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Control of Microorganisms. Definitions Conditions Influencing Antimicrobial Activity Physical Methods Chemical Agents Preservation of Microbial Cultures. Definitions. Sterilization: A treatment that kills or removes all living cells, including viruses and spores, from a substance or object

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control of microorganisms
Control of Microorganisms
  • Definitions
  • Conditions Influencing Antimicrobial Activity
  • Physical Methods
  • Chemical Agents
  • Preservation of Microbial Cultures
definitions
Definitions
  • Sterilization: A treatment that kills or removes all living cells, including viruses and spores, from a substance or object
  • Disinfection: A treatment that reduces the total number of microbes on an object or surface, but does not necessarily remove or kill all of the microbes
definitions1
Definitions
  • Sanitation: Reduction of the microbial population to levels considered safe by public health standards
  • Antiseptic: A mild disinfectant agent suitable for use on skin surfaces
  • -cidal: A suffix meaning that “the agent kills.” For example, a bacteriocidal agent kills bacteria
definitions2
Definitions
  • -static: A suffix that means “the agent inhibits growth.” For example, a fungistatic agent inhibits the growth of fungi, but doesn’t necessarily kill it.
conditions influencing antimicrobial activity
Conditions Influencing Antimicrobial Activity
  • Under most circumstances, a microbial population is not killed instantly by an agent but instead over a period of time
  • The death of the population over time is exponential, similar to the growth during log phase
conditions influencing antimicrobial activity1
Conditions Influencing Antimicrobial Activity
  • Several critical factors play key roles in determining the effectiveness of an antimicrobial agent, including:
    • Population size
    • Types of organisms
    • Concentration of the antimicrobial agent
    • Duration of exposure
    • Temperature
    • pH
    • Organic matter
    • Biofilm formation
physical methods
Physical Methods
  • Moist Heat
  • Dry Heat
  • Low Temperatures
  • Filtration
  • Radiation
physical methods moist heat
Physical Methods: Moist Heat
  • Mechanism of killing is a combinantion of protein/nucleic acid denaturation and membrane disruption
  • Effectiveness Heavily dependent on type of cells present as well as environmental conditions (type of medium or substrate)
  • Bacterial spores much more difficult to kill than vegetative cells
physical methods moist heat1
Physical Methods: Moist Heat
  • Measurements of killing by moist heat
    • Thermal death point (TDP): Lowest temperature at which a microbial suspension is killed in 10 minutes; misleading because it implies immediate lethality despite substrate conditions
    • Thermal death time (TDT): Shortest time needed to kill all organisms in a suspension at a specified temperature under specific conditions; misleading because it does not account for the logarithmic nature of the death curve (theoretically not possible to get down to zero)
physical methods moist heat2
Physical Methods: Moist Heat
  • Measurements of killing by moist heat (cont.)
    • Decimal reduction time (D value): The time required to reduced a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions
    • z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions
    • F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores
physical methods moist heat3
Physical Methods: Moist Heat
  • Calculations using D and z values
    • Given: For Clostridium botulinum spores suspended in phosphate buffer, D121 = 0.204 min
    • How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 1012 spores to 100 spores (1 spore) at 121°C?Answer: Since 1012 to 100 is 12 decimal reductions, then the time required is 12 x 0.204 min = 2.45 min
physical methods moist heat4
Physical Methods: Moist Heat
  • Calculations using D and z values (cont.)
    • Given the D value at one temperature and the z value, we can derive an equation to predict the D value at a different temperature:
physical methods moist heat5
Physical Methods: Moist Heat
  • Calculations using D and z values (cont.)
          • First, write an equation for this line. Since the y axis is on a log scale, then y = log (D). The slope of the line is -1/z; we’ll let the y intercept be equal to c. Therefore:
          • At a given temperature Ta, D = Da, so we can eliminate the “c” term
physical methods moist heat6
Physical Methods: Moist Heat
  • Calculations using D and z values (cont.)
          • We can explicitly refer to the second temperature and D value as Tb and Db, so:
physical methods moist heat7
Physical Methods: Moist Heat
  • Calculations using D and z values (cont.)
    • Given: For Clostridium botulinum spores suspended in phosphate buffer, D121 = 0.204 min and z = 10°C
    • How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 1012 spores to 100 spores (1 spore) at 111°C?Answer: To answer the question we need to know D111, which we can calculate from the formula:log(D111/0.204) = (121-111) /10D111 = 0.204(10) = 2.04 min12D111= 24.5 min
physical methods moist heat8
Physical Methods: Moist Heat
  • Calculations using D and z values (cont.)
    • Given: For Staph. aureus in turkey stuffing, D60 = 15.4 min and z = 6.8°C
    • How long would it take to reduce a population of Staph. aureus in turkey stuffing from 105 cells to 100 cells at 55°C, 60°C, and 65°C?Answers: Work it out for yourself. Here are the answers.At 55°C: 419 minAt 60°C: 77 minAt 65°C: 14.2 min
physical methods moist heat9
Physical Methods: Moist Heat
  • Methods of Moist Heat
    • Boiling at 100°C
      • Effective against most vegetative cells; ineffective against spores; unsuitable for heat sensitive chemicals & many foods
    • Autoclaving/pressure canning
      • Temperatures above 100°C achieved by steam pressure
      • Most procedures use 121.1°C, achieved at approx. 15 psi pressure, with 15 - 30 min autoclave time to ensure sterilization
      • Sterilization in autoclave in biomedical or clinical laboratory must by periodically validated by testing with spores of Clostridium or Bacillus stearothermophilus
physical methods moist heat10
Physical Methods: Moist Heat
  • Methods of Moist Heat
    • Pasteurization
      • Used to reduce microbial numbers in milk and other beverages while retaining flavor and food quality of the beverage
      • Retards spoilage but does not sterilize
      • Traditional treatment of milk, 63°C for 30 min
      • Flash pasteurization (high-temperature short term pasteurization); quick heating to about 72°C for 15 sec, then rapid cooling
physical methods moist heat11
Physical Methods: Moist Heat
  • Methods of Moist Heat
    • Ultrahigh-temperature (UHT) sterilization
      • Milk and similar products heated to 140 - 150°C for 1 - 3 sec
      • Very quickly sterilizes the milk while keeping its flavor & quality
      • Used to produce the packaged “shelf milk” that does not require refrigeration
physical methods dry heat
Physical Methods: Dry Heat
  • Incineration
    • Burner flames
    • Electric loop incinerators
    • Air incinerators used with ferementers; generally operated at 500°C
  • Oven sterilization
    • Used for dry glassware & heat-resistant metal equipment
    • Typically 2 hr at 160°C is required to kill bacterial spores by dry heat: this does not include the time for the glass to reach the required temp (penetration time) nor does it include the cooling time
physical methods low temperatures
Physical Methods:Low Temperatures
  • Refrigerator:
    • around 4°C
    • inhibits growth of mesophiles or thermophiles; psychrophiles will grow
  • Freezer:
    • “ordinary” freezer around -10 to -20°C
    • “ultracold” laboratory freezer typically -80°C
    • Generally inhibits all growth; many bacteria and other microbes may survive freezing temperatures
physical methods filtration
Physical Methods: Filtration
  • Used for physically removing microbes and dust particles from solutions and gasses; often used to sterilize heat-sensitive solutions or to provide a sterilized air flow
  • Depth filters: eg. Diatomaceous earth, unglazed porcelean
  • Membrane filters: eg. Nitrocellulose, nylon, polyvinylidene difluoride
  • HEPA filters: High efficiency particulate air filters used in laminar flow biological safety cabinets
physical methods radiation
Physical Methods: Radiation
  • Ultraviolet Radiation
    • DNA absorbs ultraviolet radiation at 260 nm wavelength
    • This causes damage to DNA in the form of thymine dimer mutations
    • Useful for continuous disinfection of work surfaces, e.g. in biological safety cabinets
physical methods radiation1
Physical Methods: Radiation
  • Ionizing Radiation
    • Gamma radiation produced by Cobalt-60 source
    • Powerful sterilizing agent; penetrates and damages both DNA and protein; effective against both vegetative cells and spores
    • Often used for sterilizing disposable plastic labware, e.g. petri dishes; as well as antibiotics, hormones, sutures, and other heat-sensitive materials
    • Also can be used for sterilization of food; has been approved but has not been widely adopted by the food industry
chemical agents
Chemical Agents
  • Phenolics
  • Alcohols
  • Halogens
  • Heavy metals
  • Quaternary Ammonium Compounds
  • Aldehydes
  • Sterilizing Gases
  • Evaluating Effectiveness of Chemical Agents
chemical agents phenolics
Chemical Agents: Phenolics
  • Aromatic organic compounds with attached -OH
  • Denature protein & disrupt membranes
  • Phenol, orthocresol, orthophenylphenol, hexachlorophene
  • Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application
  • Disagreeable odor & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage
chemical agents alcohols
Chemical Agents: Alcohols
  • Ethanol; isopropanol; used at concentrations between 70 – 95%
  • Denature proteins; disrupt membranes
  • Kills vegetative cells of bacteria & fungi but not spores
  • Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench
chemical agents halogens
Chemical Agents: Halogens
  • Act as oxidizing agents; oxidize proteins & other cellular components
  • Chlorine compounds
    • Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas)
    • Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as benchtop disinfectant
    • Halazone tablets (parasulfone dichloroamidobenzoic acid) used by campers to disinfect water for drinking
chemical agents halogens1
Chemical Agents: Halogens
  • Iodine Compounds
    • Tincture of iodine (iodine solution in alcohol)
    • Potassium iodide in aqueous solution
    • Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne
    • Used as antiseptics for cleansing skin surfaces and wounds
chemical agents heavy metals
Chemical Agents: Heavy Metals
  • Mercury, silver, zinc, arsenic, copper ions
  • Form precipitates with cell proteins
  • At one time were frequently used medically as antiseptics but much of their use has been replaced by less toxic alternatives
  • Examples: 1% silver nitrate was used as opthalmic drops in newborn infants to prevent gonorrhea; has been replaced by erythromycin or other antibiotics; copper sulfate used as algicide in swimming pools
chemical agents quaternary ammonium compounds
Chemical Agents: QuaternaryAmmonium Compounds
  • Quaternary ammonium compounds are cationic detergents
  • Amphipathic molecules that act as emulsifying agents
  • Denature proteins and disrupt membranes
  • Used as disinfectants and skin antiseptics
  • Examples: cetylpyridinium chloride, benzalkonium chloride
chemical agents aldehydes
Chemical Agents: Aldehydes
  • Formaldehyde and gluteraldehyde
  • React chemically with nucleic acid and protein, inactivating them
  • Aqueous solutions can be used as disinfectants
chemical agents sterilizing gases
Chemical Agents: Sterilizing Gases
  • Ethylene oxide (EtO)
    • Used to sterilize heat-sensitive equipment and plasticware
    • Explosive; supplied as a 10 – 20% mixture with either CO2 or dichlorofluoromethane
    • Its use requires a special EtO sterilizer to carefully control sterilization conditions as well as extensive ventilation after sterilation because of toxicity of EtO
    • Much of the commercial use of EtO (for example, plastic petri dishes) has in recent years been replaced by gamma irradiation
chemical agents sterilizing gases1
Chemical Agents: Sterilizing Gases
  • Betapropiolactone (BPL)
    • In its liquid form has been used to sterilize vaccines and sera
    • Decomposes after several hours and is not as difficult to eliminate as EtO, but it doesn’t penetrate as well as EtO and may also be carcinogenic
    • Has not been used as extensively as EtO
  • Vapor-phase hydrogen peroxide
    • Has been used recently to decontaminate biological safety cabinets
chemical agents evaluating the effectiveness
Chemical Agents:Evaluating the Effectiveness
  • Phenol Coefficient Test
    • A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhi and Staphylococcus aureus and incubated at either 20°C or 37°C
    • Samples are removed at 5 min intervals and inoculated into fresh broth
    • The cultures are incubated at 37°C for 2 days
    • The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient
chemical agents evaluating the effectiveness1
Chemical Agents:Evaluating the Effectiveness
  • Phenol Coefficient Test (cont.)
    • The reciprocal of the maximum effective dilution for the test disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient
    • For example:Suppose that, on the test with Salmonella typhiThe maximum effective dilution for phenol is 1/90The maximum effective dilution for “Disinfectant X” is 1/450The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5
chemical agents evaluating the effectiveness2
Chemical Agents:Evaluating the Effectiveness
  • Phenol Coefficient Test (cont.)
    • Phenol coefficients are useful as an initial screening and comparison, but can be misleading because they only compare two pure strains under specific controlled conditions
  • Use dilution tests and simulated in-use tests
    • Are tests designed to more closely approximate actual normal in-use conditions of a disinfectant
preservation of microbial cultures
Preservation of Microbial Cultures
  • Periodic Transfer and Refrigeration
  • Mineral Oil Slant
  • Freezing in Growth Medium
  • Drying
  • Lyophilization
  • Ultracold Freezing
preservation of microbial cultures periodic transfer and refrigeration
Preservation of Microbial Cultures:Periodic Transfer and Refrigeration
  • Stock cultures are aseptically transferred at appropriate intervals to fresh medium and incubated, then stored at 4°C until they are transferred again
  • Many labs use “agar slants;” care has to be taken to avoid contamination
  • Major problem with possible genetic changes in strains; most labs need a way to keep “long term” storage of original genetic stocks
preservation of microbial cultures mineral oil slant
Preservation of Microbial Cultures:Mineral Oil Slant
  • Sterile mineral oil placed over growth on agar slants to preserve cultures for longer period of time in the refrigerator
  • Contamination problems; messy; many organisms are sensitive to this; generally it is a poor technique and doesn’t work well
preservation of microbial cultures freezing in growth medium
Preservation of Microbial Cultures:Freezing in Growth Medium
  • Used as a “long term” storage strategy
  • Broth cultures of the organisms are frozen at -20°C
  • Often, sterile glycerin (glycerol) is added at a 25 – 50% final concentration; this helps to prevent ice crystal formation and increases viability of many organisms
preservation of microbial cultures drying
Preservation of Microbial Cultures:Drying
  • Suitable for some bacterial species
  • Samples are grown on sterile paper disks saturated with nutrient, then the disks are allowed to air dry and stored aseptically
  • Reconstituted by dropping disk into nutrient broth medium
preservation of microbial cultures lyophilization
Preservation of Microbial Cultures:Lyophilization
  • Suitable for many bacterial species as well as fungi and viruses
  • Broth cultures are placed in special ampules and attached to a vacuum pump; the vacuum removes all of the water from the cells leaving a “freeze-dried” powder
  • The culture is reconstituted by adding broth to the lyophilized powder and incubating it
  • Considered the best method of long-term storage for most bacterial species
preservation of microbial cultures ultracold freezing
Preservation of Microbial Cultures:Ultracold Freezing
  • Similar to freezing, but at very cold temperature
  • At about -70 to -80°C, in liquid nitrogen or in an ultracold freezer unit