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LECTURES IN MICROBIOLOGY Control of Microbial Growth LESSON 7 Sofronio Agustin Professor Lesson 7 Topics Controlling Microorganisms: Physical Means Chemical Means 2 Overview Various Microbial Control Methods 3 Antimicrobial Action -static agents- inhibit growth of microbes Ex: bacteriostatic, fungistatic -cidal agents - destroy or kill microbes Ex: bactericidal, fungicidal 4 Terms Sterilization Disinfection Antisepsis Sanitation Degerming 5 Sterilization Complete removal or destruction of all viable microbes including endospores. Application: Surgical instruments and commercially packaged foods. Commercial sterilization: Killing C. botulinum endospores. 6 Disinfection Use of physical or chemical agents (disinfectant) to kill vegetative bacteria and other microbes except endospores. Example: 10% Bleach solution on bench tops. 7 Antisepsis Use of physical or chemical agents to kill pathogens on living tissue. Example: Scrubbing surgical patients with chemical agents. 8 Sanitization Reduction of microbial counts to acceptable levels of public health standards. Example: Sanitization of eating utensils in restaurants. 9 Degerming Physical removal or reduction of microbes from a limited area. Ex: Washing of hands with germicidal soap. 10 Efficacy Depends On Number of microbes Target microbe characteristics Environmental factors temperature, pH, biofilms Concentration and mode of action of agent Organic matter - sputum, blood, feces 11 Factors Factors that influence the effectiveness of antimicrobial agents 12 Mode of Action Damage to cell wall Alteration of membrane function Damage to proteins Damage to nucleic acids 13 Damage to Cell Wall Effects on bacteria and fungi: Blockage of cell wall synthesis Degradation of cell wall components Reduction of its stability and integrity Ex: Penicillin, detergents, alcohols 14 Injury to Cell Membrane Effects on microbes including enveloped viruses: Bind and penetrate membrane lipids Loss of selective permeability resulting in leakage of cytoplasmic contents. Ex: Surfactants - surface active agents. 15 Surfactant Action The effect of surfactants on the cell membrane. 16 Effects on Nucleic Acids Irreversible binding to microbial DNA results in: Ceasation of transcription and translation Mutations Ex: Formaldehyde and ionizing radiation 17 Effects on Proteins Blockage of enzyme active sites prevents binding of substrate. Protein denaturation. Example: Heat, acids, alcohols, phenolics, and metallic ions. 18 Effects on Proteins Effects of heat, pH, and blocking agents on protein function. 19 Physical Methods Heat Radiation Filtration 20 Effects of Heat Moist heat: Coagulation of proteins Denaturation of proteins Dry heat: Dehydration Denaturation Oxidation (burning to ashes) 21 Effects of Heat Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min. Thermal death time (TDT): Time needed to kill all cells in a culture. Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature. 22 Moist Heat Boiling Tyndallization Pasteurization Steam under pressure 23 Boiling Water Boiling at 100 ˚C for 10-30 minutes kills most non-spore forming pathogens. Ex: Boiling of baby bottles and unsafe drinking water Hepatitis B virus needs at least 1 hour of boiling to be killed. 24 Tyndallization Intermittent use of free-flowing steam for 30 to 60 minutes. Ex: Used on heat-sensitive media, canned foods. Note: This will not destroy spores. 25 Pasteurization Pasteurization reduces spoilage caused by microbes and kills pathogens. Thermoduric microbes survive Methods : Classical: 63 ˚C for 30 minutes High Temperature Short Time: 72 ˚C for 15 seconds Ultra High Temperature: 140 ˚C for < 1 second Used in milk industry, wineries, breweries. Prevents transmission of milk-borne diseases caused by: Salmonella, Campylobacter, Listeria, Mycobacteria. 26 Steam and Pressure Used in media preparation and glassware sterilization. Ex: Autoclave and pressure cooker Autoclave setting at 121 ˚C, 15 p.s.i. pressure for at least 15 minutes effectively destroys spores. 27 Autoclave 28 Dry Heat Flaming Hot air Incineration Requires greater temperature and exposure time than moist heat 29 Hot Air Hot air oven Effective at 170˚C for 2 hrs Useful for sterilization of glasswares and oils 30 Incineration Destroys microbes to ashes or gas Bunsen flame - up to 1870˚C Ex: Sterilization of loops and needles. Furnace - 800˚C to 6500˚C Ex: Incineration of animal carcasses. 31 Incinerator An infrared incinerator using flame to burn or oxidize materials into ashes. 32 Radiation Ionizing radiation: X-rays Gamma rays Electron Beams Nonionizing radiation: Ultraviolet light Microwave -kills by heat, not especially microbicidal. 33 Radiation 34 Radiation Effects Ionizing radiation: Ejects orbital electrons from an atom High energy - penetrates liquids and solids effectively. Nonionizing radiation: Raises atoms to a higher energy level Low energy - less penetrating UV - formation of pyrimidine dimers 35 Cellular Effects of Radiation 36 Ultraviolet Radiation UV radiation cause the formation of pyrimidine dimers on DNA. 37 Applications of Radiation Ionizing radiation: Alternative sterilization method Materials sensitive to heat or chemicals Some foods (fruits, vegetables, meats) Nonionizing radiation: Alternative disinfectant Germicidal lamp in hospitals, schools, food preparation areas (inanimate objects, air, water) 38 Gamma Irradiation (a) Gamma radiation machine used to sterilize fruits, vegetables, meats, fish, and spices (b) Radora symbol 39 UV Irradiation UV treatment system used to disinfect water. 40 Filtration Removes microbes and spores from liquids and air Perforated membrane of varying pore sizes (0.22 to 0.45 um) Applications: Liquids that are sensitive to heat (e.g. serum,vaccines, media) HEPA filtration of operating rooms etc. 41 Membrane Filter (a) Membrane filtration system. (b) Membrane filter close-up. 42 Cold and Desiccation Cold temperatures - reduce microbial activity except psychrophiles. Refrigeration Freezing Desiccation - dehydration stops microbial metabolism. Lyophilization – freeze drying in a vacuum preserves microbes and vaccines. 43 Classes of Chemical Agents Phenols and Phenolics Biguanides Halogens Alcohols Heavy metals Surfactants - detergents and soaps Aldehydes Peroxygens Gases Dyes, acids, and alkali 44 Phenol and Phenolics Phenol - carbolic acid Phenolics- Lysol Bisphenols Hexachlorophene Triclosan Disrupt plasma membranes Ingredients in cutting boards, kitty litter 45 Biguanides Used as surgical hand scrubs and preoperative skin preparation Strong binding affinity to skin and mucus membranes Disrupt plasma membranes Ex: Chlorhexidine 46 Halogens Oxidizing agents Disrupt sulfhydryl groups in amino acids Iodine - topical antiseptic Tincture or Iodophor Chlorine -disinfectant and antiseptic Hypochlorous acid (HOCl) -germicidal Calcium hypochlorite used in dairy and restaurant industries 47 Alcohols Dissolve membrane lipids, denatures proteins. Used for skin degerming. Ethanol, Isopropanol 70% concentration most effective 48 Soaps and Detergents Quaternary ammonium (quats): Cationic Denature proteins and disrupt cell membrane Low-level disinfectant in the clinical setting Soaps and detergents: Fatty acids, oils, sodium or potassium salts Sanitizing and degerming agents More effective if mixed with germicides 49 Detergent Action Positively charged region of the detergent binds with bacteria and the uncharged region integrates into the cell membrane 50 Hand Scrubbing Comparison of efficacy between nongermicidal and germicidal soaps used in hand scrubbing. 51 Heavy Metals Ag, Hg, Cu Denature proteins Oligodynamic action Preservatives in cosmetics and ophthalmic solutions. Silver nitrate - Ophthalmia neonatorum prophylaxis. Silver sulfadiazine - burn patients and catheter tips. 52 Oligodynamic Action 53 Aldehydes Crosslink with functional groups of proteins on the cell surface (-NH2, -OH, -COOH, -SH) Formaldehyde, Glutaraldehyde Sterilants for surgical and dialysis instruments 54 Aldehyde Effect 55 Gases Denature proteins and affect functional groups of DNA. Sterilizes plastic materials Example: Ethylene oxide 56 Gas Sterilization Ethylene oxide sterilization chamber 57 Peroxygens Oxidizing agents - form hydroxyl free radicals Effective against anaerobes Examples: H2O2 - skin and wound cleaner Peracetic acid - quick method for sterilizing medical equipment O3 - water treatment 58 Food Preservatives Prevents spore germination Inhibits metabolism Control molds and bacteria in food and cosmetics Examples: Organic acids - sorbic, benzoic, acetic acids Calcium propionate - bread Nitrite prevents endospore formation in meat products. 59 Summary of Chemical Methods Chemical agents commonly used in healthcare 60 Efficacy of Disinfectants Principles of effective disinfection Concentration of disinfectant Presence of organic matter Temperature pH Time 61 Evaluation of Efficacy Use-Dilution Test: Metal rings dipped in test bacteria are dried. Dried rings placed in disinfectant for 10 minutes at 20oC. Rings transferred to media to determine bacterial death/survival. 62 Evaluation of Efficacy Disk-Diffusion Method (Kirby-Bauer) 63 Efficacy of Chemical Agents Chemical agent Phenolics Quats Chlorines Alcohols Glutaraldehyde Effectiveness against Endospores Poor None Fair Poor Fair Mycobacteria Good None Fair Good Good 64 Antimicrobial Resistance 65 Antimicrobial Resistance Highest resistance - bacterial spores and prions. Moderate resistance - some bacteria, protozoan cysts, fungal (sexual) spores, naked viruses. Least resistance - most bacteria, fungal (asexual) spores and hyphae, enveloped viruses, yeast, protozoan trophozoites. 66 Comparative Resistance Relative resistance of bacterial endospores and vegetative cells 67