsis
  procedures attempt to prevent sepsis

   – Antiseptics are chemicals applied to the body
     surface to destroy or inhibit vegetative pathogens

   – Tissues cannot be sterilized.


• Microbistasis is the inhibition of microbial
  growth, not the destruction of microbes
   – Used in storage (of both perishables and microbes!)
                                                           7
Practical Concerns in Microbial Control
• Method of control depends on circumstances:
  – Is the application on tissue or inanimate objects?

  – Does the application require sterilization? Disinfection?
    Is the method suitable?

  – Is the item to be reused? Can the item withstand heat,
    pressure, radiation, or chemicals?

  – Will the agent penetrate to the necessary extent?

  – Is the method cost- and labor-efficient? Is it safe?

                                                           8
       Relative Resistance of Microbes
• Highest resistance
  – bacterial endospores, prions

• Moderate resistance
  – protozoan cysts,
  – zygospores
  – naked viruses

• Least resistance
  –   most bacterial vegetative cells
  –   fungal spores and hyphae, yeast
  –   enveloped viruses
                                         9
  –   protozoan trophozoites
    Determining Microbial Death
• Death is defined as the permanent
  termination of organism’s vital processes.
  – Easier to determine in large, multicellular
    organisms
     • check for respiration, pulse, brain activity, etc.


• How is death determined in microbes?
  – vital processes are on cellular level, detection
    very difficult
     • may be completely dormant, but viable
     • living and dead cells look the same
                                                            10
               Microbial Death
• Microbial death is defined as the
  permanent loss of reproductive capability,
  even under optimum growth conditions.
  – microbes die at different rates
  – require different procedures to obtain
                                             Death phase
                                             may be
                                             prolonged




                                                   11
The effectiveness of a particular agent
   is governed by several factors
–   Number of microbes
      •   Higher load, more time
–   Nature of microbes
      •   Types, resistance, level of control desired
–   Temperature and pH
      •   System involved may dictate
–   Concentration or dosage of agent
      •   Higher levels more efficient, practicality issue
–   Mode of action of the agent
      •   Cellular target of agent
–   Presence of solvents, organic matter, or inhibitors
      •   Many substances prevent contact, interfere with
          effectiveness                                      12
                 Modes of Action
Cellular targets of physical and chemical agents:
   • cell wall
      – block synthesis, wall breaks down,
          becomes fragile, lyses easily

   •   cell membrane
       – lose of integrity, disrupts transport
       – loss of vitals, entry of harmful substances

   •   cellular synthetic processes
       – disrupt structure or function of DNA, RNA
       – prevention of replication, transcription, translation

   •   protein functions
       – interfere with active site
       – disrupt or denature proteins                        13
             Methods of Control
                --Physical--
• Microbes survive in extreme environments
  – based on adaptation and natural selection
  – we attempt to control only those living in our
    environment


• Abrupt changes in the environmental
  conditions are detrimental (to any organism)
  – even microbes do not have time to adapt
                                                     14
                           HEAT
• TDT vs. TDP
  – Minimal time at set temp. vs. minimal temp. at set
    time
    • What are the practical considerations in determining
      the time? the temperature?

  – Moist heat – lower temperatures and shorter
    exposure times effective due to:
    • coagulation and denaturation of proteins
    • destroys DNA and cell membranes
  – Dry heat – moderate to high temperatures
    • dehydration alters protein structure, removal of water
      inhibits metabolism
    • Incineration completely oxidizes cells                 15
 Heat Resistance and Thermal Death
• Bacterial endospores are most resistant
  – usually require temperatures above boiling




                                                 16
                     This week in lab!!




Bacillus subtillus




                                          17
             Moist Heat Methods
• Steam under pressure – sterilization
  – Autoclave
     15 psi @ 121oC for 15min
      • (2 atm)
  – Increasing pressure
     increases temperature of
     steam
    media, bandages, equipment
    pressure cooking used in canning foods


• Non-pressurized steam – disinfection
  – Boiling 30 minutes to destroy non-spore-
    forming pathogens
  – (water boils at 100oC at 1 atm psi)        18
                Pasteurization
• Heat is applied without destroying the flavor or
  nutritional value of food
     • ~65°C for 30 minutes (batch method)
     • ~72°C for 15 seconds (flash method)
     • 140oC for 2 seconds (ultra pasteurization)
• Pasteurization is not sterilization – kills non-
  spore-forming pathogens in dairy products
     • does not kill endospores or many nonpathogenic
       microbes
     • targets pathogenic contaminants (from handling) like
       Salmonella, Mycobacterium

• Lowers overall microbe count, increases shelf life

• Also used to stop fermentation (in beverage            19
  production) Why?
                 Tyndallization

• Intermittent application of non-pressurized
  steam achieves sterilization for substances
  that cannot withstand autoclaving
  – Steam kills vegetative cells
  – Spores germinate to vegetative form
  – Repeat steaming
     • Successive exposure kills vegetative cells


• Not considered completely reliable
                                                    20
                 Dry Heat Methods
• Requires higher temperatures or longer exposure
  time than with moist heat (to obtain same level).
   – Dehydration stabilizes proteins
   – Dry ovens – 150-180oC for 2 hours
      • Good for glassware, metallic instruments


• Incineration – use of flames (>500oC)
   – Bunsen burner @ 1870 oC, Furnaces up to 6500 oC
      • Complete oxidation of microbes and other substances
         – powders, oils (substances moisture does not penetrate well)



                                                                   21
• Drying used to preserve fruits, vegetables,
  yeasts, used with smoke on fish and
  sausage
     • absence of water inhibits enzymes,
        – sporeformers survive but do not produce toxins
     • many cells retain ability to grow when water is
       reintroduced
• Some bacteria sensitive to drying, kills some
  delicate pathogens immediately
  – Treponema pallidum (causes syphilis)
     • Keep toilet seat dry!
  – Laundry dryers or sun drying can kill pathogens
                                                           22
                            Cold
• The removal of heat is Microbiostatic
  – slows the growth of microbes, does not destroy them
• Refrigeration 0-15oC and Freezing <0oC
  – Used to preserve food, media and cultures
  – More commonly used in homes than canning

• Desiccation is the removal of water at room
  temperature

• Lyophilization - freeze drying
  – freezing rapidly under vacuum to remove water
  – Store in sealed container (keep water out)
  – Preservation of cultures
     • Tiny ice crystals, little cellular damage
                                                     23
                     Radiation
•   Electromagnetic energy from atomic activity
    – May increase shelf life by 500%
    – Used to control food-borne diseases,
      pathogens, insects
•   Ionizing radiation - gamma rays, X-rays
    – deep penetrating power
    – breaks DNA,
      causes mutation


    – ‘Cold sterilization’ - plastic medical supplies
      and food products
                                                    24
                  Radiation (cont.)
•   Non-ionizing radiation – UV light
    –    excites atoms but no ions formed
                                                 Normal DNA
    –    little penetrating power
    –    must be directly exposed
        • surface treatment only
        • Kills airborne microbes
           – also waterborne

                    Thymine dimer


•   UV light absorbed by pyrimidines, creates
    abnormal bonding of nucleotides, interferes
    with replication
    –   Effective against viruses
    –   Bacteria repair damage, endospores unaffected
                                                        25
                       Filtration
• Physical removal of microbes
  by passing a gas or liquid
  through filter
  – sterilize heat sensitive liquids:
     • blood products, vitamins, etc.


  – HEPA filters used in ventilation
    systems to control microbes
     • air in hospital isolation units
         – tuberculosis patient’s rooms
     • maximum containment labs
     • industrial clean rooms
                                          26
Chemical Agents in Microbial Control
• Desirable qualities of chemicals:
  – rapid action in low concentration
  – soluble and stable
  – broad spectrum, low toxicity
  – penetrating, noncorrosive and nonstaining
  – affordable and readily available


• No ideal germicidal agent exists
  – Consider what may come close(?)
                                                27
   Summary of commercial products




• Let’s consider a few of these…..

                                     28
                     Halogens

• Chlorine – Cl2, hypochlorites (chlorine bleach),
  chloramines
  – denatures proteins by disrupting disulfide bonds
  – unstable in sunlight, inactivated by organic matter
  – water, sewage, wastewater, inanimate objects


• Iodine - I2, iodophors (betadine)
  – denature proteins
  – milder medical & dental degerming agents,
    disinfectants, ointments
                                                          29
                        Phenolics
• Derivatives of toxic Phenol (carbolic acid)
   – One of first medical germicides
   – Too toxic for use

• Disrupt cell walls and membranes and precipitates
  proteins
   – Lysol
   – Triclosan - antibacterial additive to soaps and
     other products
   – Hibiclens – chlorhexidine
      •   Broad spectrum
      •   Preoperative scrubs
      •   Not absorbed deeply into skin
      •   Not impaired by organic matter
      •   Widely used                              Causes brain
                                                   damage 30
                    Alcohols
• Ethanol, isopropanol
  – solutions of 50-95% more effective than pure
    • Water helps
• Target cell membrane, act as surfactants
  – dissolves membrane lipids
  – coagulates proteins


• Evaporative
  – pro and con
                                                   31
            Hydrogen Peroxide
• Reactive forms of Oxygen are toxic
  – Hydroxyl free radicals damage protein and
    DNA
  – toxic to anaerobes (decompose to O2)
  – used to sterilize intricate invasive equipment
  – ozone (O3) is another form
• H 2O 2
  – Highly reactive (oxidizing agent) O2-
    (superoxide)
  – Antiseptic at low concentrations; strong
    solutions are sporicidal
                                                     32
          Detergents and Soaps
• Detergents
  – Amphipathic molecules act as surfactants
  – alter membrane permeability of some bacteria and
    fungi.
     • Dimethyl benzyl ammonium chloride
• Soaps
  – Alkaline compounds
  – Sanitizer - mechanically
    remove soil and grease
    containing microbes
  – Germicidal chemicals
    may be added
                                                       33
                     Heavy Metals
• Solutions of mercury and silver are toxic in
  low concentrations
  – kills vegetative cells by binding to proteins
    (halts metabolism)
     • Merthiolate, Metaphen (0.2% Hg in alcohol)
        – Anyone remember mercurochrome?


     • Crede Silver Nitrate
       used for gonococcal infections in
       eyes of newborns



                                                    34
                      Aldehydes
• Binds to amino acids, cross links surface
  proteins (alkylation)
  – Glutaraldehyde
     • 2% solution (Cidex) used as sterilant for heat sensitive
       instruments, rubber, plastics
        – Mild, noncorrosive, stable



  – Formaldehyde (gaseous)
     • disinfectant, preservative, toxicity limits use
     • formalin (37% aqueous solution)
        – Embalming fluid
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