VEM 022 Disinfection Lab P2 PDF

DISINFECTION VEM 022 Laboratory P2 Compiled by LJL ( August 2024 )  TERMINOLOGIES Steriliza on Steriliza on is the killing of all microorganisms in a material or on the surface of an object A surface or an object is either sterile or it is not...

DISINFECTION VEM 022 Laboratory P2 Compiled by LJL ( August 2024 )  TERMINOLOGIES Steriliza on Steriliza on is the killing of all microorganisms in a material or on the surface of an object A surface or an object is either sterile or it is not sterile, there are no grada ons in sterility Typically the last things to die when one a empts steriliza on is the highly heat- (and chemical-, etc.) resistant endospores Disinfec on Disinfec on means reducing the number of viable microorganisms present in a sample Not all disinfectants are capable of sterilizing, but, of course, all disinfectants are employed with the hope of disinfec ng Disinfectant A disinfectant is a chemical or physical agent that is applied to inanimate objects to kill microbes ti ti ti ti ti tt ti ti  An sep c Typically an an sep c is a chemical agent that is applied to living ssue to kill microbes Note that not all disinfectants are an sep cs because an an sep c addi onally must not be so harsh that it damages living ssue With this constraint imposed on an sep cs, in general an sep cs are either not as cheap or not as e ec ve at killing microbes as disinfectants ff ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti  Sani za on Sani za on is the cleaning of pathogenic microorganisms from public ea ng utensils and objects such as that done by the kitchen of a restaurant Cleansing technique that mechanically removes microbes Other terms: Sepsis – bacterial contamina on Asepsis – absence of signi cant contamina on Bactericidal – “-cidal” means to kill Bacteriosta c – “-sta c” means inhibi on of growth and mul plica on ti ti ti ti ti ti fi ti ti ti ti ti ti AIM OF STERILIZATION? Asepsis – is the prac ce to reduce or eliminate contaminants (such as bacteria, viruses, fungi, and parasites) from entering the opera ve eld in surgery or medicine to prevent infec on. Ideally, a eld is "sterile" — free of contaminants — a situa on that is di cult to a ain. However, the goal is elimina on of infec on, not sterility. ti ti fi ti ffi tt ti fi ti ti  MECHANISMS OF ACTION OF CHEMICAL AGENTS – Di erent disinfectants have di erent mechanisms of ac on – All disinfectants act by harming microorganisms in some manner – Mechanisms of harm include ! Protein denatura on ! Membrane disrup on ! Nucleic acid damage ! Inhibi on of metabolism ff ti ti ti ff ti  HOW STERILIZATION WORKS Cell wall maintains integrity of cell, when disrupted cannot prevent cell from burs ng due to osmo c e ects. Cell membrane contains the cytoplasm and controls passage of chemicals into and out of the cell. When damaged cellular contents leak out, or enter uncontrollably Viral envelope is responsible for a achment of virus to its target cells. Damage to the envelope interrupts viral replica on (i.e non-enveloped virus have greater tolerance to harsh condi ons) ti ti ff tt ti ti AN IDEAL DISINFECTANT SHOULD: Be fast ac ng even in the presence of organic substances, such as those in body uid; [resistant to inac va on] Be e ec ve against all types of infec ous agents without destroying ssues or ac ng as a poison if ingested; [broadly ac ve] Easily penetrate material to be disinfected without damaging or discoloring the material; [not poisonous or otherwise harmful] Be easy to prepare and stable even when exposed to light, heat, or other environmental factors; [penetra ng; not damaging to non-living materials; Be inexpensive and easy to obtain and use; Not have an unpleasant odor. [not unpleasant to work with] ti ff ti ti ti ti ti ti ti fl ti FACTORS INFLUENCING ABILITY TO KILL MICROBES Strength of the killing agent Exposure me of agent unto the microbe (contact me) Temperature of the environment o Rate of microbe death doubles with every 10C rise in temp Presence of organic material Environment around the area to be decontaminated (e.g pH) Type of microbe Number of microbes to be killed Material composi on ti ti ti SPAULDING CLASSIFICATION EH Spaulding believed that how an object will be disinfected depended on the object’s intended use. – CRITICAL - objects which enter normally sterile ssue or the vascular system or through which blood ows should be sterile. – SEMICRITICAL - objects that touch mucous membranes or skin that is not intact require a disinfec on process (high-level disinfec on[HLD]) that kills all microorganisms but high numbers of bacterial spores. – NONCRITICAL -objects that touch only intact skin require low-level disinfec on. ti fl ti ti ti There are two ways of controlling popula ons of microorganisms, (1) Physical methods, and (2) Chemical methods ti Chemical Disinfection 1. QUATERNARY AMMONIUM COMPOUNDS (QUATS) ! (One type of commonly employed ca onic detergent disinfectants are quaternary ammonium compounds (quats) ! Problems with the use of quats as disinfectants include ! Their inac va on by soaps ! Their inhibi on by divalent ca ons (calcium and magnesium ions) ! Their inhibi on by co on and other porous organic substances ! Their inability to kill certain species of Pseudomonas ti ti ti ti tt ti ti 2. ORGANIC ACIDS ! Various organic acids are employed especially as inhibitors of fungi and molds in foods ! For example, benzoate of soda is a sodium salt of benzoic acid, an organic acid 3. HEAVY METALS ! Various metals and metal salts are commonly employed to prevent microbial growth or kill microbes ! For example, silver nitrate has been and increasingly is again used to treat the eyes of newborns to kill any Neisseria gonorrhea that may have been acquired during passage down the birth canal ! A common example in Ohio is the treatment of ponds with copper sulfate (which is blue in water solu on) as an an -algal; note that though e ec ve at inhibi ng algal growth in ponds, it may be similarly disrup ve of algal growth ti ti ti ti ff ti 4. CHLORINE AND HYPOCHLORITE ION ! Drinking water is commonly disinfected using hypochlorite ! Hypochlorite may either be added directly (i.e., in the form of bleach) or created within water by bubbling chlorine gas through the water ! Note that chlorine is less e ec ve in the presence of signi cant organic compounds (basically because the hypochlorite ion interacts with--oxidizes-- organic compounds indiscriminately and thus is used up in the presence o excess organic compounds) ! (Bromine some mes is employed as a subs tute for chlorine) ti ff ti ti fi ff  5. HALOGENS Intermediate-level an microbial chemicals Believed that they damage enzymes via oxida on or by denaturing them Iodine tablets, iodophores (Betadine®), chlorine treatment of drinking water, bleach, chloramines in wound dressings, and bromine disinfec on of hot tubs Iodine ! Iodine is o en employed as a ncture or as an iodophor ! A ncture is an alcohol solu on of a substance, in this case iodine ! Iodine nctures may be employed as an sep cs Iodophors ! Iodophors are organic compounds that slow the release of iodine ! The iodophors addi onally serve as surfactants, thus increasing penetra on while simultaneously steadily supplying iodine over long ti ti ft ti ti ti ti ti ti ti ti ti  6. ALCOHOLS ! Alcohols work best as 70 to 99% mixtures with water ! Alcohol-water mixtures are addi onally more penetra ng than pure alcohols ! Either ethanol or isopropyl (rubbing) alcohol may be employed for disinfec ng ! Alcohols are especially appropriate for applica on to sites in which their propensity to evaporate away is convenient (e.g., to disinfect skin prior to injec on) ! The down side of alcohols is that they are not terribly penetra ng nor capable of killing endospores or other resistant cells ! Alcohols should not be applied to wounds since they can cause ssue damage ISOPROPYL ALCOHOL (70%) – Powerful disinfectant and an sep c – Mode of ac on: denatures proteins, dissolves lipids and can lead to cell membrane disintegra on – E ec vely kills bacteria and fungi – But does not inac vate spores! ff ti ti ti ti ti ti ti ti ti ti ti ti ti 7. PHENOL AND PHENOL DERIVATIVES (PHENOLICS) Intermediate- to low-level disinfectants Denature proteins and disrupt cell membranes Phenol and their deriva ves (called phenolics) are especially useful when disinfec ng materials contaminated with organic materials and remain ac ve for prolonged me Commonly used in health care se ngs, labs, and homes (Lysol, triclosan) Have disagreeable odor and possible side e ects Some phenolics are mild enough for use as an sep cs while others are too harsh or otherwise dangerous to be employed on living ssue ti tti ff ti ti ti ti ti ti 8. OXIDIZING AGENTS (HYDROGEN PEROXIDE) Peroxides, ozone, and per ace c acid kill by oxida on of microbial enzymes High-level disinfectants and an sep cs Hydrogen peroxide can disinfect and sterilize surfaces of objects Ozone treatment of drinking water Per ace c acid – e ec ve sporocide used to sterilize equipment Hydrogen peroxide (HOOH) Is a typical oxidizing agent ! Note that hydrogen peroxide is actually not a terribly e ec ve an sep c or disinfectant ! This is because bacteria and body ssues contain enzymes (catalase) that inac vate hydrogen peroxide ! On the other hand, the oxygen released upon inac va on can help oxygenate deep wounds and thus kill strict-anaerobe contaminants ti ff ti ti ti ti ti ti ti ti ff ti ti ti ti  9. ALKYLATING AGENTS – Formaldehyde, glutaraldehyde, and ethylene oxides are alkyla ng agents—they add carbon-containing func onal groups to biological molecules – Formaldehyde is employed to inac vate viruses and toxins to produce whole-killed vaccines and toxoid vaccines [whole-killed vaccines] – Glutaraldehyde is capable of sterilizing equipment, though to e ect steriliza on o en requires many hours of exposure ft ti ti ti ff ti Continuation of Alkylating agents Aldehydes Denature proteins and inac vate nucleic acids Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure) Formalin used in embalming and disinfec on of rooms and instruments ti ti Continuation of Alkylating agents Ethylene oxide A gaseous chemosterilizer that is especially useful due to its extraordinary penetra ng power; this allows it to penetrate all sorts of nooks and crannies Employed to sterilize prepackaged laboratory equipment that is otherwise destroyed by heat (e.g., plas c petri dishes) Ethylene oxide, propylene oxide, and beta-propiolactone used in closed chambers to sterilize items Denature proteins and DNA by cross-linking func onal groups Used in hospitals and dental o ces Can be hazardous to people, o en highly explosive, extremely poisonous, and are poten ally carcinogenic ti ti ffi ft ti ti 10. Surfactants Surface ac ve” chemicals that reduce surface tension of solvents to make them more e ec ve at dissolving solutes Soaps and detergents Soaps have hydrophilic and hydrophobic ends; good degerming agents but not an microbial Detergents are posi vely charged organic surfactants Quats – colorless, tasteless, harmless to humans, and an microbial; ideal for many medical and industrial applica on Low-level disinfectants ti ff ti ti ti ti ti Physical Antimicrobials Physical Methods Moist Heat Dry Heat Lyophiliza on Dessica on Pasteuriza on Radia on Filtra on Osmo c Pressure ti ti ti ti ti ti HEAT – Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes. – Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a liquid suspension will be killed in ten minutes. – Thermal Death Time (TDT): Minimal length of me in which all bacteria will be killed at a given temperature. – Decimal Reduc on Time (DRT): Time in minutes at which 90% of bacteria at a given temperature will be killed. Used in canning industry. ti ti A. DRY HEAT To e ect steriliza on dry heat typically requires higher temperatures than moist heat It also is less penetra ng and requires longer exposure Nevertheless, applica on of dry heat is cheap and easy Typically one bakes materials in an oven at 170ºC for at least one hour 160ºC for at least two hours 180ºC for at least ½ hours INCINERATION The Ul mate steriliza on Complete destruc on of paper cups, bags, wipes, soiled dressings, animal carcasses Hospital wastes ff ti ti ti ti ti ti B. MOIST HEAT – Used to disinfect, sani ze, and sterilize – Kills by denaturing proteins and destroying cytoplasmic membranes – More e ec ve than dry heat; water be er conductor of heat than air – Methods of microbial control using moist heat Boiling Autoclaving Pasteuriza on Ultrahigh-Temperature Steriliza on ff ti ti ti ti tt BOILING – Kills vegeta ve 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 me is cri cal – Water boils at lower temperatures at higher eleva ons; requires longer boiling me – Endospores, protozoan cysts, and some viruses can survive boiling BOILING TEMPERATURE IS LINKED TO PRESSURE – 100 C at 1 ATM – 121 oC at 2 ATM (1 barr) – 132 C at 3 ATM (2 barr) – 80 C at 0.5 ATM (Mont Blanc) – 70 C at 0.35 ATM (Mont Everest) – 40 C at 0.02 ATM (Mechanical vacuum) ti ti ti ti ti  PASTEURISATION – Pasteuriza on is the applica on of moist heat of less-than boiling temperatures to foods to prevent the growth of pathogenic microorganisms in the vegeta ve state (food-spoiling organisms as well as various heat-labile pathogens) – First used with milk: 72°C for 15 to 20 seconds – Or 62 C for 30 minutes – Hea ng to 80°C for 1 minute will kill most vegeta ve organisms – Milk and beer AUTOCLAVE – An autoclave is a high pressure device used to allow the applica on of moist heat above the normal-atmosphere boiling point of water – Exposure to 121ºC for 15 minutes is typically su cient to sterilize, at 15 psi – Again, the material must be 121ºC before the clock starts – Large items, large volumes, and items that are poorly penetrated by steam may take much longer than 15 minutes to sterilize – Steam under pressure STEAM QUALITY IS IMPORTANT! Ideal: – Saturated steam: 98% steam, 2% water vapor Not Ideal: – Dry Steam: superheated ti ti ti ffi ti ti ti OTHER PHYSICAL METHODS OF MICROBIAL CONTROL 1. FILTRATION – Removal of microbes by passage of a liquid or gas through a screen like material with small pores. Used to sterilize heat sensi ve materials like vaccines, enzymes, an bio cs, and some culture media. – High E ciency Par culate Air Filters (HEPA): Used in opera ng rooms and burn units to remove bacteria from air. – Membrane Filters: Uniform pore size. Used in industry and research. Di erent sizes: – 0.22 and 0.45um Pores: Used to lter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses. – 0.01 um Pores: Retain all viruses and some large proteins. ffi ti fi ti ti ff ti ti  2. IONIZING RADIATION – Ionizing radia on is radia on that ionizes water; this temporarily turns water into an oxidizing agent – Di erent types of ionizing radia on display di erent degrees of penetra on – One poten al applica on of ionizing radia on is as an an microbial in food preserva on Ionizing Radia on: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). – Dislodge electrons from atoms and form ions. – Cause muta ons in DNA and produce peroxides. – Used to sterilize pharmaceu cals and disposable medical supplies. Food industry is interested in using ionizing radia on. – Disadvantages: Penetrates human ssues. May cause gene c muta ons in humans. Microwave Radia on: Wavelength ranges from 1 millimeter to 1 meter. – Heat is absorbed by water molecules. – May kill vegeta ve cells in moist foods. – Bacterial endospores, which do not contain water, are not damaged by microwave radia on. – Solid foods are unevenly penetrated by microwaves. – Trichinosis outbreaks have been associated with pork cooked in microwaves. ff ti ti ti ti ti ti ti ti ti ti ti ti ff ti ti ti ti ti ti ti  ANTISEPTICS Chemical agents that slow or stop the growth of micro-organisms (germs) on external surfaces of the body and help prevent infec ons. An sep cs should be dis nguished from an bio cs that destroy micro-organisms inside the body, and from disinfectants, which destroy micro-organisms found on inanimate (non-living) objects. O en referred to as skin disinfectants. Most chemical agents can be used as both an an sep c and a disinfectant. The purpose for which it is used is determined by its concentra on. For example hydrogen peroxide 6% solu on is used for cleansing wounds, while stronger solu ons (>30%) are used in industry as a bleach and oxidising agent. ft ti ti ti ti ti ti ti ti ti ti ti RANKING OF MICROORGANISMS’ RESISTANCE TO DISINFECTION (from least to greatest) Vegeta ve bacteria < Enveloped Viruses < Vegeta ve Fungal Cells < Small non-enveloped virus < Fungal spores < Mycobacteria < Endospores SPOROCIDAL AGENTS Glutaraldehyde Formaldehyde Other aldehydes Chlorine-releasing agents Iodine and iodophores Peroxygens Ethylene oxide P-propiolactone ti ti INACTIVATION OF PRIONS – Steam steriliza on with NaOH – Alkaline cleaner (pH 2.2, 1 hr 23°C) – Copper plus perace c acid – Vapourized hydrogen peroxide ti ti WHAT IS AN INDICATOR OF STERILIZATION CONTROL? – On paper – Self -contained – Sealed ampulla (spores + broth) – Spores suspension – Tube witness (point of fusion)  EXCESSIVE USE OF CHEMICALS CAN CREATE RESISTANT MICROBES?? – Li le evidence that extensive use of products containing an sep c and disinfec ng chemicals adds to human or animal health – The use of such products promotes the development of resistant microbes STERILIZATION IS SUCCESSFUL WITH PROPER HYGIENE – Avoid cleanrooms when ill – Frequent bathing and shampooing – Avoid cosme cs such as face powder, hair sprays, perfumes and a ershave – Clothing should be clean, nonfrayed and nonlin ng – Avoid smoking tt ti ti ti ti ft ti