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Microbial Control
Outline
•    History
•    Definition of terms
•    Pattern of microbial death
•    Antimicrobial agents
     –  Physical
     –  Chemical
History



Ring- a - ring of rosies
 Pocketful of posies
  Atichoo ! Atichoo !
   We all fall down
Bubonic Plague or the
                Black Death
•  Epidemic swept thru Europe in the Middle
   Ages (13th and 14th centuries)
•  40 million people were killed
  –  About 1/3 of the population of the continent
•  Etiological agent:
  –  Yersinia pestis     Gram (-) rod
•  2 Vectors
  –  Rat
  –  Flea
Yersinia pestis - Gram (-) bacillus
Vectors - Rat and Flea
Bubonic Plague Infection

1. Flea bite with Yersinia pestis
2. Bacteria multiply in the bloodstream
  Bacteremia
3. Bacteria localize in lymph nodes,
especially axillary and groin areas
4. Hemorrhaging occurs in lymph nodes,
resulting in reddish rash that will eventually
become “black and blue” swellings or Buboes
(hence the name Bubonic Plague or Black
Death)
Bubonic Plague Infection
5. If untreated, about 50 % Mortality Rate
6. If bacteria spread to the lungs, it becomes
Pneumonic Plague and is now highly
contagious (Almost a 99 % Mortality Rate)
History



                            Ring- a - ring of rosies
                               Pocketful of posies
                                Atichoo ! Atichoo !
                                   We all fall down
A pouch of sweet smelling herbs or posies were carried due to
the belief that the disease was transmitted by bad smells
Humans vs. Microbes
•  Most of History, microbes have been
   winning the battle

•  In the last 100 yrs or so the battle has
   swung in our favor
  –  Why?
     •  Because of our increasing knowledge of how to
        Control Microbial Growth
Smallpox




     Variola virus
Eradicated in 1977 (Somalia)
Terms :
Sterilization vs. Disinfection
•  Sterilization
  –  destroying all forms of life
•  Disinfection
  –  destroying pathogens or unwanted
     organisms
Disinfectant vs. Antiseptic
•  Disinfectant
  –  antimicrobial agent used on inanimate objects
•  Antiseptic
  –  antimicrobial agent used on living tissue
cidal    vs. static
•  Bactericidal - kills bacteria
•  Bacteristatic - inhibits bacterial growth

•    Fungicidal
•    Fungistatic
•    Algacidal
•    Algastatic
—    Commercial Sterilization: Killing C. botulinum endospores
Microbial death
“a microbe is defined DEAD if it does not
  grow when inoculated into culture medium
  that would normally support its growth”

Death is defined as the inability of the
 organisms to form a visible colony
                     –James Jay
                     (Modern Food Microbiology 6th ed)
“It takes more
                          time to kill a
                          large population
                          of bacteria than
                          it does to kill a
                          small
                          population,
                          because only a
                          fraction of
                          organisms die
                          during a given
                          time interval.




Bacterial populations die at a
  constant logarithmic rate
Conditions Influencing Effectiveness
   of Antimicrobial Agent Activity
1.  Population size
  –    Larger population requires a longer time to die

2.  Population composition
  –    Microorganisms vary markedly on susceptibility
  –    Vegetative versus Spores
  –    Young versus Mature cells

5.  Concentration or Intensity of an Antimicrobial Agent
  –    The more concentrated an agent the more rapidly
       microbes can be destroyed
  –    BUT sometimes an agent may be more effective at lower
       concentrations (e.g. 70% alcohol)
Conditions Influencing Effectiveness
  of Antimicrobial Agent Activity
 3.  Duration of Exposure
   –    The longer the exposure to an agent the more they will be
        killed


 6.  Temperature
   –    An increase in temperature at which a chemical acts often
        enhances it activity
   –    Example: acids used in high T = more effective


 7.  Local environment
   –    pH, organic matter, etc
   –    Controls or Protects the pathogen
Targets of Antimicrobial Agents

1. Cell membrane

2. Enzymes & Proteins

3. DNA & RNA
Methods of microbial control
•  Physical
  –  Heat
  –  Filtration
  –  Radiation
•  Chemical
HEAT
•    Flaming
•    Incineration
•    Hot-air sterilization
•    Moist-air sterilization


                        Hot-air       Autoclave

Equivalent treatments   170˚C, 2 hr   121˚C, 15 min
HOW DOES HEAT KILL
     MICROBES
•  MOIST HEAT
  –  Kill effectively by degrading nucleic acids and by
     denaturing enzymes and other essential proteins
  –  May also disrupt cell membranes


•  DRY HEAT
  –  Microbial death results from the oxidation of cell
     constituents and denaturation of proteins
MOIST HEAT
STERILIZATION
•  Thermal Death Point (TDP)
  –  The lowest temperature at which a microbial
     suspension in killed in 10 minutes


•  Thermal Death Time (TDT)
  –  The shortest time needed to kill all organisms
     in a microbial suspension at a specific
     temperature and under defined conditions
•  Decimal Reduction Time or D value
      –  Time required to kill 90% of the
         microorganisms or spores in a sample at a
         specified temperature
      –  Time required for the line to drop by one log
         cycle or tenfold
      –  Used to estimate the relative resistance of a
         microbe to different temperatures

However, such a destruction is logarithmic and it is theoretically
NOT POSSIBLE to “completely destroy” microbes in a sample
The z Value
                     and
                 The F Value

•  z Value
  –  The increase in temperature required to reduce D
     to 1/10 its value or to reduce it by one log cycle


•  F value
  –  Time in minutes at a specific temperature needed
     to kill a population of cells or spores
  –  Usually 121°C
APPLICATION:
 After a food have been canned, it must be heated to
eliminate the risk of botulism arising from the presence of
                     Clostridium spores

Example:
IF the D value = 0.204 minutes
It would take 12D or 2.5 minutes to
reduce the spore number by heating at
the specified temperature
APPLICATION:
•  If the z value for Clostridium spores is 10°C
•  It takes a 10°C change in temperature to alter
   the D value tenfold

•  THUS: if the cans are to be processed at
   111°C rather than 121°C, the D value would
   increase by tenfold t 2.04 minutes
•  The 12D value = 24.5 minutes
THE USE OF PHYSICAL
METHODS: FILTRATION
•  Applicable for heat-sensitive materials that needs sterilization

•  2 types of filters
    –  Depth filters: consist of fibrous or granular materials that have been
       bonded into a thick layer filled with twisting channels of small diameter
    –  Membrane filters: porous membranes; 0.2 µm pore sizes

•  Laminar flow hood versus biological safety cabinets (HEPA filters)
    –  High Efficiency Particulate Air
    –  Remove 99.97% particles
    –  for sterilizing AIR
THE USE OF PHYSICAL
 METHODS: RADIATION
•  IONIZING RADIATION
   •    X rays, gamma rays, electron beams
   •    Excellent as a sterilizing agent and penetrates deep into objects


•  NON-IONIZING RADIATION
    –  UV (about 260nm)
    –  Quite lethal but does not penetrate glass, dirt films, water and
       other substances very effectively


•  Microwaves: kill by heat not usually antimicrobial
MicrobialControlpart1

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MicrobialControlpart1

  • 2. Outline •  History •  Definition of terms •  Pattern of microbial death •  Antimicrobial agents –  Physical –  Chemical
  • 3. History Ring- a - ring of rosies Pocketful of posies Atichoo ! Atichoo ! We all fall down
  • 4. Bubonic Plague or the Black Death •  Epidemic swept thru Europe in the Middle Ages (13th and 14th centuries) •  40 million people were killed –  About 1/3 of the population of the continent •  Etiological agent: –  Yersinia pestis Gram (-) rod •  2 Vectors –  Rat –  Flea
  • 5. Yersinia pestis - Gram (-) bacillus Vectors - Rat and Flea
  • 6. Bubonic Plague Infection 1. Flea bite with Yersinia pestis 2. Bacteria multiply in the bloodstream Bacteremia 3. Bacteria localize in lymph nodes, especially axillary and groin areas
  • 7. 4. Hemorrhaging occurs in lymph nodes, resulting in reddish rash that will eventually become “black and blue” swellings or Buboes (hence the name Bubonic Plague or Black Death)
  • 8. Bubonic Plague Infection 5. If untreated, about 50 % Mortality Rate 6. If bacteria spread to the lungs, it becomes Pneumonic Plague and is now highly contagious (Almost a 99 % Mortality Rate)
  • 9. History Ring- a - ring of rosies Pocketful of posies Atichoo ! Atichoo ! We all fall down A pouch of sweet smelling herbs or posies were carried due to the belief that the disease was transmitted by bad smells
  • 10. Humans vs. Microbes •  Most of History, microbes have been winning the battle •  In the last 100 yrs or so the battle has swung in our favor –  Why? •  Because of our increasing knowledge of how to Control Microbial Growth
  • 11. Smallpox Variola virus Eradicated in 1977 (Somalia)
  • 12. Terms : Sterilization vs. Disinfection •  Sterilization –  destroying all forms of life •  Disinfection –  destroying pathogens or unwanted organisms
  • 13. Disinfectant vs. Antiseptic •  Disinfectant –  antimicrobial agent used on inanimate objects •  Antiseptic –  antimicrobial agent used on living tissue
  • 14. cidal vs. static •  Bactericidal - kills bacteria •  Bacteristatic - inhibits bacterial growth •  Fungicidal •  Fungistatic •  Algacidal •  Algastatic
  • 15. —  Commercial Sterilization: Killing C. botulinum endospores
  • 16. Microbial death “a microbe is defined DEAD if it does not grow when inoculated into culture medium that would normally support its growth” Death is defined as the inability of the organisms to form a visible colony –James Jay (Modern Food Microbiology 6th ed)
  • 17. “It takes more time to kill a large population of bacteria than it does to kill a small population, because only a fraction of organisms die during a given time interval. Bacterial populations die at a constant logarithmic rate
  • 18. Conditions Influencing Effectiveness of Antimicrobial Agent Activity 1.  Population size –  Larger population requires a longer time to die 2.  Population composition –  Microorganisms vary markedly on susceptibility –  Vegetative versus Spores –  Young versus Mature cells 5.  Concentration or Intensity of an Antimicrobial Agent –  The more concentrated an agent the more rapidly microbes can be destroyed –  BUT sometimes an agent may be more effective at lower concentrations (e.g. 70% alcohol)
  • 19.
  • 20. Conditions Influencing Effectiveness of Antimicrobial Agent Activity 3.  Duration of Exposure –  The longer the exposure to an agent the more they will be killed 6.  Temperature –  An increase in temperature at which a chemical acts often enhances it activity –  Example: acids used in high T = more effective 7.  Local environment –  pH, organic matter, etc –  Controls or Protects the pathogen
  • 21.
  • 22. Targets of Antimicrobial Agents 1. Cell membrane 2. Enzymes & Proteins 3. DNA & RNA
  • 23. Methods of microbial control •  Physical –  Heat –  Filtration –  Radiation •  Chemical
  • 24. HEAT •  Flaming •  Incineration •  Hot-air sterilization •  Moist-air sterilization Hot-air Autoclave Equivalent treatments 170˚C, 2 hr 121˚C, 15 min
  • 25. HOW DOES HEAT KILL MICROBES •  MOIST HEAT –  Kill effectively by degrading nucleic acids and by denaturing enzymes and other essential proteins –  May also disrupt cell membranes •  DRY HEAT –  Microbial death results from the oxidation of cell constituents and denaturation of proteins
  • 27. •  Thermal Death Point (TDP) –  The lowest temperature at which a microbial suspension in killed in 10 minutes •  Thermal Death Time (TDT) –  The shortest time needed to kill all organisms in a microbial suspension at a specific temperature and under defined conditions
  • 28. •  Decimal Reduction Time or D value –  Time required to kill 90% of the microorganisms or spores in a sample at a specified temperature –  Time required for the line to drop by one log cycle or tenfold –  Used to estimate the relative resistance of a microbe to different temperatures However, such a destruction is logarithmic and it is theoretically NOT POSSIBLE to “completely destroy” microbes in a sample
  • 29. The z Value and The F Value •  z Value –  The increase in temperature required to reduce D to 1/10 its value or to reduce it by one log cycle •  F value –  Time in minutes at a specific temperature needed to kill a population of cells or spores –  Usually 121°C
  • 30.
  • 31. APPLICATION: After a food have been canned, it must be heated to eliminate the risk of botulism arising from the presence of Clostridium spores Example: IF the D value = 0.204 minutes It would take 12D or 2.5 minutes to reduce the spore number by heating at the specified temperature
  • 32. APPLICATION: •  If the z value for Clostridium spores is 10°C •  It takes a 10°C change in temperature to alter the D value tenfold •  THUS: if the cans are to be processed at 111°C rather than 121°C, the D value would increase by tenfold t 2.04 minutes •  The 12D value = 24.5 minutes
  • 33. THE USE OF PHYSICAL METHODS: FILTRATION •  Applicable for heat-sensitive materials that needs sterilization •  2 types of filters –  Depth filters: consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter –  Membrane filters: porous membranes; 0.2 µm pore sizes •  Laminar flow hood versus biological safety cabinets (HEPA filters) –  High Efficiency Particulate Air –  Remove 99.97% particles –  for sterilizing AIR
  • 34.
  • 35. THE USE OF PHYSICAL METHODS: RADIATION •  IONIZING RADIATION •  X rays, gamma rays, electron beams •  Excellent as a sterilizing agent and penetrates deep into objects •  NON-IONIZING RADIATION –  UV (about 260nm) –  Quite lethal but does not penetrate glass, dirt films, water and other substances very effectively •  Microwaves: kill by heat not usually antimicrobial