2Terminology for Microbial Control Sterilization- removal or destruction of all forms of microbial lifeCommercial sterilization- subjects canned food to only enough heat to destroy the endospores of Clostridium botulinumDisinfection- is the destruction of vegetative pathogens on a surface, usually with chemicalsSpores and viruses are not necessarily destroyedAntisepsis- is the chemical disinfection of living tissue, such as skin or mucous membranes
3Terminology for Microbial Control Asepsis- is the absence of pathogens on an object or area, as in antiseptic surgeryDegerming (degermation)- is the removal of transient microbes from the skin by mechanical cleansing or by antisepticSanitation- is the reduction of microbial populations on objects to safe public health levelsA biocide or germicide- kills microorganismsFungicides kill fungi, virucides kill virusesSuffix – cide means the killer of a specified microorganismSuffix- stat used in this way indicates only that the substance inhibits – for example bacteriostasisExample for asepsis is handwashing Example for bacteriostatic is a refrigerator
4Rate of Microbial Death Bacterial populations killed by heat or chemicals tend to die at constant rates—for example, 90% every 10 minutes. Plotted logarithmically, these figures form straight descending lines.
5Factors that influence effectiveness of an antimicrobial treatment: 1. Number of microbes-more cells, more time needed to kill all2. Environmental influences-organics often inhibit chemical agents (blood, feces, vomit)-temperature (disinfectants work better in warm temperatures)-pH (heat is more effective in an acid pH)Environmental- any medium containing fats and protein will tend to protect bacteria
6Factors that influence effectiveness of an antimicrobial treatment: 3. Time of exposure-Chemical antimicrobials require a certain amount of exposure; same agent may need longer on resistant organisms or spores-with heat, lower temps require longer to kill4. Microbial characteristics-resistance genes, protective structures(e.g.capsules) etc. can inhibit action-bioflims prevent penetrationsusceptibility to different agents varies among microbes
7Type and Age of MicrobeBacteria- susceptible to protein denaturing BUT mycobacteria is not because of its hydrophobic coatGrowth cycles- physiologically young bacteria(early in growth cycle) susceptible to heatEndospores more resistant to heat the older they get
8Properties of Materials: Plastic and rubber- not heat tolerantCutting edges of surgical instruments- no moistureor corrosive chemicalsEdges can become pitted due to rust and corrosion, microbes can hide thereFabrics- no chemical disinfection; some too fragile
9Actions of Microbial Control Agents 1. Alteration of membrane permeabilityThe plasma membrane controls the passage of nutrients and wastes into and out of the cell.Damage to the plasma membrane causes:leakage of cellular contentsinterferes with cell growth.= Leak lysis, deathCell lysis is a process in which a cell is broken down or destroyed
10Actions of Microbial Control Agents 2. Damage to proteins and Nucleic AcidsEnzymes and other proteins are essential for cell functionDenatures proteinsEnzymes (no reactions)Proteins necessary for bacteria metabolismHydrogen bonds are brokenCovalent bonds are also broken3. Damage to nucleic acidsPrevent replication, transcription, or translationHydrogen bonds hold proteins in the characteristic 3-dimensional shape required for their functions. Heat and certain chemicals break these bonds and the shape is lost. This is called denaturation.DNA and RNA carry the cell’s genetic information and function in protein synthesis. Damage to these by heat, radiation, or chemicals usually kills the cell.
11Physical Methods of Microbial Control -to disinfect objects, food, and solutions common methods:-Temperature: kill or inhibit growthHeatLow Temps.-Filtration: physical removal-Desiccation: inhibit growth-Osmotic pressure-Radiation: kill
12Methods involving Heat: Heat: Common food preservationDenatures protein (changes shape)Thermal Death Point (TDP) = lowest temp at which all microbes in liquid suspension will be killed in 10 minThermal Death Time (TDT)= minimal length of time for all microbes in liquid suspension to be killed at given temp*Both are different for different species due to microbial variation in heat toleranceDecimal Reduction Time (DRT) = Time in minutes in which 90% of bacteria at a given temperature will be killedConcept of equivalent treatments:With any heat treatment, the higher the temperature used the shorter the exposure time needed to achieve the same effect
13Methods involving Heat: 1. Moist heat will always kill faster than dry heat at the same temperatureKills microbes by coagulation of cell proteinsA. Boiling (100°C) kills vegetative forms of bacterial pathogens, many viruses, and fungi within 10 minutesSome mo’s and viruses are resistantEndospores (up to 20 hrs.) and some viruses (30 minutes) survive boiling for longer times.This occurs faster in the presence of water, so moist heat requires lower temperatures and less time of exposure than dry heat.Edospores-dormant stage of some bacteria that allow them to survive harsh conditions-ex. Extreme heat
14Methods involving Heat: B. AutoclavesMoist Heat (steam) and pressure for sterilizationAchieves higher temps. than boilingPreferred method (sealed chamber, air is exhausted, and steam under pressure is injected) for all materials that can withstand itKills all organisms and their endospores in about minutesAn autoclave is shown in Figure 1.1 and 1.2Here we use an autoclave to sterilize culture media. In hospitals, doctors’ offices, dentists’ offices, etc. this methods is used to sterilize medical equipment.Materials being autoclaved are often wrapped in paper, so that after sterilization the outside of the package can be handled without contaminating the sterile item inside.The size of the container, the volume of a liquid, and the type of wrapping can influence the time and temperature required for sterilization.
15Autoclave Figure 1.1 Figure 1.2 Autoclave tape Autoclave tape works by changing color after exposure to temperatures commonly used in sterilization processes, typically 121°C in a steam autoclave. It is important to note that the presence of autoclave tape that has changed color on an item does not ensure that the product is sterile, as the tape will change color upon exposure only. For steam sterilization to occur, the entire item must completely reach and maintain 121°C for minutes with proper steam exposure to ensure sterilization. Diagonal shaped more leat, words Autoclaved lead freeFigure 1.2Autoclave tape
16Methods involving Heat: C. PasteurizationLouis PasteurMild HeatingKills most pathogensKills bacteria that cause spoilagePreserves taste in productLowers bacterial numbersPasteurization or pasteurisation is a process of heating a food, which is usually a liquid, to a specific temperature for a predefined length of time and then immediately cooling it after it is removed from the heat. This process slows spoilage caused by microbial growth in the food.Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food. Instead, it aims to reduce the number of viable pathogens so they are unlikely to cause disease (assuming the pasteurized product is stored as indicated and is consumed before its expiration date).
17Methods involving Heat: 2. Dry HeatKills by burning to ashes or by oxidationFlaming- we use this on loops in labsIncineration- burning of contaminated paperHot air sterilizationHot ovensMainly used for items not suitable for autoclavingOily substances, powdersLarge amounts of glassware
18Low TemperatureLower temp inhibits growth, rapid freezing limits moisture (bacteriostatic)Refrigerator temperatures (0° to 7°C) slow the metabolic rate of microbes; howeverPsychrotrophic species still grow slowly.Some organisms grow at temperatures slightly below freezing, but microbes at the usual temperatures of freezer compartments are completely dormant.
19Filtration Liquids Heat- sensitive materials Small pores prevent passage of bacteriaHigh-efficiency particulate air filters (HEPA)Operating roomsSpecial clean roomsMasksUsed with solutions for: renal dialysis, heart bypass machines, I.V.’sCatch particles autoclaves miss
20DesiccationMicrobes require water for growth, and adequately dried (desiccated) foods will not support their growth, therefore inhibiting growthAbsence of WaterLyophilization- rapid freeze dryingUsed for blood products , serum products, enzymes, culturesAvoids ice crystal formation; cells burst when water expandsSpecies vary in their susceptibility. Dry surroundings kill some vegetative cells in one hour or less. Others can survive for months or even years. Bacteria that produce endospores are extremely resistant to drying. Many viruses are also resistant to drying.
21Osmotic PressureHigh salt or sugar concentrations cause water to leave the cell; this is an example of osmosis.Generally, molds and yeasts resist osmotic pressures better than bacteria.
22Radiation Ionization Radiation includes X rays, gamma rays, and high-energy electron beamsvery short wavelengths and high levels of energyPenetrate deeplyIonizes water to form hydroxyl radicalsThese destroy cell components, especially DNAKills: vegetative cells, viruses, most endospores with adequate exposureApplications: food preservation, sterilization of pharmaceuticals, medical supplies, mailRecently, approval has been granted for use of low level radiation of fruits and meats. The post office is now using this method to sterilize some mail. This process does involve the use of dangerous radiation and can only be used in a properly shielded room, so it is mostly used in factories where widescale use of the setup makes it economical.
23Radiation Nonionizing radiation Have a longer wavelength and less energyUltraviolet (UV) light is the common exampleCauses the formation of thymine dimers, which interferes with DNA replication and formation of mRNA.UV lamps are used in hospitals and in food serviceThis method does not sterilize, but it does reduce bacterial growthPenetrating power is very low, so any type of covering protects microbes.Sunlight has some weak antimicrobial effects (biocidal), but the wavelengths of sunlight are too long to work well.
24Types of Chemical Agents 1. Phenols and PhenolicsA. Phenol (carbolic acid)Irritating to skin and mucous membranesBad odorRarely used todayB. PhenolicsChemicals derived from phenolChemically altered to make it less irritating and more effectiveDamages plama mbs., inactivates enzymes, denature proteinsOften used as disinfectants as they remain active in the presence of organic matterIts main use now is in throat lozenges and sprays, but the concentration is so low that there is little antimicrobial effect, although there is some local anesthetic action. Some throat sprays may have a concentration above 1% and these may show antibacterial action.
25Types of Chemical Agents C. Bisphenolscontain 2 phenolic groups connected by a bridgeHexachlorophenepHisoHex is an examplePrescription antibacterial lotionUsed in nurseries to control gram + bacteriaSkin bacteria: Staphylococcus and StreptococcusTriclosanFound in antibacterial soapEffective against G+ and G- bacteria
26Types of Chemical Agents 2. BiguanidesChlorhexidine is an exampleHibiclens soapUsed on skin and mucous membranesSimilar to phenolics but less toxicDisrupt plasma mb.Broad spectrumEffective against most vegetative bacteria and fungi, but not against endospores and many viruses.Damaging to eyes
27Types of Chemical Agents 3. Halogens- Effective alone or in compoundsA. Iodine (I2)One of the oldest and most effectiveVery effective on: all bacteria, many endospores, fungi and some virusesCombines with amino acids in proteins and denatures proteinsAlters plasma mb.Negative AspectsStainingSometimes irritating to the skinMay trigger allergiesApplicationsSkin disinfection, wound treatment, water treatment
28Types of Chemical Agents B. Chlorine (Cl2)Action: forms hypochlorus acid with water → oxidizing agent, denatures proteinsBroad spectrum: bacteria, fungi, some endospores, some virusesPositive aspects: -effective against all vegetative cells including Mycobacterium-cost effectiveNegative aspects: -action inhibited by organics-can form carcinogenic compoundsApplications: water and sewage treatment, surface and instrument disinfectionSodium hypochlorite (NaClO): Is active ingredient of bleachThe U.S. military uses tablets of sodium dichloroisocyanurate for fielddisinfection of water
29Types of Chemical Agents C. Alcohols- frequently used for skin degermingKill bacteria, fungi, but not endospores or naked viruses.Act by denaturing proteins and disrupting cell membranes.Used to mechanically wipe microbes off skin before injections or blood drawing, instrument disinfectionNot good for open wounds, because cause proteins to coagulate and leave bacteria unharmedEthanol (ethyl alcohol): Optimum concentration is 70%.Isopropanol: Rubbing alcoholBetter disinfectant than ethanolUsual concentration is 90%Also cheaper and less volatile.
30Types of Chemical Agents 5. Heavy metals and their compoundsOligodynamic action- Very tiny amounts are effectiveIncludes- silver, mercury, copper, zincBind sulfur groups causing inactivation or denaturing of proteinsA. Silver:1% silver nitrate used to protect infants against gonorrheal eye infections until recently.B. MercuryOrganic mercury compounds like merthiolate andmercurochrome are used to disinfect skin wounds.C. CopperCopper sulfate is used to kill algae in pools and fish tanks (fungicidal, algicidal)D. ZincUsed in mouthwashesSuperficial fungal and bacterial infectionsHowever, because these drops often cause the baby's eyes to be irritated, most hospitals now use erythromycin ointment instead
31Types of Chemical Agents 6. Surface-Acting AgentsDisrupt plasma membrane & denature proteinsDecrease surface tensionInclude soaps and detergentsWashing with soap breaks up the oily film that covers skin and allows microbes and dirt to be washed away7. Quaternary Ammonium CompoundsWidely used surface active agentsDenature proteins & disrupt cell membranesCationic (positively charge) detergentsEffective against gram positive bacteria, less effective against gram-negative bacteria.Also destroy fungi, amoebas, and enveloped viruses.
32Types of Chemical Agents 8. AldehydesThese can act very effectively against microbesInactivate proteinsAction: cross-link (thus inactivate) nucleic acids and proteinsHigh activity (sterilization)biocidal including endosporesPositive aspectsachieves sterilizationNegative aspectsunstable-toxic-volatile with noxious fumesApplications: specimen preservation (embalming), vaccine sterilization
33Types of Chemical Agents 9. Gaseous SterilizersChemicals that sterilize in a chamber similar to an autoclave.Denature proteins, by replacing functional groups with alkyl groupsA. Ethylene OxideKills all microbes and endospores , but requires exposure of 4 to 18 hours.Toxic and explosive in pure form.Highly penetrating.Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.
34Types of Chemical Agents 10. PeroxygensOxidize cellular componentsA. Hydrogen PeroxideCommon household antisepticNot good for open wounds because quickly broken down by catalase present in human cellsused in deep wounds because it releases oxygen as it breaks down, which makes conditions unfavorable for anaerobic bacteriaEffective in disinfection of inanimate objects (kills endospores)Sporicidal at higher temperaturesUsed by food industry and to disinfect contact lenses
35Types of Chemical Agents B. Benzoyl PeroxideMain ingredient in many acne treatmentsMay be used in treating wound infections caused by anaerobesC. Peracetic AcidOne of the most effective liquid sporicides availableSterilant :Kills bacteria and fungi in less than 5 minutesKills endosporesand viruses within 30 minutesUsed widely in disinfection of food and medical instrument because it does not leave toxic residues