Disinfectants b


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Disinfectants b

  1. 1. Control of Microorganisms <ul><li>Definitions </li></ul><ul><li>Conditions Influencing Antimicrobial Activity </li></ul><ul><li>Physical Methods </li></ul><ul><li>Chemical Agents </li></ul><ul><li>Preservation of Microbial Cultures </li></ul>
  2. 2. Definitions <ul><li>Sterilization: A treatment that kills or removes all living cells, including viruses and spores, from a substance or object </li></ul><ul><li>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 </li></ul>
  3. 3. Definitions <ul><li>Sanitation: Reduction of the microbial population to levels considered safe by public health standards </li></ul><ul><li>Antiseptic: A mild disinfectant agent suitable for use on skin surfaces </li></ul><ul><li>-cidal: A suffix meaning that “the agent kills.” For example, a bacteriocidal agent kills bacteria </li></ul>
  4. 4. Definitions <ul><li>-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. </li></ul>
  5. 5. Conditions Influencing Antimicrobial Activity <ul><li>Under most circumstances, a microbial population is not killed instantly by an agent but instead over a period of time </li></ul><ul><li>The death of the population over time is exponential, similar to the growth during log phase </li></ul>
  6. 6. Conditions Influencing Antimicrobial Activity <ul><li>Several critical factors play key roles in determining the effectiveness of an antimicrobial agent, including: </li></ul><ul><ul><li>Population size </li></ul></ul><ul><ul><li>Types of organisms </li></ul></ul><ul><ul><li>Concentration of the antimicrobial agent </li></ul></ul><ul><ul><li>Duration of exposure </li></ul></ul><ul><ul><li>Temperature </li></ul></ul><ul><ul><li>pH </li></ul></ul><ul><ul><li>Organic matter </li></ul></ul><ul><ul><li>Biofilm formation </li></ul></ul>
  7. 7. Physical Methods <ul><li>Moist Heat </li></ul><ul><li>Dry Heat </li></ul><ul><li>Low Temperatures </li></ul><ul><li>Filtration </li></ul><ul><li>Radiation </li></ul>
  8. 8. Physical Methods: Moist Heat <ul><li>Mechanism of killing is a combinantion of protein/nucleic acid denaturation and membrane disruption </li></ul><ul><li>Effectiveness Heavily dependent on type of cells present as well as environmental conditions (type of medium or substrate) </li></ul><ul><li>Bacterial spores much more difficult to kill than vegetative cells </li></ul>
  9. 9. Physical Methods: Moist Heat <ul><li>Measurements of killing by moist heat </li></ul><ul><ul><li>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 </li></ul></ul><ul><ul><li>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) </li></ul></ul>
  10. 10. Physical Methods: Moist Heat <ul><li>Measurements of killing by moist heat (cont.) </li></ul><ul><ul><li>Decimal reduction time ( D value): The time required to reduce a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions </li></ul></ul><ul><ul><li>z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions </li></ul></ul><ul><ul><li>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 </li></ul></ul>
  11. 11. Physical Methods: Moist Heat <ul><li>Calculations using D and z values </li></ul><ul><ul><li>Given: For Clostridium botulinum spores suspended in phosphate buffer, D 121 = 0.204 min </li></ul></ul><ul><ul><li>How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 10 12 spores to 10 0 spores (1 spore) at 121°C? Answer: Since 10 12 to 10 0 is 12 decimal reductions, then the time required is 12 x 0.204 min = 2.45 min </li></ul></ul>
  12. 12. Physical Methods: Moist Heat <ul><li>Calculations using D and z values (cont.) </li></ul><ul><ul><li>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: </li></ul></ul>
  13. 13. Physical Methods: Moist Heat <ul><li>Calculations using D and z values (cont.) </li></ul><ul><ul><ul><ul><ul><li>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: </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>At a given temperature T a , D = D a , so we can eliminate the “c” term </li></ul></ul></ul></ul></ul>
  14. 14. Physical Methods: Moist Heat <ul><li>Calculations using D and z values (cont.) </li></ul><ul><ul><ul><ul><ul><li>We can explicitly refer to the second temperature and D value as T b and D b , so: </li></ul></ul></ul></ul></ul>
  15. 15. Physical Methods: Moist Heat <ul><li>Calculations using D and z values (cont.) </li></ul><ul><ul><li>Given: For Clostridium botulinum spores suspended in phosphate buffer, D 121 = 0.204 min and z = 10°C </li></ul></ul><ul><ul><li>How long would it take to reduce a population of C. botulinum spores in phosphate buffer from 10 12 spores to 10 0 spores (1 spore) at 111°C? Answer: To answer the question we need to know D 111 , which we can calculate from the formula : log( D 111 /0.204) = (121-111) /10 D 111 = 0.204(10) = 2.04 min 12 D 111 = 24.5 min </li></ul></ul>
  16. 16. Physical Methods: Moist Heat <ul><li>Calculations using D and z values (cont.) </li></ul><ul><ul><li>Given: For Staph. aureus in turkey stuffing, D 60 = 15.4 min and z = 6.8°C </li></ul></ul><ul><ul><li>How long would it take to reduce a population of Staph. aureus in turkey stuffing from 10 5 cells to 10 0 cells at 55°C, 60°C, and 65°C? Answers: Work it out for yourself. Here are the answers. At 55°C: 419 min At 60°C: 77 min At 65°C: 14.2 min </li></ul></ul>
  17. 17. Physical Methods: Moist Heat <ul><li>Methods of Moist Heat </li></ul><ul><ul><li>Boiling at 100°C </li></ul></ul><ul><ul><ul><li>Effective against most vegetative cells; ineffective against spores; unsuitable for heat sensitive chemicals & many foods </li></ul></ul></ul><ul><ul><li>Autoclaving/pressure canning </li></ul></ul><ul><ul><ul><li>Temperatures above 100°C achieved by steam pressure </li></ul></ul></ul><ul><ul><ul><li>Most procedures use 121.1°C, achieved at approx. 15 psi pressure, with 15 - 30 min autoclave time to ensure sterilization </li></ul></ul></ul><ul><ul><ul><li>Sterilization in autoclave in biomedical or clinical laboratory must by periodically validated by testing with spores of Clostridium or Bacillus stearothermophilus </li></ul></ul></ul>
  18. 18. Physical Methods: Moist Heat <ul><li>Methods of Moist Heat </li></ul><ul><ul><li>Pasteurization </li></ul></ul><ul><ul><ul><li>Used to reduce microbial numbers in milk and other beverages while retaining flavor and food quality of the beverage </li></ul></ul></ul><ul><ul><ul><li>Retards spoilage but does not sterilize </li></ul></ul></ul><ul><ul><ul><li>Traditional treatment of milk, 63°C for 30 min </li></ul></ul></ul><ul><ul><ul><li>Flash pasteurization (high-temperature short term pasteurization); quick heating to about 72°C for 15 sec, then rapid cooling </li></ul></ul></ul>
  19. 19. Physical Methods: Moist Heat <ul><li>Methods of Moist Heat </li></ul><ul><ul><li>Ultrahigh-temperature (UHT) sterilization </li></ul></ul><ul><ul><ul><li>Milk and similar products heated to 140 - 150°C for 1 - 3 sec </li></ul></ul></ul><ul><ul><ul><li>Very quickly sterilizes the milk while keeping its flavor & quality </li></ul></ul></ul><ul><ul><ul><li>Used to produce the packaged “shelf milk” that does not require refrigeration </li></ul></ul></ul>
  20. 20. Physical Methods: Dry Heat <ul><li>Incineration </li></ul><ul><ul><li>Burner flames </li></ul></ul><ul><ul><li>Electric loop incinerators </li></ul></ul><ul><ul><li>Air incinerators used with ferementers; generally operated at 500°C </li></ul></ul><ul><li>Oven sterilization </li></ul><ul><ul><li>Used for dry glassware & heat-resistant metal equipment </li></ul></ul><ul><ul><li>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 </li></ul></ul>
  21. 21. Physical Methods: Low Temperatures <ul><li>Refrigerator: </li></ul><ul><ul><li>around 4°C </li></ul></ul><ul><ul><li>inhibits growth of mesophiles or thermophiles; psychrophiles will grow </li></ul></ul><ul><li>Freezer: </li></ul><ul><ul><li>“ ordinary” freezer around -10 to -20°C </li></ul></ul><ul><ul><li>“ ultracold” laboratory freezer typically -80°C </li></ul></ul><ul><ul><li>Generally inhibits all growth; many bacteria and other microbes may survive freezing temperatures </li></ul></ul>
  22. 22. Physical Methods: Filtration <ul><li>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 </li></ul><ul><li>Depth filters: eg. Diatomaceous earth, unglazed porcelean </li></ul><ul><li>Membrane filters: eg. Nitrocellulose, nylon, polyvinylidene difluoride </li></ul><ul><li>HEPA filters: High efficiency particulate air filters used in laminar flow biological safety cabinets </li></ul>
  23. 23. Physical Methods: Radiation <ul><li>Ultraviolet Radiation </li></ul><ul><ul><li>DNA absorbs ultraviolet radiation at 260 nm wavelength </li></ul></ul><ul><ul><li>This causes damage to DNA in the form of thymine dimer mutations </li></ul></ul><ul><ul><li>Useful for continuous disinfection of work surfaces, e.g. in biological safety cabinets </li></ul></ul>
  24. 24. Physical Methods: Radiation <ul><li>Ionizing Radiation </li></ul><ul><ul><li>Gamma radiation produced by Cobalt-60 source </li></ul></ul><ul><ul><li>Powerful sterilizing agent; penetrates and damages both DNA and protein; effective against both vegetative cells and spores </li></ul></ul><ul><ul><li>Often used for sterilizing disposable plastic labware, e.g. petri dishes; as well as antibiotics, hormones, sutures, and other heat-sensitive materials </li></ul></ul><ul><ul><li>Also can be used for sterilization of food; has been approved but has not been widely adopted by the food industry </li></ul></ul>
  25. 25. Chemical Agents <ul><li>Phenolics </li></ul><ul><li>Alcohols </li></ul><ul><li>Halogens </li></ul><ul><li>Heavy metals </li></ul><ul><li>Quaternary Ammonium Compounds </li></ul><ul><li>Aldehydes </li></ul><ul><li>Sterilizing Gases </li></ul><ul><li>Evaluating Effectiveness of Chemical Agents </li></ul>
  26. 26. Chemical Agents: Phenolics <ul><li>Aromatic organic compounds with attached -OH </li></ul><ul><li>Denature protein & disrupt membranes </li></ul><ul><li>Phenol, orthocresol, orthophenylphenol, hexachlorophene </li></ul><ul><li>Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application </li></ul><ul><li>Disagreeable odor & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage </li></ul>
  27. 27. Chemical Agents: Alcohols <ul><li>Ethanol; isopropanol; used at concentrations between 70 – 95% </li></ul><ul><li>Denature proteins; disrupt membranes </li></ul><ul><li>Kills vegetative cells of bacteria & fungi but not spores </li></ul><ul><li>Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench </li></ul>
  28. 28. Chemical Agents: Halogens <ul><li>Act as oxidizing agents; oxidize proteins & other cellular components </li></ul><ul><li>Chlorine compounds </li></ul><ul><ul><li>Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas) </li></ul></ul><ul><ul><li>Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as benchtop disinfectant </li></ul></ul><ul><ul><li>Halazone tablets (parasulfone dichloroamidobenzoic acid) used by campers to disinfect water for drinking </li></ul></ul>
  29. 29. Chemical Agents: Halogens <ul><li>Iodine Compounds </li></ul><ul><ul><li>Tincture of iodine (iodine solution in alcohol) </li></ul></ul><ul><ul><li>Potassium iodide in aqueous solution </li></ul></ul><ul><ul><li>Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne </li></ul></ul><ul><ul><li>Used as antiseptics for cleansing skin surfaces and wounds </li></ul></ul>
  30. 30. Chemical Agents: Heavy Metals <ul><li>Mercury, silver, zinc, arsenic, copper ions </li></ul><ul><li>Form precipitates with cell proteins </li></ul><ul><li>At one time were frequently used medically as antiseptics but much of their use has been replaced by less toxic alternatives </li></ul><ul><li>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 </li></ul>
  31. 31. Chemical Agents: Quaternary Ammonium Compounds <ul><li>Quaternary ammonium compounds are cationic detergents </li></ul><ul><li>Amphipathic molecules that act as emulsifying agents </li></ul><ul><li>Denature proteins and disrupt membranes </li></ul><ul><li>Used as disinfectants and skin antiseptics </li></ul><ul><li>Examples: cetylpyridinium chloride, benzalkonium chloride </li></ul>
  32. 32. Chemical Agents: Aldehydes <ul><li>Formaldehyde and gluteraldehyde </li></ul><ul><li>React chemically with nucleic acid and protein, inactivating them </li></ul><ul><li>Aqueous solutions can be used as disinfectants </li></ul>
  33. 33. Chemical Agents: Sterilizing Gases <ul><li>Ethylene oxide (EtO) </li></ul><ul><ul><li>Used to sterilize heat-sensitive equipment and plasticware </li></ul></ul><ul><ul><li>Explosive; supplied as a 10 – 20% mixture with either CO 2 or dichlorofluoromethane </li></ul></ul><ul><ul><li>Its use requires a special EtO sterilizer to carefully control sterilization conditions as well as extensive ventilation after sterilation because of toxicity of EtO </li></ul></ul><ul><ul><li>Much of the commercial use of EtO (for example, plastic petri dishes) has in recent years been replaced by gamma irradiation </li></ul></ul>
  34. 34. Chemical Agents: Sterilizing Gases <ul><li>Betapropiolactone (BPL) </li></ul><ul><ul><li>In its liquid form has been used to sterilize vaccines and sera </li></ul></ul><ul><ul><li>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 </li></ul></ul><ul><ul><li>Has not been used as extensively as EtO </li></ul></ul><ul><li>Vapor-phase hydrogen peroxide </li></ul><ul><ul><li>Has been used recently to decontaminate biological safety cabinets </li></ul></ul>
  35. 35. Chemical Agents: Evaluating the Effectiveness <ul><li>Phenol Coefficient Test </li></ul><ul><ul><li>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 </li></ul></ul><ul><ul><li>Samples are removed at 5 min intervals and inoculated into fresh broth </li></ul></ul><ul><ul><li>The cultures are incubated at 37°C for 2 days </li></ul></ul><ul><ul><li>The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient </li></ul></ul>
  36. 36. Chemical Agents: Evaluating the Effectiveness <ul><li>Phenol Coefficient Test (cont.) </li></ul><ul><ul><li>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 </li></ul></ul><ul><ul><li>For example: Suppose that, on the test with Salmonella typhi The maximum effective dilution for phenol is 1/90 The maximum effective dilution for “Disinfectant X” is 1/450 The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5 </li></ul></ul>
  37. 37. Chemical Agents: Evaluating the Effectiveness <ul><li>Phenol Coefficient Test (cont.) </li></ul><ul><ul><li>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 </li></ul></ul><ul><li>Use dilution tests and simulated in-use tests </li></ul><ul><ul><li>Are tests designed to more closely approximate actual normal in-use conditions of a disinfectant </li></ul></ul>
  38. 38. Preservation of Microbial Cultures <ul><li>Periodic Transfer and Refrigeration </li></ul><ul><li>Mineral Oil Slant </li></ul><ul><li>Freezing in Growth Medium </li></ul><ul><li>Drying </li></ul><ul><li>Lyophilization </li></ul><ul><li>Ultracold Freezing </li></ul>
  39. 39. Preservation of Microbial Cultures: Periodic Transfer and Refrigeration <ul><li>Stock cultures are aseptically transferred at appropriate intervals to fresh medium and incubated, then stored at 4 °C until they are transferred again </li></ul><ul><li>Many labs use “agar slants;” care has to be taken to avoid contamination </li></ul><ul><li>Major problem with possible genetic changes in strains; most labs need a way to keep “long term” storage of original genetic stocks </li></ul>
  40. 40. Preservation of Microbial Cultures: Mineral Oil Slant <ul><li>Sterile mineral oil placed over growth on agar slants to preserve cultures for longer period of time in the refrigerator </li></ul><ul><li>Contamination problems; messy; many organisms are sensitive to this; generally it is a poor technique and doesn’t work well </li></ul>
  41. 41. Preservation of Microbial Cultures: Freezing in Growth Medium <ul><li>Used as a “long term” storage strategy </li></ul><ul><li>Broth cultures of the organisms are frozen at -20 °C </li></ul><ul><li>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 </li></ul>
  42. 42. Preservation of Microbial Cultures: Drying <ul><li>Suitable for some bacterial species </li></ul><ul><li>Samples are grown on sterile paper disks saturated with nutrient, then the disks are allowed to air dry and stored aseptically </li></ul><ul><li>Reconstituted by dropping disk into nutrient broth medium </li></ul>
  43. 43. Preservation of Microbial Cultures: Lyophilization <ul><li>Suitable for many bacterial species as well as fungi and viruses </li></ul><ul><li>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 </li></ul><ul><li>The culture is reconstituted by adding broth to the lyophilized powder and incubating it </li></ul><ul><li>Considered the best method of long-term storage for most bacterial species </li></ul>
  44. 44. Preservation of Microbial Cultures: Ultracold Freezing <ul><li>Similar to freezing, but at very cold temperature </li></ul><ul><li>At about -70 to -80 °C, in liquid nitrogen or in an ultracold freezer unit </li></ul>