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Using Chemicals to Destroy Microorganisms and Viruses Chapter 5 Approaches to Control • Control mechanisms either physical or chemical – May be a combination of both – Physical methods • • • • Heat Irradiation Filtration Mechanical removal – Chemical methods • Use a variety of antimicrobial chemicals • Chemical depends on circumstances and degree of control required Approaches to Control • Principles of control – Sterilization • Removal of ALL microorganisms – Sterile item is absolutely free of microbes, endospores and viruses • Can be achieved through filtration, heat, chemicals and irradiation – Disinfection • Eliminates most pathogens – Some viable microbes may exist • Disinfectants = used on inanimate objects and surfaces • Antiseptics = used on living tissues Approaches to Control • Principles of control – Pasteurization • Brief heat treatment used to reduce organisms that cause food spoilage – Surfaces can also be pasteurized – Decontamination • Treatment to reduce pathogens to level considered safe to handle – Degerming • Mechanism uses to decrease number of microbes in an area – Particularly the skin (antiseptics) Approaches to Control • Principles of control – Sanitized • Implies a substantially reduced microbial population – This is not a specific level of control – Preservation • Process used to delay spoilage of perishable items – Often includes the addition of growth-inhibiting ingredients Approaches to Control • Situational considerations – Microbial control methods are highly variable • Depends on situation and degree of control required – – – – Daily life Hospital Microbiology laboratories Food and food production facilities – Water treatment Approaches to Control • Daily life – Washing and scrubbing with soaps and detergents achieves routing control • Hand washing single most important step to achieving control – Soap acts as wetting agent • Aids in mechanical removal of microorganisms – Removes numerous organisms from outer layer of skin » Normal flora usually unaffected because it resides in deeper layers Approaches to Control • Hospitals – Minimizing microbial population very important • Due to danger of nosocomial infections – Patients are more susceptible to infection – Pathogens more likely found in hospital setting » Numerous organisms develop antimicrobial resistance due to high concentrations of antibiotics • Instruments must be sterilized to avoid introducing infection to deep tissues Approaches to Control • Microbiology laboratories – Use rigorous methods of control • To eliminate microbial contamination to experimental samples and environment – Aseptic technique and sterile media used for growth » Eliminates unwanted organisms – Contaminated material treated for disposal » Eliminates contamination of environment Approaches to Control • Food and food production facilities – Retention of quality enhanced through prevention of microbial growth and contamination • Achieved through physical removal and chemical destroying organisms • Heat treatment most common and most reliable mechanism • Irradiation approved to treat certain foods • Chemicals prevent spoilage – Risk of toxicity Approaches to Control • Water treatment facilities – Ensures drinking water is safe – Chlorine generally used to disinfect water • Can react with naturally occurring chemicals – Form disinfection by-products (DBP) » Some DBP linked to long-term health risks • Some organisms resistant to chemical disinfectants Selection of Antimicrobial Procedure • Selection of effective procedure is complicated – Ideal method does not exist • Each has drawbacks and procedural parameters • Choice of procedure depends on numerous factors – Type of microbe – Extent of contamination • Number of organisms – Environment – Risk of infection – Composition of infected item Selection of Antimicrobial Procedure • Type of microorganism – Most critical consideration • Is organism resistant or susceptible to generally accepted methods? • Resistant microbes include – Bacterial endospores » Resistant to heat, drying and numerous chemicals – Protozoan cysts and oocysts » Generally excreted in feces and cause diarrheal disease – Mycobacterium species » cell wall structure initiates resistance – Pseudomonas species » Can grow in presence of many chemical disinfectants – Naked viruses » Lack envelope and are more resistant to chemical killing Bacterial endospores Clostridium botulinum – causes botulism, resists boiling, but autoclaving kills Protozoans Giardia lamblia and Cryptosporidium parvum Cause digestive problems Selection of Antimicrobial Procedure • Number of organisms initially present – Time it takes to kill it directly affected by population size • Large population = more time – Commercial effectiveness is gauged by decimal reduction time • a.k.a D value • Time required to kill 90% of population under specific conditions – Washing reduces time required to reach disinfection or sterilization Selection of Antimicrobial Procedure • Environmental conditions – Environmental conditions strongly influence effectiveness • pH, temperature and presence of organic materials can increase or decrease effectiveness – Most chemicals are more effective at higher temperatures and lower pH – Effectiveness can be hampered by the presence of organism molecules » Can interfere with penetration of antimicrobial agent Selection of Antimicrobial Procedure • Potential risk of infection – Medical items categorized according to potential risk of disease transmission • Critical items = come in contact with body tissues – Needles and scalpels • Semicritical instruments = contact mucous membranes but do not penetrate body tissues – Endoscope • Non-critical instruments = contact unbroken skin only – Show little risk of transmission – stethoscope Selection of Antimicrobial Procedure • Composition of the item – Some sterilization and disinfection methods inappropriate for certain items • Heat inappropriate for plastics and other heat sensitive items Heat as Control • Heat treatment most useful for microbial control – Relatively fast, reliable, safe and inexpensive • Heat can be used to sterilize or disinfect • Methods include – Moist heat – Dry heat Heat as Control • Moist heat – Destroys through irreversible coagulation of proteins – Moist heat includes • Boiling • Pasteurization • Pressurized steam Heat as Control • Boiling (100° C) – Destroys most microorganisms and viruses – Not effective means of sterilization • Does not destroy endospores • Pasteurization – – – – Pasteur developed to avoid spoilage of wine Does not sterilize but significantly reduces organisms Used to increase shelf life of food Most protocols employ HTST method • Heated to 72°C and held for 15 seconds – Other protocol UHT • Heated to 140°C - 150°C, held for several seconds then rapidly cooled Heat as Control • Pressurized steam – Autoclave used to sterilize using pressurized steam • Heated water steam increased pressure • Preferred method of sterilization – Achieves sterilization at 121°C and 15psi in 15 minutes • Effective against endospores • Flash autoclaving sterilizes at 135°C and 15psi in 3 minutes • Prions destroyed at 132°C and 15psi for 4.5 hours • Dry heat Heat as Control – Not as effective as moist heat • Sterilization requires longer times and higher temperatures – 200°C for 1.5 hours vs. 121°C for 15 minutes – Incineration method of dry heat sterilization • Oxidizes cell to ashes • Used to destroy medical waste and animal carcasses • Flaming laboratory inoculation loop incinerates organism – Results in sterile loop Other Physical Methods of Control • Heat sensitive materials require other methods of microbial control – Filtration – Irradiation – High-pressure treatment Other Physical Methods of Control • Filtration – Membrane filtration used to remove microbes from fluids and air – Liquid filtration • Used for heat sensitive fluids • Membrane filters allow liquids to flow through – Traps microbes on filter • Depth filters trap microbes using electrical charge – Filtration of air • High efficiency particulate air (HEPA) filter removes nearly all microbes from air – Filter has 0.3µm pores to trap organisms Other Physical Methods of Control • Radiation – Electromagnetic radiation • Energy released from waves – Based on wavelength and frequency » Shorter wavelength, higher frequency = more energy • Range of wavelength is electromagnetic spectrum • Radiation can be ionizing or non-ionizing Other Physical Methods of Control • Ionizing radiation – Radiation able to strip electrons from atoms – Three sources • Gamma radiation • X-rays • Electron accelerators – Causes damage to DNA and potentially to plasma membrane – Used to sterilize heat resistant materials • Medical equipment, surgical supplies, medications • Some endospores can be resistant Other Physical Methods of Control • Ultraviolet radiation – Non-ionizing radiation • Only type to destroy microbes directly • Damages DNA – Causes thymine dimers – Used to destroy microbes in air, drinking water and surfaces – Limitation • Poor penetrating power – Thin films or coverings can limit effect Other Physical Methods of Control • High pressure processing – Used in pasteurization of commercial foods • Does not use high temperatures • Employs high pressure – Up to 130,000 psi • Destroys microbes by denaturing proteins and altering cell membrane permeability Chemicals as Control • Chemicals can be used to disinfect and sterilize – Called germicidal chemicals • Reacts with vital cell sites – Proteins – DNA – Cell membrane Chemicals as Control • Potency of chemicals – Formulations generally contain more than one antimicrobial agent – Regulated by • FDA – Antiseptics • EPA – Disinfectants – Germicidal agents grouped according to potency • Sterilants = – Destroy all microorganisms • High-level disinfectants – Destroy viruses and vegetative cells, – Not endospores • Intermediate-level disinfectants – Kill vegetative cells fungi, most viruses, – Not endospores • Low-level disinfectants – Removes fungi, vegetative bacteria and enveloped viruses, – Not mycobacteria, naked viruses or endospores Chemicals as Control • Selecting appropriate chemical – Points to consider • Toxicity – Benefits must be weighed against risk of use • Activity in presence of organic material – Many germicides inactivated in presence of organic matter • Compatibility with material being treated – Liquids cannot be used on electrical equipment Chemicals as Control • Selecting appropriate chemical – Points to consider • Residue – Residues can be toxic or corrosive • Cost and availability • Storage and stability – Concentrated stock relieves some storage issues • Environmental risk – Is germicidal agent harmful to environment Chemicals as Control • Classes of chemicals – Germicides represent a number or chemical families • • • • • • • • • • Alcohols Aldehydes Biguanides Ethylene oxide Halogens Metals Ozone Peroxides Phenolics Quaternary ammonium compounds Preservation of Perishable Products • Preservation extends shelf-life of many products – Chemicals are often added to prevent or slow growth of microbes • Other methods include – Low temperature storage – Freezing – Reducing available water Chemicals as Control • Chemical preservatives – Numerous chemicals are used as preservatives • Formaldehyde, Quats, and phenols – Weak organic acids often used as food preservatives • Benzoic, ascorbic and propionic acids • Used in bread, cheese and juice • Mode of action – Alter cell membrane function – Interfere with energy transformation – Nitrates and nitrites used in processed meats • Inhibits germination of endospores and growth of vegetative cells • Have been shown to be potent carcinogen Chemicals as Control • Low temperature storage – Microbial growth is temperature dependent • Low temperatures slow down or stop enzymatic reactions of mesophiles and thermophiles – Some psychrophiles still able to grow – Freezing as means of food preservation • Essentially stops microbial growth • Irreversibly damages cell – Kills up to 50% of microbes » Remaining cells still pose potential threat Chemicals as Control • Reducing water availability – Decreasing water availability accomplished by salting or drying food. • Addition of salt increases environmental solutes – Causes cellular plasmolysis • Numerous bacteria can continue to grow in high salt environments – Staphylococcus aureus can survive in high salt concentrations • Desiccation or drying is often supplemented by other methods – Salting • Lyophilization (freeze drying) – Widely used to preserve foods like coffee, milk and meats