Lect.6 (chapter 5 microbial control)


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Lect.6 (chapter 5 microbial control)

  1. 1. Dr. Adel Ahmed Ali El-Morsi B.Sc. (General BotanyB.Sc. (General Botany , (, ( MansouraMansoura University, 1995University, 1995 M.Sc. Microbiology (virology), Mansoura University, 1999M.Sc. Microbiology (virology), Mansoura University, 1999 Ph.D. Microbiology (virology), Mansoura University, 2005Ph.D. Microbiology (virology), Mansoura University, 2005 General MicrobiologyGeneral Microbiology B204B204
  2. 2. General Microbiology (General Microbiology (B204B204)) Prepared by Dr. Adel Ahmed Ali El-MorsiPrepared by Dr. Adel Ahmed Ali El-Morsi
  3. 3. Controlling Microorganisms  Reduce or destroy undesirable microbes in a given area Effected in two basic ways:Effected in two basic ways: 1. By Killing Microorganisms 2. By inhibiting the Growth of Microorganisms Usually involves the use of:Usually involves the use of: 1. Physical Agents 2. Chemical Agents
  4. 4. Microbial Control AA BB CC DD EE
  5. 5.  SterilizationSterilization  Sterile • Inanimate objects free of all life • Sterilization is the removing of all microorganisms in a material or on the surface of an object (an extreme level of cleanliness) • A surface or an object is either sterile or it is not sterile, there are no gradations in sterility • Typically the last things to die when one attempts sterilization is the highly heat- (and chemical-, etc.) resistant endospores • Once something is sterilized, it will remain sterile if properly sealed. SterilizationSterilization –– a process that destroys all viablea process that destroys all viable microbes, including viruses and endosporesmicrobes, including viruses and endospores Terminology of Microbial ControlTerminology of Microbial Control  PreventionPrevention Control of Growth to Prevent Infection and Spoilage
  6. 6.  Disinfection/disinfectantsDisinfection/disinfectants •Disinfection means reducing the number of viable microorganisms present in a sample •Disinfection / Reducing Growth Nonliving Surfaces •A disinfectant is a chemical or physical agent that is applied to inanimate objects to kill microbes •Not all disinfectants are capable of sterilizing, but, of course, all disinfectants are employed with the hope of disinfecting. •While disinfection is not as extreme as sterilization, but it is considered to be an adequate level of cleanliness for most situations.  DisinfectionDisinfection destroy vegetative pathogens not endospores Disinfection – a process to destroy vegetativeDisinfection – a process to destroy vegetative pathogens, not endospores; inanimate objectspathogens, not endospores; inanimate objects Examples of disinfectants include iodine solution, copper sulfate, ozone, and chlorine gas.
  7. 7.  Sepsis refers to microbial contamination.  Asepsis is the absence of significant contamination. • Aseptic surgery techniques prevent microbial contamination of wounds.  Antisepsis/antiseptic Antiseptic • Typically an antiseptic is a chemical agent that is applied to living tissue to kill microbes • Note that not all disinfectants are antiseptics because an antiseptic additionally must not be so harsh that it damages living tissue • In general antiseptics are either not as cheap or not as effective at killing microbes as disinfectants Antiseptic – disinfectants applied directly to exposed body surfaces AntisepsisAntisepsis • Antisepsis / Reducing Growth on Living Tissue • Antiseptics are generally less toxic than disinfectants because they must not cause too much damage to the host living tissue. • Examples:iodine,70%ethanol,3%hydrogen peroxide.
  8. 8.  SanitizationSanitization • cleansing technique that mechanically removes microbes to safe levels • Sanitization is the cleaning of pathogenic microorganisms from public eating utensils and objects such as that done by the kitchen of a restaurant • Subject to High Temperature Washing (Dishwashers) Sanitization – any cleansing technique that mechanically removes microbes  PasteurizationPasteurization • not sterilization (removes unwanted organisms) • Pasteurization uses mild temperatures (63°C for 30 minutes or 71°C for 15 seconds) to kill pathogens and reduce levels of non-pathogenic organisms that cause milk and other foods to spoil
  9. 9.  Microbicidal agentsMicrobicidal agents (cidal means kill) • Causes microbial death • Bactericide, Sporocide, Fungicide,Viricide • Suffix-cidal • An antimicrobial that kills a microorganism (or, more specifically, a bacterium) is said to be bactericidal  MicrobistasisMicrobistasis (static means inhibition of growth and multiplication) • Prevents microbial growth • Bacteriostatic, Fungistatic • Suffix –stasis/-static • Bacteriostatic means that the antimicrobial inhibits bacterial growth but does not kill the bacteria; consequently, removal or dilution of the antimicrobial can result in a resurgence of bacterial growth  DegermingDegerming ((Swab the Skin) removing organisms from an object’s surface Degermation – mechanically removing microbes form surface (skin) such as surgical hand scrubbing, or wiping skin with alcohol prior to venapuncture
  10. 10.  Sterilization vs. DisinfectionSterilization vs. Disinfection • Sterilization: destroying all forms of life • Disinfection: destroying pathogens or unwanted organisms  Disinfectant vs. AntisepticDisinfectant vs. Antiseptic • Disinfectant: antimicrobial agent used on inanimate objects • Antiseptic: antimicrobial agent used on living tissue Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)  cidal vs. staticcidal vs. static • Bactericidal - kills bacteria • Bacteristatic - inhibits bacterial growth Fungicidal, Fungistatic, Algacidal, Algastatic Terms for Microbial ControlTerms for Microbial Control
  11. 11.  Sterilization:Sterilization: Removal (destruction ) of all microbial life Commercial Sterilization: Heat Treatment of Canned Foods e.g. Killing C. botulinum endospores  Disinfection:Disinfection: Removal of pathogens (destruction of vegetative pathogens)  Antisepsis:Antisepsis: Removal of pathogens from living tissue (disinfection of living tissue)  Sepsis: refers to microbial contamination.  Degerming:Degerming: Removal of microbes from a limited area  Sanitization:Sanitization: Lower microbial counts on eating utensils  Biocide/GermicideBiocide/Germicide: Kills microbes  Bacteriostasis:Bacteriostasis: Inhibiting, not killing, microbes TerminologyTerminology
  12. 12. Microbial DeathMicrobial Death  Involves permanent loss of reproductive capability • even under optimum growth conditions  Still alive, but can’t reproduce!! • Not viable in environment  Hard to detect  Loss of movement. BB
  13. 13. Rate of Microbial DeathRate of Microbial Death  Bacteria usually die at a constant logarithmic rate  Plotted Logarithmically This Will Give a Straight Line
  14. 14.  Any method (agent) which can reduce or destroy undesirable microbes in a given area through killing and /or inhibiting using:- • Physical • Chemical • Mechanical CC
  15. 15. Cellular Targets of ControlCellular Targets of Control  Mode of action of antimicrobials:  Cell wallCell wall  Cell membraneCell membrane  Cellular synthetic processes (DNA, RNA)Cellular synthetic processes (DNA, RNA)  Proteins (Proteins (Enzymes)Enzymes) Targets of Antimicrobial AgentsTargets of Antimicrobial Agents Control Agents Act ByControl Agents Act By Mechanisms of action for Antimicrobial control agents  Alternation of membrane permeability  Damage to proteins  Damage to nucleic acids  Interfere with metabolic pathways
  16. 16.  Many types of chemical and physical microbial controls  Modes of action fall into two basic categories 1.1. Alteration of cell walls or cytoplasmic membranesAlteration of cell walls or cytoplasmic membranes 2.2. Interference with protein and nucleic acid structureInterference with protein and nucleic acid structure Action of Antimicrobial AgentsAction of Antimicrobial Agents  Alteration of Membrane Permeability - Susceptibility of membrane is due to its lipid and protein composition - Control Agents can alter permeability  Damage to Proteins and Nucleic Acids - Break hydrogen and covalent bonds in proteins - Interfere with DNA, RNA, Protein Synthesis
  17. 17.  Cell wall maintains integrity of cell • When disrupted, cannot prevent cell from bursting due to osmotic effects  Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell • When damaged, cellular contents leak out  Viral envelope responsible for attachment of virus to target cell • Damage to envelope interrupts viral replication  Nonenveloped viruses have greater tolerance of harsh conditions Alteration of Cell Walls and MembranesAlteration of Cell Walls and Membranes  Protein function depends on 3-D shape • Extreme heat or certain chemicals denature proteins  Chemicals, radiation, and heat can alter or destroy nucleic acids • Can produce fatal mutants • Can halt protein synthesis through action on RNA Damage to Proteins and Nucleic AcidsDamage to Proteins and Nucleic Acids
  18. 18.  Ideally, agents should be:Ideally, agents should be: • Inexpensive • Fast-acting • Stable during storage • Control all microbial growth while being harmless to humans, animals, and objects Selection of Microbial Control MethodsSelection of Microbial Control Methods Practical Concerns for Microbial ControlPractical Concerns for Microbial Control  Does the application require sterilization?  Is the item to be reused?  Can the item withstand heat, pressure, radiation, or chemicals?  Is the method suitable?  Will the agent penetrate to the necessary extent?  Is the method cost- and labor-efficient & is it safe?
  19. 19. 1. Nature of site to be treated 2. Degree of susceptibility of microbes involved 3. Environmental conditions that pertain Factors Affecting the Efficacy ofFactors Affecting the Efficacy of Antimicrobial methodsAntimicrobial methods DD
  20. 20.  Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects  Method and level of microbial control based on site of medical procedure Site to Be TreatedSite to Be Treated
  21. 21. Organismal differencesOrganismal differences  Microorganisms differ in their susceptibility to antimicrobial agents."  Often what fails to be killed by a disinfectant are endospores though certain viruses and some vegetative bacteria are also highly resistant to disinfection  Also, the same organism may differ in susceptibility depending on growth phase with actively growing organisms typically more susceptible to disinfection than not-growing cultures Microbial Characteristics and ControlMicrobial Characteristics and Control
  22. 22. Level of ResistanceLevel of Resistance 1. Endospores 2. Mycobacteria 3. Fungal Spores 4. Small Non-enveloped Viruses - Polio, Rotavirus, Rabies 5. Vegetative Fungal Cells 6. Enveloped Viruses - Herpes, Hepatitis B & C, HIV 7. Vegetative Bacteria Susceptibilities VarySusceptibilities Vary  Endospores are Difficult to Kill
  23. 23. Relative Resistance of MicrobesRelative Resistance of Microbes  Highest resistanceHighest resistance • Bacterial endospores & prions  Moderate resistanceModerate resistance • Pseudomonas sp. • Mycobacterium tuberculosis • Staphylococcus aureus • Protozoan cysts  Least resistanceLeast resistance • most vegetative cells • fungal spores • enveloped viruses • yeast • protozoan trophozoites
  24. 24. Relative Susceptibility of MicroorganismsRelative Susceptibility of Microorganisms  Effectiveness of germicides classified as high, intermediate, or low • High-levelHigh-level kill all pathogens, including endospores • Intermediate-levelIntermediate-level kill fungal spores, protozoan cysts, viruses and pathogenic bacteria • Low-levelLow-level germicides kill vegetative bacteria, fungi, protozoa, and some viruses
  25. 25. Environmental conditions that pertainEnvironmental conditions that pertain  Increasing temperatures increases the efficacy of most chemical antimicrobials  The converse of this statement is that relatively cold temperatures result in relatively poor disinfection
  26. 26. Environmental conditions that pertain (cont.(  Concentration effects: Generally, the use of more disinfectant provides better killing than the use of less disinfectant  The fewer organisms present, the shorter the time needed to achieve sterility. Thoroughly cleaning objects before attempting to sterilize them is a practical application of this principle. Clearing objects of tissue debris and blood is also important because such organic matter impairs the effectiveness of many chemical agents.
  27. 27. Factors That Affect Death RateFactors That Affect Death Rate 1. Number of microbes 2. Nature of microbes in the population. (Microbial characteristics e.g. glycocalyx, cell wall, resistance) 3. Environment. (organic matter, temperature, concentration, biofilms & pH ) 4. Concentration or dosage of agent. 5. Mode of action of the agent. 6. Presence of solvents, organic matter, or inhibitors. 7. Time of exposure (Time to Kill in Proportion to the Population Size). Effectiveness of antimicrobial treatment depends on:
  28. 28. DD Why is prevention best?
  29. 29. 1. Heat – Moist verse Dry (Exposure to extremes of heat) 2. Cold temperatures (Exposure to extremes of cold) 3. Desiccation 4. Radiation 5. Osmotic pressure Physical Methods of Microbial ControlPhysical Methods of Microbial Control  Not for use on living organisms  Somehow, alter membrane permeability and / or structure of proteins and nucleic acids
  30. 30. HeatHeat  Most Frequent and Widely Used.  Always Consider 1. Type of Heat 2. Time of Application 3. Temperature  Endospores are the most heat resistant of all cells.  Effects of high temperatures • Denaturation of proteins • Interference with integrity of cytoplasmic membrane and cell walls • Disruption of structure and function of nucleic acids
  31. 31. Thermal Death MeasurementsThermal Death Measurements  Thermal death point (TDP):Thermal death point (TDP): • lowest temperature required to kill all microbes in a sample in 10 minutes  Thermal death time (TDT):Thermal death time (TDT): • shortest length of time required to kill all microbes at a specified temperature Heat  Decimal reduction time (DRT):Decimal reduction time (DRT): • Minutes to kill 90% of a population at a given temperature
  32. 32. Thermal Death PointThermal Death Point  Thermal Death Point (TDP) / Lowest Temp to Kill All the Bacteria in a Broth in 10 Minutes. • This aspect of thermal death is useful in purifying water via boiling.  Boiling: Kills Many Vegetative Cells and Inactivates Viruses Within 10 Minutes (30 Minutes to be Safe) but has no effect on spores
  33. 33. Thermal Death TimeThermal Death Time  Thermal Death Time (TDT) / Time Span Required to Kill All the Bacteria in a Broth at a Given Temperature.  It was developed for food canning and has found applications in cosmetics and pharmaceuticals.
  34. 34. Decimal Reduction TimeDecimal Reduction Time (DRT(DRT((  Decimal Reduction Time (DRT) / Length of Time in Which 90% of a Bacterial Population will be Killed at a given Temperature  Reduces the number of organisms to 1/10 the initial level.  Used in Commercial Sterilization.
  35. 35.  Used to disinfect, sanitize, and sterilize  Kills by denaturing proteins, destruction of DNA and destroying cytoplasmic membranes.  More effective than dry heat; water better conductor of heat than air. Moist heat is also more penetrating than dry heat  Methods of microbial control using moist heat • Boiling • Autoclaving • Pasteurization • Ultrahigh-Temperature Sterilization Moist HeatMoist Heat
  36. 36.  Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses within 10 minutes at sea level  Temperature cannot exceed 100ºC at sea level; steam carries some heat away  Boiling time is critical  Water boils at lower temperatures at higher elevations; requires longer boiling time  Endospores, protozoan cysts, and some viruses can survive boiling BoilingBoiling • Hepititis (20 min) • Some spores may survive boiling water for up to 20 hrs
  37. 37. AutoclavingAutoclaving  Pressure applied to boiling water prevents steam from escaping  Boiling temperature increases as pressure increases  Autoclave conditions – 121ºC, 15 psi, 15 minutes AutoclaveAutoclave
  38. 38.  Pressure applied to boiling water prevents steam from escaping  Boiling temperature increases as pressure increases  Autoclave conditions – 121ºC, 15 psi, 15 minutes  Volume, Contact, Wrapping, Testing AutoclavingAutoclaving Autoclave Tape
  39. 39. AutoclavingAutoclaving  Steam Under Pressure  121° C for 15 Minutes at 15 lb/in2  Heat-labile Substances will be Denatured (proteins)  Steam Must Contact the Material
  40. 40.  Pasteurization is the application of moist heat of less-than boiling temperatures to foods to prevent the growth of food- spoiling organisms as well as various heat-labile pathogens. without destroying the food flavor or value  batch method 63°C - 66°C for 30 minutes  flash method 71.6°C for 15 seconds Pasteurization  Pasteur’s method  Pasteurization is a process used in preserving heat sensitive foods such as milk, ice cream, yogurt, fruit juices, beer, and other beverages.
  41. 41.  Pasteurization is not a method of sterilization, which is why pasteurized foods will eventually spoil if given enough time.  Pasteurization extends the shelf life of a product and reduces the level of pathogens in the product.  Not sterilization; heat-tolerant and heat- loving microbes survive • These do not cause spoilage prior to consumption • These are generally not pathogenic • kills non-spore-forming pathogens and lowers overall microbe count • does not kill endospores or many nonpathogenic microbes
  42. 42. Ultrahigh-Temperature SterilizationUltrahigh-Temperature Sterilization  A new method called ultrahigh temperature (UHT) sterilization involves heating at 140°C for 1-3 seconds, then rapid cooling.  Treated liquids can be stored at room temperature  Milk that has been treated in this way can be kept at room temperature for 2 months with only minimal changes in flavor.
  43. 43.  Used for materials that cannot be sterilized with or are damaged by moist heat  Denatures proteins and oxidizes metabolic and structural chemicals  Requires higher temperatures or longer time than moist heat  Nevertheless, application of dry heat is cheap and easy  higher temperatures than moist heat Dry HeatDry Heat
  44. 44. Dry Heat  1. Direct Flaming1. Direct Flaming • Bunsen burner (1870o C) • Dehydrates cells and removes water • Inoculating Loop and Needle 100% effective  2. Incineration (2. Incineration ( <<12001200oo C )C ) • Burns and Physically Destroys Organisms combusts & dehydrates cells • Loops: needles, inoculating wires (1500°C) • Disposable wastes (paper cups, bags, dressings) • Glassware  3. Hot Air Sterilization (dry ovens)3. Hot Air Sterilization (dry ovens) • coagulate proteins • used on substances that would be damaged by moist heat sterilization and objects that Won’t Melt e.g. glassware, metal, dressings or powders (i)   171ºC for at least one hour (ii)  160ºC for at least two hours (iii) 121ºC for at least 16 hours
  45. 45. 22..Cold (Low) TemperaturesCold (Low) Temperatures  Microbistatic (Not Bactericidal) • slows the growth of microbes  Decreasing Temperature Decreases Chemical Activity  refrigeration 0-15o C  freezing <0o C  used to preserve food, media and cultures
  46. 46.  Refrigeration is a great short term solution, it decreases microbial metabolism, growth, and reproduction i.e. it merely slows the growth of organisms rather than preventing it • Chemical reactions occur slower at low temperatures • Liquid water not available  Psychrophilic microbes can multiply in refrigerated foods  Refrigeration halts growth of most pathogens  Slow freezing more effective than quick freezing  Organisms vary in susceptibility to freezing Refrigeration and FreezingRefrigeration and Freezing  For organisms that survive the freezing process, freezing constitutes a reasonably good long-term preservation method with prevention of deterioration increasing as temperatures are lowered  Lower temperatures result in greater long-term storage  (- 20ºC, -80ºC, -180ºC)
  47. 47. 33..DesiccationDesiccation  Gradual removal of water from cells (Dehydration)  Leads to metabolic disruption/inhibition  Stops Growth / Microbes Are Still Viable  not effective microbial control • many cells retain ability to grow when water is reintroduced  Viruses and Endospores Can Resist Desiccation
  48. 48.  Lyophilization used for long term preservation of microbial cultures • Prevents formation of damaging ice crystals. • Freeze-drying involves freezing something and then evacuating air so that boiling occurs at low temperatures; this desiccates material thereby preventing deterioration and spoilage. • Liquid nitrogen or frozen carbon dioxide (dry ice). LyophilizationLyophilization (Freeze-drying(Freeze-drying((
  49. 49. Electromagnetic spectrum, energy without mass travelling in waves at the speed of light, 3x105km/sec, Efficiency is Dependent on the Wavelength, Intensity, and Duration (shorter wavelength means great energy and can penetrate further).  Radiation damages DNA  Two types used: • Ionizing radiation: Wavelengths shorter than 1 nm (X rays, gamma rays, electron beams) • Non-ionizing radiation: Wavelengths greater than 1 nm (UV) • (Microwaves kill by heat; not especially antimicrobial) 44..RadiationRadiation  Radiation described as ionizing or nonionizing according to effects on cellular chemicals.
  50. 50.  Wavelengths shorter than 1 nm – electron beams, gamma rays, and X rays.  are more penetrating but are more difficult and expensive to use.  Ionizing radiation is radiation that ionizes water; this temporarily turns water into an oxidizing agent. Ionizing RadiationIonizing Radiation  Create ions by ejecting electrons from atoms they strike.  Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions; hydroxide ions denature other molecules (DNA). Types of Ionizing RadiationTypes of Ionizing Radiation  Electron beamsElectron beams – effective at killing but do not penetrate well • Used to sterilize spices, meats, microbiological plastic ware, and medical and dental supplies  Gamma raysGamma rays – penetrate well but require hours to kill microbes • Used to sterilize meats, spices, and fresh fruits and vegetables  X-raysX-rays require too much time to be practical for growth control
  51. 51.  Food irradiation is a process whereby the food is exposed to high levels of radiation in order to kill insects, bacteria and mold, and make the food last longer on the store shelves.  One potential application of ionizing radiation is as an antimicrobial is in food preservation and other industrial processes.  Used on substances that could be damaged by heat • plastic petri dishes, plastic syringes, catheters, surgical gloves
  52. 52.  Usually use cobalt-60 which has a half-life of 5 years.  Materials which are sterilized using this type of radiation do not become radioactive, and controversy exists on whether or not irradiation of food changes its nutritional value.  In some cases the taste of the food is changed, similar to how milk changes taste once it is pasteurized.  Studies have shown that irradiating microorganisms like E. coli and Salmonella may give rise to even more dangerous, radiation-resistant strains of bacteria.  Under laboratory conditions scientists found that one particular type of bacteria can survive a radiation dose five times what the FDA will allow for beef.  Radiation is completely ineffective against viruses, and does absolutely nothing to clean the food of waste products and other unsanitary matter often left on beef, chicken, and lamb as the result of slaughterhouse conditions.  India, children blood tests showed chromosome damage after being fed freshly irradiated wheat for six weeks.
  53. 53. Nonionizing RadiationNonionizing Radiation  Excites electrons and causes them to make new covalent bonds • Affects 3-D structure of proteins and nucleic acids  UV light causes pyrimidine (Thymine) dimers in DNA •Interfere with replication
  54. 54. UV RadiationUV Radiation  The most lethal type of radiation is ultraviolet radiation with a wavelength of 260 nm. This is the wavelength most actively absorbed by DNA.  Due to its poor penetration, does not penetrate plastic, glass or proteinaceous matter, UV radiation is only useful for disinfecting outer surfaces of objects , transparent fluids, air and liquids.  Used to reduce microbial populations • hospital rooms, nurseries and operating rooms  This type of radiation is also harmful if someone is directly exposed to it (for extended periods of time), as it may damage the skin and eyes. Aquarium lightAir and surface microbial controlEgg surface microbial controlEgg surface microbial control
  55. 55. MicrowavesMicrowaves  Kill Microbes Indirectly with Heat Do Not Microwave Baby BottlesDo Not Microwave Baby Bottles  Inside milk may be hotter than outer bottle.  Heating the bottle in a microwave can cause slight changes in the milk: Inactivates Antibodies (Breast Milk), Denatures Protein and Destroys Vitamins  Microwaving changes food nutrients.  Increases Leukocytes in Blood (Sign of Infection and Poisoning)  Decreases Erythrocytes and Iron Stores and Results in Anemia.  Increases Cholesterol.  Causes Production of Radiolytic Compounds (Mutated Compounds) Which Depress the Immune System and are Carcinogenic.
  56. 56. 55..Osmotic PressureOsmotic Pressure  High concentrations of salt or sugar in foods to inhibit growth (jam, jerky, pickled food, salted fish); Sugar Curing / Salting  Cells in a hypertonic solution of salt or sugar lose water Causes plasmolysis i.e. shrinkage of cytoplasm (Water in cell is drawn out by osmosis); cell desiccates  Fungi have greater ability than bacteria to survive hypertonic environments (May Still Get Some Mold or Yeast Growth)
  57. 57. DD
  58. 58. FiltrationFiltration  The passage of a liquid or gas through a filter with pores small enough to retain microbes (25µm to less than 0.01µm). • physical removal of microbes • passing a gas or liquid through filter • organisms above a certain size trapped in the pores  Especially important to sterilize (Removes microorganisms) solutions which would be denatured by heat (culture media, enzymes, vaccines, antibiotics, injectable drugs, amino acids, vitamins.)  used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms • Air can be filtered using a high- efficiency particulate air (HEPA) filter
  59. 59. HEPA FiltersHEPA Filters  HEPA filters are High-Efficiency Particulate Air filters designed for the filtration of small particles. Certified HEPA filters must capture a minimum of 99.97% of 0.3 microns contaminants.  Filtration is the primary method of eliminating pathogens from the air supply i.e. used to filter the air flowing into aseptic environments ( e.g. operating rooms) and out of potentially contaminated ones (e.g., containment facilities) 1. Operating Rooms 2. Burn Units 3. Fume Hoods 4. Isolation Rooms 5. Bio-cabinets 6. Pharmaceutical Manufacturing Facilities
  60. 60. DD
  61. 61. Effectiveness of Chemical DisinfectantsEffectiveness of Chemical Disinfectants 1. Type of Microbe1. Type of Microbe - G + More Susceptible to Disinfectants - Pseudomonands Can Grow in Disinfectants and Antiseptics - M. tuberculosis is Resistant to Many Disinfectants - Endospores Most Resistant 2. Environment2. Environment • Concentration of disinfectant • Organic matter • pH • Time • Contact with microbetime
  62. 62.  Phenol Coefficient  Use-Dilution Test • Metal cylinders (rings) • Serial dilution (test tubes)  Diffusion Method • Disk diffusion • Cup plate diffusion  In-Use Test Methods for Evaluating Antimicrobial agentMethods for Evaluating Antimicrobial agent Testing for Drug SusceptibilityTesting for Drug Susceptibility
  63. 63.  Old Standard Test  The phenol coefficient is the value obtained by dividing the highest dilution of the test solution by the highest dilution of phenol that sterilizes the given culture of bacteria under standard conditions of time and temperature.)  Greater than 1.0 indicates that agent is more effective than phenol  Has been replaced by newer methods Phenol CoefficientPhenol Coefficient
  64. 64.  Especially useful for determining the ability of disinfectants to kill microorganisms dried onto a typical clinical surface (stainless steel)  Metal cylinders dipped into broth cultures of bacteria and dried  Contaminated cylinder immersed into dilution of disinfectant for 10 minutes  Cylinders removed, washed, and placed into tube of medium for 48 h  Most effective agent entirely prevents growth at highest dilution Use-Dilution TestUse-Dilution Test Three microbes are used:Salmonella choleraesuis, Staphylococcus aureus andPseudomonas aeruginosa
  65. 65. Dilution testsDilution tests  Minimum inhibitory concentration (MIC) • smallest concentration of drug that visibly inhibits growth  In vitro activity of a drug is not always correlated with in vivo effect • If therapy fails, a different drug, combination of drugs, or different administration must be considered  Best to chose a drug with highest level of selectivity but lowest level toxicity
  66. 66. Diffusion MethodDiffusion Method  A method that requires less manipulation to judge the efficacy of disinfectants.  Here filter paper is soaked with disinfectant and then simply placed on the agar surface of a petri dish that has been inoculated with a lawn of test organism.  The clear area around the disk following incubation is used as an indication of disinfectant efficacy.  Essential for groups of bacteria commonly showing resistance  Kirby-Bauer disk diffusion test
  67. 67.  Swabs taken from objects before and after application of disinfectant or antiseptic  Swabs inoculated into growth medium and incubated  Medium monitored for growth  Accurate determination of proper strength and application procedure for each specific situation In-Use TestIn-Use Test
  68. 68. Chemical Control MethodsChemical Control Methods (Microbial Agents(  Major Categories  Phenols and Phenolics  Biguanides (Chlorhexidine)  Halogens  Alcohols  Oxidizing agents  Heavy Metals and Their Compounds  Surface-Active Agents (Surfactants) • Detergents & soaps • Quaternary Ammonium Compounds  Chemical Food Preservatives  Aldehydes  Gaseous Agents and aerosols  Antibiotics (Antimicrobics)
  69. 69. Phenol and Phenolics - Another Name for Carbolic Acid / Lysol / Pine-Sol - Joseph Lister - Exert Influence By 1. Injuring Plasma membranes 2. Inactivating Enzymes 3. Denaturing Proteins  Phenolics are Long Lasting, Good for Blood and Body Fluids  Phenols are effective antibacterial agents, and they are also effective against fungi and many viruses but No Effect on Spores  Phenols have a characteristic pine-tar odor and turn milky in water.  Phenols can be toxic to pets especially cats and pigs.
  70. 70. BiguanidesBiguanides - Damage Plasma Membranes - Caution: Can Damage Eyes – Avoid Splashing - Only Operates in Narrow pH Range (5-7) - Example / Chlorhexidine ChlorhexidineChlorhexidine A surfactant & protein denaturant with broad microbicidal properties Not sporicidal Used as skin degerming agents for preoperative scrubs, skin cleaning & burns
  71. 71. HalogensHalogens ((Intermediate-level antimicrobial chemicals)) - Can be Used Alone or in Solution - Inactivated by Sunlight - They damage enzymes - Can be Corrosive to Metal - Can Irritate Mucus Membranes ChlorineChlorine -- Purifies Drinking Water  Cl2, hypochlorites (chlorine bleach), chloramines  Denaturation of proteins by disrupting disulfide bonds  Can be sporicidal  Forms an Acid Which is Bactericidal  Acts as a Disinfectant in Gaseous Form or in Solution as Calcium Hypochlorite Chlorine compounds are good disinfectants on clean surfaces, but are quickly inactivated by dirt. Chlorine can be irritating to skin and corrosive to metal. Inexpensive / Chlorox Never Mix with Other Cleaning Agents!
  72. 72. HalogensHalogens IodineIodine – combines with Amino Acids  I2 /Iodophor/Organic Molecule/Betadine  Inactivates Enzymes / Denature proteins  Can be sporicidal Iodine is normally considered to be the least toxic of the disinfectants. Milder medical & dental degerming agents, disinfectants, ointments Iodine products can stain clothing and porous surfaces.
  73. 73. • Denature Proteins (coagulating proteins of vegetative bacterial cells and fungi) • Dissolve Lipids and disrupt cytoplasmic membranes • Evaporates both advantageous and disadvantageous • Fast Acting, No Residue, Flammable • Alcohols should not be applied to wounds since they can cause tissue damage • Swabbing of skin with 70% ethanol prior to injection Wet Disinfectants Ethyl, isopropyl in solutions of 50-90% Act as surfactants Not sporicidal Good for enveloped viruses AlcoholsAlcohols ((Intermediate-level disinfectants))
  74. 74. Oxidizing AgentsOxidizing Agents ((High-level disinfectants and antiseptics) oxidizing agents are active against bacteria, bacterial spores, viruses, and fungi at quite low concentrations. Peroxides H2O2(skin), ozone O3 (swim pools), and peracetic acid (dialysis equipment),bleach (sodium hypochlorite), bromine kill by oxidation of microbial enzymes Hydrogen peroxide can disinfect and sterilize surfaces of objects Ozone treatment of drinking water Peraceticacid effective sporocide used to sterilize equipment Hydrogen PeroxideHydrogen Peroxide Weak (3%) to strong (35%) Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O2 gas • toxic to anaerobes Strong solutions are sporicidal in increasing concentrations
  75. 75. Types of DisinfectantsTypes of Disinfectants Heavy Metals and Their CompoundsHeavy Metals and Their Compounds Heavy Metals. Ag, Hg, Cu • Oligodynamic action Ions are antimicrobial because they alter the 3-D shape of proteins, inhibiting or eliminating their function (Denature/inactivate proteins) Denature proteins by breaking disulfide bonds Low-level bacteriostatic and fungistatic agents i.e. Not sporicidal Example Solutions of silver & mercury / Silvadene Ointment 1% silver nitrate to prevent blindness (antiseptic for eyes, wounds( Zinc chloride (ZnCl2) mouthwashes Thimerosal(mercury-containingcompound)used to preserve vaccines Copper controls algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks; interferes with chlorophyll (CuSO(CuSO44 algicide)algicide) • Indian tradition of storage of river water in brass containers as a way to prevent disease. The river water may have up to 1 million fecal bacteria per ml. That count could be reduced to undetectable by 2 days of storage
  76. 76. Surfactants are substances that are soluble in water but are able to dissolve lipids ie. “Surface active” chemicals that reduce surface tension of solvents to make them more effective at dissolving solutes Soaps and detergentsSoaps and detergents • Soaps have hydrophilic and hydrophobic ends; good degerming agents but not antimicrobial • Soaps Have Limited Germicidal Action but Assist in the Removal of Organisms by Scrubbing • Detergents are positively charged organic surfactants • Acid-Anionic Detergents / Dairy SurfactantsSurfactants Surface-Active AgentsSurface-Active Agents Soap Degerming Acid-anionic detergents Sanitizing Quarternary ammonium compounds Cationic detergents Bactericidal, Denature proteins, disrupt plasma membrane
  77. 77. Quaternary Ammonium CompoundsQuaternary Ammonium Compounds (QUATS)(QUATS) • Cationic Detergents Attached to NH4 + • Ammonia compounds act as surfactants • Most Effective on Gram-Positive Bacteria • Mouthwashes and Sore Throat Remedies QUATS are generally odorless, colorless, nonirritating, and deodorizing. Can be inactivated in the presence of some soaps or soap residues and their antibacterial activity is reduced in the presence of organic material. Disruption of plasma membrane, denaturation of proteins Examples Benzalkonium chloride (aka Zephiran), Roccal: lab disinfectant and Cetylpyridinium chloride (Cepacol)
  78. 78. Chemical Food PreservativesChemical Food Preservatives Salts Sugar Dry Organic Acids • Inhibit metabolism • Sorbic acid, benzoic acid, calcium propionate • Control molds and bacteria in foods and cosmetics Nitrate and Nitrite Salts / Meats /To Prevent Germination of Clostridium botulinum endospores Antibiotics. Nisin and natamycin prevent spoilage of cheese
  79. 79. Types ofTypes of DisinfectantsDisinfectants AldehydesAldehydes Compounds containing terminal –CHO groups e.g. Formaldehyde and Glutaraldehyde - Most Effective of all Chemical - Disinfectants - Carcinogenic - Oxidize Molecules Inside Cells Cross-link with functional groups (–NH2, –OH, –COOH, —SH) amino, hydroxyl, sulfhydryl, and carboxyl groups to denature proteins and inactivate nucleic acids Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure), 2% solution (Cidex) used as sterilant for heat sensitive instruments Formaldehyde: Disinfectant to rooms and instruments, preservative, toxicity limits use
  80. 80. Ethylene oxide not Ethyl oxide, propylene oxide, beta- propiolactone & chlorine dioxide used in closed chambers to sterilize items Strong alkylating agents, sporicidal Denature proteins and DNA by cross-linking functional groups Use in sterilize hospital equipment, disposable lab plasticwareand dental offices. Can be hazardous to people, often highly explosive, extremely poisonous, and are potentially carcinogenic The most effective method of gas chemical sterilization presently available is the use of ethylene oxide (ETO) gas. ETO gas sterilization should be used only for material and supplies that will not withstand sterilization by steam under pressure. Gaseous Agents & AerosolsGaseous Agents & Aerosols
  81. 81. Types of DisinfectantsTypes of Disinfectants AntibioticsAntibiotics - Used to Preserve Cheese - Used in Feed Given to Food Animals - Nisin - Natamycin Growing Problem Indiscriminant and Inappropriate Use Super Bugs 1. Methicillin Resistant S. aureus 2. Vancomycin Resistant Enterococcus 3. Multidrug Resistant M. tuberculosis This is Why it is so Important to Order Sensitivities In one study, three out of four patients seen in the emergency room for skin infections had Staphylococcal aureus infections and over 50% had MRSA infections. That equates to 12 million MRSA infections each year in the USA. MRSA
  82. 82. Origins of Antimicrobial DrugsOrigins of Antimicrobial Drugs Antibiotics • Common metabolic products of aerobic spore-forming bacteria & fungi • bacteria in genera Streptomyces & Bacillus • molds in genera Penicillium & Cephalosporium Inhibiting other microbes in the same habitat • antibiotic producers have less competition for nutrients & space Typically used for treatment of disease …but Some are used for antimicrobial control outside the body
  83. 83. Ideal Antimicrobial DrugIdeal Antimicrobial Drug…..…..  Selectively toxic to microbe  Not host cells  Microbicidal, not microbistatic  Soluble  Potent  No antimicrobial resistance  Remains active  Readily delivered to site of infection  Expense  Not allergen
  84. 84. ChemotherapyChemotherapy  Antimicrobial • Control infection  Antibiotic • Produced by the natural metabolic processes of microorganisms • Can inhibit or destroy other microorganisms  Semisynthetic • Chemically modified drugs in lab  Synthetic • Synthesized compounds in lab  Narrow spectrum • Effective against limited microbial types • Target a specific cell component that is found only in certain microbes  Broad spectrum • Effective against wide variety microbial types • Target cell components common to most pathogens
  85. 85. Selectively ToxicSelectively Toxic  Should kill or inhibit microbial cells without simultaneously damaging host tissues  Complete selective toxicity • Difficult to achieve • Characteristics of the infectious agent become more similar to the vertebrate host cell • More side effects are seen  toxic dose of a drug • The concentration causing harm to the host  therapeutic dose • the concentration eliminating pathogens in the host  Together, the toxic and therapeutic doses are used to formulate the chemotherapeutic index
  86. 86. Targets of Antimicrobial DrugsTargets of Antimicrobial Drugs 1. Inhibition of cell wall synthesis 2. Inhibition of nucleic acid synthesis, structure or function 3. Inhibition of protein synthesis 4. Disruption of cell membrane structure or function
  87. 87. 11..Bacterial Cell WallBacterial Cell Wall Most bacterial cell walls contain peptidoglycan Penicillin and cephalosporin block synthesis of peptidoglycan • Causes the cell wall to lyse Penicillins do not penetrate the outer membrane • less effective against gram- negative bacteria Broad spectrum penicillins and cephalosporins • cross the cell walls of gram- negative bacteria
  88. 88. 22..Inhibit Nucleic Acid SynthesisInhibit Nucleic Acid Synthesis May block synthesis of nucleotides, inhibit replication, or stop transcription sulfonamides and trimethoprim • block enzymes required for tetrahydrofolate synthesis • needed for DNA & RNA synthesis
  89. 89. 33..Drugs that Block Protein SynthesisDrugs that Block Protein Synthesis Ribosomes • eukaryotes differ in size and structure from prokaryotes Aminoglycosides (streptomycin, gentamicin) • insert on sites cause misreading of mRNA Tetracyclines • block attachment of tRNA and stop further synthesis
  90. 90. 44..Disrupt Cell Membrane FunctionDisrupt Cell Membrane Function Damaged membrane • dies from disruption in metabolism or lysis These drugs have specificity for a particular microbial group • based on differences in types of lipids in their cell membranes Polymyxins • interact with phospholipids • cause leakage, particularly in gram-negative bacteria Amphotericin B and nystatin • form complexes with sterols on fungal membranes • causes leakage
  91. 91. Drug ResistanceDrug Resistance (Development of Resistant Microbes(Development of Resistant Microbes(( Microorganisms begin to tolerate an amount of drug that would ordinarily be inhibitory • due to genetic versatility or variation • intrinsic and acquired Little evidence that extensive use of products containing antiseptic and disinfecting chemicals adds to human or animal health The use of such products promotes the development of resistant microbes
  92. 92. Mechanisms of Drug ResistanceMechanisms of Drug Resistance Drug inactivation by acquired enzymatic activity • penicillinases Decreased permeability to drug or increased elimination of drug from cell • acquired or mutation Change in drug receptors • mutation or acquisition Change in metabolic patterns • mutation of original enzyme
  93. 93. Side Effects of DrugsSide Effects of Drugs 5% of all persons taking antimicrobials will experience a serious adverse reaction to the drug • Toxicity to organs • Allergic responses • Suppression and alteration of microflora Considerations in Selecting an Antimicrobial DrugConsiderations in Selecting an Antimicrobial Drug 1. Identify the microorganism causing the infection • Specimens should be taken before antimicrobials initiated 2. Test the microorganism’s susceptibility (sensitivity) to various drugs in vitro when indicated 3. Overall medical condition of the patient
  94. 94. What about virusesWhat about viruses?!?!??!?!? Do not destroy their target pathogen Instead they inhibit their development • Inhibit virus before enters cell • Viral-associated proteins • Stop it from entering the cell • Stop it from reproducing • Prevent from exiting the cell