2 Terminology of Microbial Control Sterilization(a) Sterilization is the killing of all microorganisms in a material or on the surface of an object(b) A surface or an object is either sterile or it is not sterile, there are no gradations in sterility(c) Typically the last things to die when one attempts sterilization is the highly heat- (and chemical-, etc.) resistant endosporesAsepticDegerming
3 Terminology of Microbial Control (cont.) Disinfection/disinfectants(a) Disinfection means reducing the number of viable microorganisms present in a sample(b) A disinfectant is a chemical or physical agent that is applied to inanimate objects to kill microbes(c) Not all disinfectants are capable of sterilizing, but, of course, all disinfectants are employed with the hope of disinfectingAntisepsis/antiseptic Antiseptic(a) Typically an antiseptic is a chemical agent that is applied to living tissue to kill microbes(b) Note that not all disinfectants are antiseptics because an antiseptic additionally must not be so harsh that it damages living tissue(c) With this constraint imposed on antiseptics, in general antiseptics are either not as cheap or not as effective at killing microbes as disinfectants
4 Terminology of Microbial Control (cont.) SanitizationSanitization is the cleaning of pathogenic microorganisms from public eating utensils and objects such as that done by the kitchen of a restaurantPasteurizationSuffix –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 growthSuffix-cidalAn antimicrobial that kills a microorganism (or, more specifically, a bacterium) is said to be bactericidal
6 Action of Antimicrobial Agents Many types of chemical and physical microbial controlsModes of action fall into two basic categoriesAlteration of cell walls or cytoplasmic membranesInterference with protein and nucleic acid structure
7 Alteration of Cell Walls and Membranes Cell wall maintains integrity of cellWhen disrupted, cannot prevent cell from bursting due to osmotic effectsCytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cellWhen damaged, cellular contents leak outViral envelope responsible for attachment of virus to target cellDamage to envelope interrupts viral replicationNonenveloped viruses have greater tolerance of harsh conditions
8 Damage to Proteins and Nucleic Acids Protein function depends on 3-D shapeExtreme heat or certain chemicals denature proteinsChemicals, radiation, and heat can alter or destroy nucleic acidsCan produce fatal mutantsCan halt protein synthesis through action on RNA
9 Selection of Microbial Control Methods Ideally, agents should be:InexpensiveFast-actingStable during storageControl all microbial growth while being harmless to humans, animals, and objects
10 Factors Affecting the Efficacy of Antimicrobial methods Nature of site to be treatedDegree of susceptibility of microbes involvedEnvironmental conditions that pertain
11 Site to Be TreatedHarsh chemicals and extreme heat cannot be used on humans, animals, and fragile objectsMethod and level of microbial control based on site of medical procedure
12 Organismal 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 disinfectionAlso, the same organism may differ in susceptibility depending on growth phase with actively growing organisms typically more susceptible to disinfection than not-growing cultures
14 Relative Susceptibility of Microorganisms Effectiveness of germicides classified as high, intermediate, or lowHigh-level kill all pathogens, including endosporesIntermediate-level kill fungal spores, protozoan cysts, viruses and pathogenic bacteriaLow-level germicides kill vegetative bacteria, fungi, protozoa, and some viruses
15 Environmental conditions that pertain Increasing temperatures increases the efficacy of most chemical antimicrobialsThe converse of this statement is that relatively cold temperatures result in relatively poor disinfectionThe 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.
16 Environmental conditions that pertain (cont.) Concentration effects: Generally, the use of more disinfectant provides better killing than the use of less disinfectant
18 Methods for Evaluating Disinfectants and Antiseptics Phenol CoefficientUse-Dilution TestDisk-Diffusion MethodIn-Use Test
19 Phenol CoefficientEvaluating the efficacy of disinfectants and antiseptics by determining the ratio of agent’s ability to control microbes to that of phenolGreater than 1.0 indicates that agent is more effective than phenolHas been replaced by newer methods
20 Use-Dilution TestEspecially 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 driedContaminated cylinder immersed into dilution of disinfectant for 10 minutesCylinders removed, washed, and placed into tube of medium for 48 hMost effective agent entirely prevents growth at highest dilution
21 Disk-Diffusion Method A method that requires less manipulation to judge the efficacy of disinfectantsHere 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 organismThe clear area around the disk following incubation is used as an indication of disinfectant efficacy
23 In-Use TestSwabs taken from objects before and after application of disinfectant or antisepticSwabs inoculated into growth medium and incubatedMedium monitored for growthAccurate determination of proper strength and application procedure for each specific situation
24 Physical Methods of Microbial Control Exposure to extremes of heatExposure to extremes of coldDesiccationFiltrationOsmotic pressureRadiation
25 Heat-Related Methods Effects of high temperatures Denaturation of proteinsInterference with integrity of cytoplasmic membrane and cell wallsDisruption of structure and function of nucleic acidsThermal death point – lowest temperature that kills all cells in broth in 10 minutesThermal death time – time to sterilize volume of liquid at set temperature
27 Moist Heat Used to disinfect, sanitize, and sterilize Kills by denaturing proteins and destroying cytoplasmic membranesMore effective than dry heat; water better conductor of heat than air. Moist heat is also more penetrating than dry heatMethods of microbial control using moist heatBoilingAutoclavingPasteurizationUltrahigh-Temperature Sterilization
28 BoilingKills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses within 10 minutes at sea levelTemperature cannot exceed 100ºC at sea level; steam carries some heat awayBoiling time is criticalWater boils at lower temperatures at higher elevations; requires longer boiling timeEndospores, protozoan cysts, and some viruses can survive boiling
29 AutoclavingPressure applied to boiling water prevents steam from escapingBoiling temperature increases as pressure increasesAutoclave conditions – 121ºC, 15 psi, 15 minutesVolumeContactWrappingTesting
33 Sterile Services Department A sterile services department (SSD) is vital for an effective infection program. Dirty, recyclable equipment should be collected from the wards and transferred to the SSD, where it is washed, inspected, sterilized, packed, and dispatched back to the wards.
34 Sterile Services Department (cont.) Workflow of the SSD - in the ward, dirty re-usable instruments are collected and put into clearly labelled containers and delivered to the SSD. The dirty instruments are then received in the dirty area of the SSD. All equipment is first washed in hot water and detergent. The equipment is then inspected for cleanliness and damage. Instruments are then packed into individual trays for ward use and autoclaved and/or disinfected as required. The packaged trays are then inspected to ensure that they are dry and then sorted for ward collection. The sterile packs should be stored in well-ventilated rooms ready for dispatch to the wards. Collections should be regular and there should be a written record of receipt and delivery.
36 Pasteurization Pasteur’s method 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 pathogensPasteur’s methodToday, also used for milk, ice cream, yogurt, and fruit juicesNot sterilization; heat-tolerant and heat-loving microbes surviveThese do not cause spoilage prior to consumptionThese are generally not pathogenic
37 Pasteurization Milk Batch method – 30 minutes at 63ºC Flash pasteurization – 72ºC for 15 secondsUltrahigh-temperature pasteurization – 134ºC for 1 second
38 Ultrahigh-Temperature Sterilization 140ºC for 1 second, then rapid coolingTreated liquids can be stored at room temperatureChemical degradation
39 Dry HeatUsed for materials that cannot be sterilized with or are damaged by moist heatDenatures proteins and oxidizes metabolic and structural chemicalsRequires higher temperatures or longer time than moist heatNevertheless, application of dry heat is cheap and easyTypically one bakes materials in an oven at(i) 171ºC for at least one hour(ii) 160ºC for at least two hours(iii) 121ºC for at least 16 hoursIncineration – ultimate means of sterilizationLoops (1500°C), carcasses, dressings
40 Refrigeration and Freezing 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 itChemical reactions occur slower at low temperaturesLiquid water not availablePsychrophilic microbes can multiply in refrigerated foodsRefrigeration halts growth of most pathogens (Listeria, Yersinia)Slow freezing more effective than quick freezingOrganisms vary in susceptibility to freezing
41 Refrigeration 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 loweredLower temperatures result in greater long-term storage:(i) -20ºC is the temperature of a standard freezer(ii) -80ºC is a good temperature for long-term storage of microorganisms(iii)-180ºC is the temperature of liquid nitrogen, a common medium for long-term storage of microorganisms
42 Desiccation and Lyophilization (Freeze-drying) Drying inhibits growth due to removal of water; only microbiostatic. Desiccation prevents organism metabolism, and is a good means of preventing organism growthDesiccation is an effective means of organism storage for those organisms capable of remaining viable upon desiccationLyophilization used for long term preservation of microbial culturesPrevents formation of damaging ice crystalsFreeze-drying involves freezing something and then evacuating air so that boiling occurs at low temperatures; this desiccates material thereby preventing deterioration and spoilageLiquid nitrogen or frozen carbon dioxide (dry ice)
44 FiltrationUsed to sterilize ophthalmic solutions, antibiotics, vaccines, liquid vitamins, enzymes, culture media eg, tissue cultureMembrane filters (nitrocellulose) with pore sizes 25µm to less than 0.01µmHigh-efficiency particulate air (HEPA) filters are used to filter the air flowing into aseptic environments ( e.g. operating rooms) and out of potentially contaminated ones (e.g., containment facilities)
48 Osmotic PressureHigh concentrations of salt or sugar in foods to inhibit growth (jam, jerky, pickled food, salted fish)Water in cell is drawn out by osmosis; cell desiccatesFungi have greater ability than bacteria to survive hypertonic environments
49 RadiationElectromagnetic radiation (enrgy without mass travelling in waves at the speed of light, 3x105km/sec)Shorter wavelength equals more energy and greater penetrationRadiation described as ionizing or nonionizing according to effects on cellular chemicals
51 Ionizing RadiationWavelengths shorter than 1 nm – electron beams, gamma rays, and X raysIonizing radiation is radiation that ionizes water; this temporarily turns water into an oxidizing agentCreate ions by ejecting electrons from atoms they strikeIons disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions; hydroxide ions denature other molecules (DNA)One potential application of ionizing radiation is as an antimicrobial is in food preservation
52 Ionizing RadiationElectron beams – effective at killing but do not penetrate wellUsed to sterilize some microbiological plastic ware, and medical and dental suppliesGamma rays – penetrate well but require hours to kill microbesUsed to sterilize some medical instrumentsX-rays require too much time to be practical for growth control
54 Nonionizing Radiation Wavelengths greater than 1 nmExcites electrons and causes them to make new covalent bondsAffects 3-D structure of proteins and nucleic acidsUV light causes thymine dimers in DNAUV light does not penetrate wellSuitable for disinfecting air, transparent fluids, and surfaces of objects
55 Chemical Methods of Microbial Control Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNAEffect varies with temperature, length of exposure, and amount of organic matterAlso varies with pH, concentration, and age of chemicalTend to be more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa
56 General RulesCertain rules should be followed when using disinfectants.The manufacturer’s instructions should be followed. The expiry date should be checked and the optimum dilution used.Articles to be disinfected should be washed and cleaned before disinfection.The disinfectant container should not be refilled without sterilizing between each use and not be topped up.Disinfectants should be supplied ready for use from the pharmacy and empty containers should be returned to the pharmacy. Empty containers should not be discarded or used to store any other solutions as this is potentially dangerous. Open containers of disinfectant should not be tolerated as there is a serious risk of contamination with multiple antibiotic-resistant bacteria, such as Pseudomonas species and spores.
57 General RulesDisinfectants should be used according to instructions at the optimum dilution. Some disinfectants may be rapidly inactivated by organic matter, therefore any object that is to be disinfected should be cleaned thoroughly with warm water and detergent prior to disinfection.When disinfectants are indicated for use on surfaces, they should be applied by wiping rather than bathing as bathing wastes disinfectants.Sustained-action disinfectant should be used for hand hygiene by staff and for cleaning the skin and mucous membranes of patients. Alcohol preparations over 40% are not recommended because of the risk of fire when use in conjunction with diathermy.In general, hard surfaces do not require disinfectants - warm water with detergent is usually sufficient to remove all organic contamination. The exceptions are where persistence of potentially dangerous pathogens, such as HIV or HBV is suspected, when the surface should be wiped by a disinfectant afterwards.
58 Chemical Methods of Microbial Control Major CategoriesPhenolsAlcoholsHalogensOxidizing agentsSurfactantsHeavy MetalsAldehydesGaseous AgentsAntimicrobics
59 Phenol and Phenolics Intermediate- to low-level disinfectants Denature proteins and disrupt cell membranesEffective in presence of organic matter and remain active for prolonged timeCommonly used in health care settings, labs, and homes (Lysol, triclosan)Have disagreeable odor and possible side effects
61 Alcohols Intermediate-level disinfectants Denature proteins and disrupt cytoplasmic membranesEvaporate rapidly – both advantageous and disadvantageousSwabbing of skin with 70% ethanol prior to injectionAlcohols should not be applied to wounds since they can cause tissue damageTinctures
62 Halogens Intermediate-level antimicrobial chemicals Believed that they damage enzymes via oxidation or by denaturing themIodine tablets, iodophores (Betadine®), chlorine treatment of drinking water, bleach, chloramines in wound dressings, and bromine disinfection of hot tubs
64 Oxidizing AgentsPeroxides, ozone, and peracetic acid kill by oxidation of microbial enzymesHigh-level disinfectants and antisepticsHydrogen peroxide can disinfect and sterilize surfaces of objectsCatalase neutralizes; not useful for treating open woundsOzone treatment of drinking waterPeracetic acid – effective sporocide used to sterilize equipment
65 SurfactantsSurfactants 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 solutesSoaps and detergentsSoaps have hydrophilic and hydrophobic ends; good degerming agents but not antimicrobialDetergents are positively charged organic surfactantsQuats – colorless, tasteless, harmless to humans, and antimicrobial; ideal for many medical and industrial application (benzalkonium chloride)Low-level disinfectantsPseudomonas
67 Heavy MetalsIons are antimicrobial because they alter the 3-D shape of proteins, inhibiting or eliminating their function (cysteine)Low-level bacteriostatic and fungistatic agents1% silver nitrate to prevent blindness caused by N. gonorrhoeaeThimerosal used to preserve vaccinesCopper controls algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks; interferes with chlorophyll
69 Aldehydes Compounds containing terminal –CHO groups Cross-link with amino, hydroxyl, sulfhydryl, and carboxyl groups to denature proteins and inactivate nucleic acidsOnly one liquid chemical, if properly used, is capable of rendering an item sterile. That chemical is glutaraldehyde. The item to be sterilized must be totally submerged in the glutaraldehyde solution for 10 hours. Before immersion, the item must be thoroughly cleansed and rinsed with sterile water or sterile normal saline. It should be noted that this chemical is extremely caustic to skin, mucous membranes, and other tissues i.e.Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure)Formalin used in embalming and disinfection of rooms and instruments
70 Gaseous AgentsEthylene oxide, propylene oxide, and beta-propiolactone used in closed chambers to sterilize itemsDenature proteins and DNA bycross-linking functional groupsUsed in hospitals and dental officesCan be hazardous to people, often highly explosive, extremely poisonous, and are potentially carcinogenicThe 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.
71 Development of Resistant Microbes Little evidence that extensive use of products containing antiseptic and disinfecting chemicals adds to human or animal healthThe use of such products promotes the development of resistant microbes
72 Waste disposalAll hospital waste should be disposed of so that it presents no risk of injury or contamination.Clinical waste is generated during routine patent care and surgery. It is potentially dangerous and should be clearly labelled as high risk. Examples of clinical waste include dressings, body fluids, pathology waste, IV needles and syringes, drainage bags etc. Laboratory waste should also come under the high risk category and should be autoclaved before leaving the department and labelled biohazard.Other hospital waste include non-clinical waste and kitchen waste. The latter should be disposed of properly because it is a potential source of pests and vermin and thus may pose an indirect threat to the hospital. Provision must also be made for the safe disposal of radioactive waste. A simple colour-coding system should be used to separate waste so that the different components can be treated safely.In general, non-clinical waste is taken to a compactor and clinical waste to an incinerator or if not possible, to a lime-pit. Under no circumstances should clinical waste, needles or syringes be sent to the municipal dump.All staff handling clinical waste should be adequately trained especially with regards for the protocol of action in case of accidents. All staff must be provided with adequate protective clothing and replacement garments. Hepatitis B immunization should be offered to all staff and proper records kept for such immunization.
73 Waste disposalIt is totally unjustified to recycle needles and syringes. Broken glass should be included in the sharps category. To dispose safely of broken glass, thick gloves should be worn and the pieces collected with a newspaper. The glass is then wrapped securely in the paper and either disposed of into the sharps container or if not practicable, into a cardbox box which is then marked and sealed.Sharps containers must be leak and puncture-proof. Have a handle that allows lifting, a non-reopenable lid, and carry a biohazard sign. They should be sealed and replaced when it is no more than two-thirds full. Sharps containers are treated as clinical waste and should be put into clinical waste bags before being incinerated.In the event of an inoculation or contamination accident, it must be documented by the senior manager and reported to the Infection Control Team so that appropriate action can be taken.There must be clearly defined policy on the action to be taken after the injury and all staff should be aware of the policy.
74 General Use ArticlesBedpans and Urinals - gloves should be worn to empty the bedpan and its contents directly into the bedpan disinfector. Alternatively, the contents may be flushed down the sluice or toilet and then washed thoroughly with warm water and detergent to remove all signs of organic contamination and dried. The bedpan disinfector is the preferred method and it functions at no less than 80oC for 1 minute.Washing bowls - these must be washed thoroughly between each patient and stored and inverted to dry. Fresh water and towels should be used for each patient.Towels, soaps, hairbrushes, shaving brushes etc. - all these items should be for individual use only and should never be shared.Crockery and cutlery - each patient should have an individual set. the crockery and cutlery should be washed in very hot water (>60oC) and detergent. Disposable crockery is only necessary in cases of strict isolation such as rabies.Mattresses and pillows - they are a major source of cross-infection. Wet mattresses must be changed. Contaminated mattresses should be washed with warm water and detergent. Mattresses should be covered with an impervious layer so that they can be cleaned thoroughly between patients. Damaged mattresses should be discarded as they may easily trap microorganisms.Thermometers - they should be washed in warm water and dried before being wiped over with a swab soaked in 70% isopropyl alcohol. They should never be soaked in disinfectants.Trolley tops - they should be washed with warm water and detergent
75 General Use Articles Endoscopy Unit Fibre-optic endoscopes are usually heat-labile and therefore require chemical disinfection. 2% glutaraldehyde should be used under strict controlled conditions as required by the COSHH regulations. Protoscopes and sigmoidoscopes may be disposable or reusable. The latter must be cleaned, sterilized, or disinfected by heat or 2% glutaraldehyde.Disposable EquipmentSyringes and needles - disposable syringes and needles should be used. Under no circumstances should fine-bore needles be recycled because they cannot be cleaned. If syringes are to be recycled, it must be well controlled. After thorough cleaning, the syringes must be autoclaved or processed in ethylene oxide.Administration sets - administration sets for IV fluids must be disposableUrinary catheters and drainage bags - these should be single-use and disposable
76 ICUs, Operating Theatres Ventilatory circuits - multiple-use circuits must be heat-disinfected for at least 80oC for 3 minutes or sterilized by autoclave or ethylene oxide between each patient. Filters may be used between circuits and if properly maintained, a ventilated patient may use the same circuit for 4-5 days before disinfection.Humidifiers - humidifiers are a common source of viruses and Gram-negative bacilli and should be emptied daily and refilled with distilled water. They should be disinfected when contaminated or when the respiratory circuit is changed. Routine heat disinfection is essential after each patient use and if humidification is required for a prolonged period, the humidifier should be cleaned thoroughly, dried, and filled daily with distilled water.Endotracheal suction catheters - they are usually disposable but may be used up to 24 hours on the same patient.Endotracheal tubes - these may be recycled after thorough cleaning and autoclaving. Disposable endotracheal tubes are available but are more expensive.Ambu-bags - they are extremely difficult to disinfect and become contaminated very quickly. Heat is the most reliable method and 2% glutaraldehyde is a less acceptable alternative.Oxygen-delivery face masks - these can be disposable or reusable. If reused, they should be washed thoroughly and wiped over with 70% isopropyl alcohol.Suction and drainage bottles - these are usually disposable, with a self-dealing inner container held in a clear outer plastic container. The outer container should be heat-disinfected or autoclaved after each use. Non-disposable bottles must be changed every 24 hours or sooner if full. They must then be washed and autoclaved. Recyclable connector tubing should be cleaned thoroughly and sterilized. The system must be closed and risk to staff from body fluids should be minimal.
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