Control of Microbes Physical and chemical methods to destroy or reduce microbes in a given area

2 Terms Sterilization – a process that destroys all viable microbes, including viruses & endospores Sterilization – a process that destroys all viable microbes, including viruses & endospores Disinfection – a process to destroy vegetative pathogens, not endospores Disinfection – a process to destroy vegetative pathogens, not endospores Sanitization – any cleansing technique that mechanically removes microbes Sanitization – any cleansing technique that mechanically removes microbes Degermation – reduces the number of microbes Degermation – reduces the number of microbes

3 Relative Resistance of Microbes Highest resistance Highest resistance Bacterial endospores Bacterial endospores Moderate resistance Moderate resistance Pseudomonas sp. Pseudomonas sp. Mycobacterium tuberculosis Mycobacterium tuberculosis Staphylococcus aureus Staphylococcus aureus Protozoan cysts Protozoan cysts Least resistance Least resistance most vegetative cells most vegetative cells Fungal spores Fungal spores enveloped viruses enveloped viruses Yeast Yeast Protozoan trophozoites Protozoan trophozoites

4 Factors that influence action of antimicrobial agents: 1. Number of microbes 2. Nature of microbes in the population 3. Temperature & pH of environment 4. Concentration or dosage of agent 5. Mode of action of the agent 6. Presence of solvents, organic matter, or inhibitors

5 Cellular Targets 1. Cell wall 2. Cell membrane 3. Cellular synthetic processes (DNA, RNA) 4. Proteins

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8 Practical Concerns Does the application require sterilization? Does the application require sterilization? Is the item to be reused? Is the item to be reused? Can the item withstand heat, pressure, radiation, or chemicals? Can the item withstand heat, pressure, radiation, or chemicals? Is the method suitable (dangerous)? Is the method suitable (dangerous)? Will the agent penetrate to the necessary extent? Will the agent penetrate to the necessary extent? Is the method cost- and labor-efficient & is it safe? Is the method cost- and labor-efficient & is it safe?

9 Methods of Physical Control 1. Heat 2. Cold temperatures 3. Desiccation 4. Radiation 5. Filtration

10 1. Heat Moist heat – use of hot water or steam Mode of action – denaturation of proteins, destruction of membranes & DNA Mode of action – denaturation of proteins, destruction of membranes & DNA Sterilization Sterilization autoclave 15 psi/121 o C/10-40min autoclave 15 psi/121 o C/10-40min intermittent sterilization – unpressurized steam at 100 o C min for 3 days intermittent sterilization – unpressurized steam at 100 o C min for 3 days Disinfection Disinfection Pasteurization <100 o C for seconds; kills Salmonella, Listeria & overall microbe count Pasteurization <100 o C for seconds; kills Salmonella, Listeria & overall microbe count (NOT STERILIZED!) Boiling at 100 o C for 30 minutes to destroy non-spore-forming pathogens Boiling at 100 o C for 30 minutes to destroy non-spore-forming pathogens

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12 Pasteurization Flash – 71.6 o C (161 o F) for 15 seconds Flash – 71.6 o C (161 o F) for 15 seconds Ultra – 82 o C (179 o F) for 3 seconds Ultra – 82 o C (179 o F) for 3 seconds UHT – 138 o C (280 o F) 1-2 seconds UHT – 138 o C (280 o F) 1-2 seconds

13 Dry heat uses higher temperatures than moist heat, can also sterilize incineration – o C combusts & dehydrates cells incineration – o C combusts & dehydrates cells dry ovens – o C- coagulate proteins dry ovens – o C- coagulate proteins Used for dry powders, pipettes, oily substances, syringes Used for dry powders, pipettes, oily substances, syringes Will not erode sharp objects Will not erode sharp objects

14 2. Cold Temperatures Microbiostatic – slows the growth of microbes Microbiostatic – slows the growth of microbes refrigeration 0-15 o C & freezing <0 o C refrigeration 0-15 o C & freezing <0 o C Can be used to preserve certain food, media and cultures Can be used to preserve certain food, media and cultures

15 3. Desiccation Gradual removal of water from cells, leads to metabolic inhibition Gradual removal of water from cells, leads to metabolic inhibition Used to keep some foods viable for longer periods of time Used to keep some foods viable for longer periods of time Pretzels, meat, fruits, pasta, rice, milk, etc Pretzels, meat, fruits, pasta, rice, milk, etc Not effective microbial control – many cells retain ability to grow when water is reintroduced Not effective microbial control – many cells retain ability to grow when water is reintroduced

16 4. Radiation 1.Ionizing radiation – removes electrons, deep penetrating power, breaks up DNA, UV light creates thymine pyrimidines, which interfere with replication gamma rays, X-rays, cathode rays (Gram + ) gamma rays, X-rays, cathode rays (Gram + ) used to sterilize medical supplies & food products (vitamins, drugs, sutures) used to sterilize medical supplies & food products (vitamins, drugs, sutures) 2. Non-ionizing radiation – does not remove electrons, little penetrating power, used to sterilize air, water & solid surfaces radio, microwave, infrared

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19 5. Filtration Physical removal of microbes by passing a gas or liquid through filter Physical removal of microbes by passing a gas or liquid through filter Inorganic: porcelain or ground glass in flask Inorganic: porcelain or ground glass in flask Organic: diotomaceous earth Organic: diotomaceous earth Membrane: cellulose acetate or polycarbonate Membrane: cellulose acetate or polycarbonate HEPA: high efficiency particulate air filter HEPA: high efficiency particulate air filter Used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms Used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms

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21 Chemical Control 1. Halogens 2. Phenolics 3. Chlorhexidine 4. Alcohols 5. Hydrogen peroxide 6. Detergents & soaps 7. Heavy metals 8. Aldehydes

22 1. Halogens Chlorine – Cl 2, hypochlorites (chlorine bleach), chloramines (ammonia added) Chlorine – Cl 2, hypochlorites (chlorine bleach), chloramines (ammonia added) Denaturation of proteins by disrupting disulfide bonds Denaturation of proteins by disrupting disulfide bonds Can be sporicidal in high concentrations Can be sporicidal in high concentrations Iodine - I 2, iodophors (betadine) Iodine - I 2, iodophors (betadine) Denature proteins Denature proteins Can be sporicidal in high concentrations Can be sporicidal in high concentrations Milder medical & dental degerming agents, disinfectants, ointments Milder medical & dental degerming agents, disinfectants, ointments

23 2. Phenolics Disrupt cell membranes & precipitating proteins; bactericidal, fungicidal, virucidal, not sporicidal Disrupt cell membranes & precipitating proteins; bactericidal, fungicidal, virucidal, not sporicidal (expensive, pungent odor, very caustic) (expensive, pungent odor, very caustic) Phenol derivative – cresol, less toxic Phenol derivative – cresol, less toxic creosote creosote Bisphenols Bisphenols Lysol Lysol Triclosan- antibacterial additive to soaps Triclosan- antibacterial additive to soaps Dial Dial

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25 3. Chlorhexidine Hibiclens, Hibitane, Oral B mouthwash Hibiclens, Hibitane, Oral B mouthwash A surfactant & protein denaturant with broad microbicidal properties A surfactant & protein denaturant with broad microbicidal properties Not sporicidal Not sporicidal Used as skin degerming agents for preoperative scrubs, skin cleaning & burns Used as skin degerming agents for preoperative scrubs, skin cleaning & burns

26 4. Alcohols Ethyl, isopropyl in solutions of 50-90% Ethyl, isopropyl in solutions of 50-90% Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi Not sporicidal Not sporicidal

27 5. Hydrogen peroxide Weak (3%) to strong (25%) Weak (3%) to strong (25%) Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O 2 gas – toxic to anaerobes Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O 2 gas – toxic to anaerobes Strong solutions are sporicidal Strong solutions are sporicidal

28 6. Detergents & soaps Quaternary ammonia cpds (cyclobutane pyrimidine dimers) act as surfactants that alter membrane permeability of some bacteria & fungi Quaternary ammonia cpds (cyclobutane pyrimidine dimers) act as surfactants that alter membrane permeability of some bacteria & fungi Not sporicidal Not sporicidal Soaps- mechanically (sanitization) remove soil and grease containing microbes Soaps- mechanically (sanitization) remove soil and grease containing microbes

29 7. Heavy metals Used by Romans and Greeks (on skin) Used by Romans and Greeks (on skin) Solutions of silver & mercury kill vegetative cells in low concentrations by inactivating proteins Solutions of silver & mercury kill vegetative cells in low concentrations by inactivating proteins Not sporicidal Not sporicidal Mercury Mercury Mercurochrome Mercurochrome Merthiolate Merthiolate Copper Copper Copper Sulfate used to clean water, mixed with lime and used on fungal infections (vineyards 1882) Copper Sulfate used to clean water, mixed with lime and used on fungal infections (vineyards 1882) Silver Silver Silver nitrate used in newborn eyes (Gonnorhea) Silver nitrate used in newborn eyes (Gonnorhea)

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31 8. Aldehydes Glutaraldehyde & formaldehyde kill by alkylating protein & DNA Glutaraldehyde & formaldehyde kill by alkylating protein & DNA Known as “Cold sterilization” Known as “Cold sterilization” Glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments Glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments Formaldehyde - disinfectant, preservative, toxicity limits use Formaldehyde - disinfectant, preservative, toxicity limits use Formalin 37gr/100ml of water (embalming fluid) Formalin 37gr/100ml of water (embalming fluid)

32 Gases & aerosols Ethylene oxide (ETO), propylene oxide, betapropiolactone & chlorine dioxide Ethylene oxide (ETO), propylene oxide, betapropiolactone & chlorine dioxide Strong alkylating agents, sporicidal Strong alkylating agents, sporicidal Gas autoclave Gas autoclave

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